Enhancer of zeste

DEVELOPMENTAL BIOLOGY

Embryonic

The E(Z) transcript has a strong maternal component, but this mRNA is degraded from 2 to 4 hours after fertilization. Similarly, the amount of zygotic transcript increases from 4 to 8 hours, and later drops off. This indicates that E(z) is required only transiently for gene silencing (Jones, 1993).

Effects of Mutation or Deletion

Embryos produced from E(z) mutant females display strong posteriorly directed homeotic transformations in which virtually all segments bear eight abdominal segment-like denticles. Embryos from mutant females were selected to assure no effect would be due to the mother's E(z) gene product. The developmental results are due to the loss of control of Abdominal B expression in more anterior segments. Higher Abdominal B levels in the anterior transform these segments into a posterior phenotype. Introducing a trithorax mutation into E(z) mutant offspring reverses this phenotype (Jones, 1993).

P elements are a group of mobile DNA elements found in Drosophila. They transpose by a nonreplicative cut and paste mechanism that is controlled by a regulatory state known as P cytotype. The existence of a state prohibitive for P-element transposition was initially recognized when reciprocal crosses were performed between strains that contain P elements (P strains) and those that lack P elements (M strains). If a P strain male is crossed to an M strain female, the P elements are mobilized in the germ line of the progeny, resulting in a series of abnormalities called hybrid dysgenesis. However, the progeny of a cross between an M strain male and a P strain female are normal, indicating that P strain females are able to repress P-element transposition in the germ line of their offspring. These reciprocal cross experiments led to the definition of a repressive state for P-element transposition, called P cytotype, and a permissive state for transposition, called M cytotype. Genetic experiments also indicate that P cytotype has both a maternal effect and is maternally inherited. Maternally derived cytoplasm is sufficient to confer the repressive state to offspring for one generation but maternal inheritance of chromosomal P elements is required for the maintenance of P cytotype through multiple generations (Roche, 1998 and references).

Molecular characterization of P strains reveals the existence of two types of P elements: full-length 2.9-kb elements and internally deleted P elements. A typical P strain contains 40-50 P elements: only approximately one-third of these elements are full-length. Full-length P elements encode two polypeptides, an 87-kD transposase protein and a truncated 66-kD repressor protein. Transposase production, and hence transposition, occurs only in the germ line due to the restricted splicing of the P-element third intron (IVS3) to this tissue. Retention of IVS3 in the germ line and the soma results in the production of the 66-kD repressor protein. Additional proteins that repress transposition in vivo are encoded by internally deleted elements, such as the KP element. The maternal component of P cytotype is thought to arise from a deposition of P-element repressor proteins into unfertilized oocytes, as observed for the P strains Harwich and pi2. The zygotic component of P cytotype could be due to a requirement for continuous repressor production by chromosomal P elements inherited by subsequent generations (Roche, 1998 and references).

An important component of P cytotype is transcriptional repression of the P-element promoter. Both the 66-kD and KP repressor proteins bind to a site within the P-element termini that overlaps the P-element promoter TATA element and the transposase binding site, suggesting that these proteins might affect P-element transcription. Indeed, transcriptional repression of P-lacZ enhancer trap elements in the germ line and the soma occurs in a P cytotype-dependent manner. In addition, the transposase protein inhibits the binding of TFIID to the P-element promoter TATA element, which results in transcriptional repression of the P-element promoter in vitro. Other mechanisms of P cytotype regulation may involve antisense P element RNA, the formation of inactive transposase-repressor protein heteromultimers, or a competition between transposase and repressor proteins for binding to their common site at the P-element termini (Roche, 1998 and references).

A role for chromatin structure in P cytotype transcriptional repression was proposed because heterologous promoters contained within P-element ends are repressed by P cytotype. For example, germ-line-expressed hsp83 or vasa-IVS3-beta-geo reporter transgenes are transcriptionally repressed in a P cytotype-dependent manner. Neither the hsp83 nor vasa promoter contains binding sites for P-element protein products, suggesting that P cytotype transcriptional repression may occur through a chromatin-based transcriptional silencing mechanism. Repression of a P[white] transgene by zeste¹ is enhanced in a P-element background and may be explained by an influence of P-element products on chromatin organization. Alterations of chromatin structure might also be responsible for the suppression of the phenotype of cytotype-dependent vestigial (vg) alleles in the presence of P cytotype. Therefore, P cytotype transcriptional repression may not occur solely by a simple repressor-operator interaction (Roche, 1998 and references).

The ability of a strain containing P elements to exhibit P cytotype is strongly determined by the genomic position of the repressor-producing elements. A study in which 17 inbred (over 100 generations of inbreeding) P-element-containing fly lines were examined for P cytotype resulted in the identification of only three lines that could repress hybrid dysgenesis. All three lines contained at least one complete P element that was located at cytological position 1A, at the tip of the X chromosome. Other studies of the distribution of P elements in natural Drosophila populations, and in local transposition experiments using a minichromosome, demonstrated that 1A is a hotspot for P-element insertion. By outcrossing and recombination, a strain was created from one of the inbred P lines that contained only two P elements, both of which were located at cytological position 1A. This strain, Lk-P(1A), completely represses transposition in the germ line and does not have a strong ability to induce hybrid dysgenesis, even though both elements are complete (Roche, 1998 and references).

A study of the P strain Lk-P(1A) was undertaken to learn more about the role of chromatin in P cytotype repression. This strain contains two full-length P elements inserted in the heterochromatic telomere-associated sequences (TAS elements) at cytological location 1A. Mutations in the Polycomb group gene (Pc-G gene) Enhancer of zeste [E(z)], whose protein product binds at 1A, result in a loss of Lk-P(1A) cytotype control. E(z) mutations also affect the trans-silencing of heterologous promoters between P-element termini by P-element transgenes inserted in the TAS repeats. The maternal effect of E(z) mutations on Lk-P(1A) P cytotype suggests that a complex required for P cytotype repression is established early in development and is stably maintained throughout the life cycle of the fly. These data suggest that pairing interactions between P elements, resulting in exchange of chromatin structures, may be a mechanism for controlling the expression and activity of P elements (Roche, 1998).

These experiments also reveal a new aspect of P cytotype repression. P-element repression may involve interactions between P elements inserted at different genomic positions to allow for the spread of inactive chromatin structures. Pairing interactions between P elements may be mediated solely by sequence homology. The conversion of sequences from one P element to an element on a homologous or nonhomologous chromosome, or to the site of a P-element-induced break, suggests that homology searching mechanisms do exist in Drosophila. Homology searching might permit sequence-dependent interactions between P elements in different genomic locations. Indeed, the variegated expression of P-element transgene arrays might be a result of pairing interactions between the repeated P-element transgenes. Alternatively, maternally-inherited proteins, such as the 66-kD repressor protein, might be involved in pairing interactions between P elements. The transposase-mediated trans-cleavage of the P-element ends, when contained on separate plasmids, indicates that interactions between P-element-encoded products bound to different transposons can occur. Although the P-lacZ or P[wA] elements cannot encode the 66-kD protein, they are capable of encoding an N-terminal 144 amino acid P-element peptide. This putative peptide would contain the DNA-binding and leucine zipper dimerization domains of the KP and 66-kD repressor proteins and could mediate interactions between the different P elements by dimerization of bound monomers. However, the 144 amino acid P-element peptide would be fused to ß-galactosidase in the case of the P-lacZ elements at 1A and none of the P-lacZ strains exhibit ß-galactosidase activity in the ovary (Roche, 1998 and references).

E(Z), like the 66-kD repressor protein, is maternally inherited and could also be mediating interactions between the different P elements. The Pc-G genes have been implicated in trans-silencing interactions, such as the z¹-w interaction and the cosuppression of repeated w-Adh transgenes. Whatever the mechanism, interactions between the ß-geo reporter transgene and the 1A (or 100F) P elements, could allow a chromatin complex to spread to the site of the reporter transgene. It is possible that E(Z) and HP1 are components of this complex. The repressive chromatin complex could stably repress expression of the reporter transgene throughout the life cycle of the fly. The heterochromatic TAS repeats might promote formation of a repressive protein complex which spreads, which may explain why only P elements inserted within the TAS repeats silence the reporter transgene. It is also possible that pairing interactions between P elements may result in silencing by altering the subnuclear localization of regions of the genome (Roche, 1998 and references).

It is becoming clear that P cytotype repression of P elements will be a good model system for studying gene regulation at a global chromosome level. By analyzing the silencing interactions of P elements, it may be possible to learn more about how chromosome structure and nuclear architecture influence gene expression, not only in Drosophila, but also in other organisms that silence repeated sequences (Roche, 1998).

The proteins encoded by two groups of conserved genes, the Polycomb and trithorax groups, have been proposed to maintain, at the level of chromatin structure, the expression pattern of homeotic genes during Drosophila development. To identify new members of the trithorax group, a collection of deficiencies were screened for intergenic noncomplementation with a mutation in ash1, a trithorax group gene. Five of the noncomplementing deletions uncover genes previously classified as members of the Polycomb group. This evidence suggests that there are actually three groups of genes that maintain the expression pattern of homeotic genes during Drosophila development. The products of the third group appear to be required to maintain chromatin in both transcriptionally inactive and active states. Six of the noncomplementing deficiencies uncover previously unidentified trithorax group genes. One of these deficiencies removes 25D2-3 to 26B2-5. Within this region, there are two allelic, lethal, P-insertion mutations that identify one of these new trithorax group genes. The gene has been called little imaginal discs based on the phenotype of mutant larvae. The protein encoded by the little imaginal discs gene is the Drosophila homolog of human retinoblastoma binding protein 2 (Gilde, 2000).

When heterozygous, trithorax mutations cause either no transformations or an extremely low frequency of transformations of the third thoracic segment to the second segment. However, when homozygous, trithorax mutations cause transformations of the first and third thoracic segments to the second segment and anterior transformations of the abdominal segments. Other genes in which mutations cause similar phenotypes have been classified as members of the trithorax group. Trithorax group genes have been identified by several approaches. Two of the trithorax group genes, ash1 and ash2, were identified as pupal lethal mutations that disrupt imaginal disc development. Most of the other trithorax group genes were identified in a genetic screen for dominant suppressors of the adult phenotypes of dominant Polycomb or Antennapedia mutations. Like mutations in Polycomb group genes, mutations in trithorax group genes show intergenic noncomplementation, i.e., heterozygosis for recessive mutations in two different trithorax group genes can cause an adult mutant phenotype. The phenotype can include partial transformations of the first and third thoracic segments to the second thoracic segment and partial anterior transformations of the abdominal segments. The similar phenotypes of mutations in trithorax group genes and their intergenic noncomplementation has suggested that the products of these genes also act via multimeric protein complexes. Indeed, a 2-MD complex has been detected in embryos that contains the products of the trithorax group genes, brahma. However, this complex does not contain the products of the trithorax group gene ash1, which is in a different 2-MD complex, which also contains the product of the trithorax gene, nor does this complex contain the product of ash2, which is in a 0.5-MD complex. Taking advantage of the phenomenon of intergenic noncomplementation, a large fraction of the Drosophila genome was screened to look for new trithorax group genes. Females heterozygous for an ash1 mutation were crossed to males heterozygous for one of 133 deficiencies and the progeny doubly heterozygous for the ash1 mutation and the deficiency for homeotic transformations were examined. In this way regions of the genome with candidate trithorax group genes were identified (Gilde, 2000).

Six of the deficiencies uncovered genes that were previously classified in the Polycomb group. They were so classified, because they either enhanced the Polycomb mutant phenotype or caused a phenotype like Polycomb mutants. This result was quite unexpected because the antagonism between trithorax and Polycomb group genes suggested that loss of function of Polycomb group genes should suppress trithorax mutant phenotypes, while these deficiencies showed an enhancement of trithorax group mutant phenotypes. Nevertheless it is likely that the Polycomb group genes uncovered by these deficiencies are responsible for the observed intergenic noncomplementation with ash1^RE418. It was thought possible that the observed intergenic noncomplementation is specific for ash1 mutations rather than general for mutations in trithorax group genes. This possibility was excluded for four of the five genes by showing that E(Pc)¹, Psc¹, Su(z)2¹, Asx^XF23, Asx³, and Asx¹³ also show intergenic noncomplementation with trx^b11 and/or brm² and increase the penetrance of two different double mutants: ash1^{VF101 trxb11} and brm² trx^e2. It has also been reported that Asx mutations show intergenic noncomplementation with mutations in trithorax group genes. In some of these cases, the different mutant alleles tested give inconsistent results. For example, both Scm^D1 and Scm^m56 show intergenic noncomplementation with ash1^VV183 and enhance the phenotype of the ash1^VF101 trx^b11 double mutant, whereas Scm³⁰² does not enhance the phenotype of ash1^VV183 and suppresses the phenotype of ash1^VF101 trx^b11. It is supposed that this difference is due to differences in the specific alterations of the Scm protein caused by these mutations (Gilde, 2000).

Until now the antagonism of function between the products of Polycomb group genes and trithorax group genes has been demonstrated unidirectionally by the suppression of Polycomb group mutant phenotypes by mutations in trithorax group genes. Advantage was taken of the intergenic noncomplementation of mutations in trithorax group genes to assay suppression of trithorax group mutant phenotypes by mutations in genes previously classified as Polycomb group genes. Among ash1^VF101;trx^b11 and brm²;trx^e2 double heterozygotes, 52% and 35%, respectively, of adult flies express transformations of the third thoracic segment to the second thoracic segment. Most mutations in seven of the genes that have been classified as members of the Polycomb group (Polycomb, polyhomeotic, pleiohomeotic, Polycomb-like, multi sex combs, extra sex combs, and Super sex combs) suppress the penetrance of these transformations, in both of these double heterozygotes. Moreover, most mutations in these genes do not show intergenic noncomplementation with mutations in any of the three trithorax group genes that have been tested. It is suggested that these genes represent the Polycomb group defined here as genes in which loss-of-function mutations enhance the dominant phenotype caused by Polycomb mutations and suppress the phenotype caused by heterozygosity for double mutations in trithorax group genes such as ash1^VF101;trx^b11 and brm²;trx^e2 (Gilde, 2000).

The zeste (z) gene encodes a transcription factor that binds DNA in a sequence-specific manner. The z¹ mutation causes reduced white gene transcription. Mutations in three genes identified as dominant modifiers of the zeste-white interaction, Enhancer of zeste, Suppressor of zeste-2, and Sex comb on midleg, can also cause phenotypes like mutations in Polycomb group genes. Here it is shown that mutations in these three genes also behave as mutations in trithorax group genes: they show intergenic noncomplementation with mutations in trithorax group genes and/or increase the penetrance of ash1^VF101;trx^b11 and/or brm²;trx^e2 heterozygotes. Moreover, mutations in three other genes identified as suppressors of the zeste-white interaction, Suppressor of zeste-4, Suppressor of zeste-6, and Suppressor of zeste-7, may show intergenic noncomplementation with mutations in trithorax group genes and/or increase the penetrance of ash1^VF101;trx^b11 heterozygotes. The biochemical mechanism by which mutations in these genes modify the zeste-white interaction is not known. However, it is thought to be significant that many of the genes identified as Suppressors of zeste behave as if they are both trithorax and Polycomb group genes; that Enhancer of Polycomb is a suppressor of zeste, and that sex combs extra is an enhancer of zeste (Gilde, 2000).

It is proposed that the six genes (previously classified as Polycomb group genes) belong in a distinct group; in these genes, loss-of-function or antimorphic mutations show intergenic noncomplementation with mutations in trithorax group genes and increase the penetrance caused by double heterozygosis of mutations in trithorax group genes. It is proposed that this group be called the ETP (Enhancers of trithorax and Polycomb mutations) group. Loss-of-function mutations in this group of genes not only enhance the dominant phenotype caused by Polycomb mutations, as do mutations in Polycomb group genes but they also enhance the phenotype caused by heterozygosity for double mutations in trithorax group genes, such as ash1^VF101;trx^b11 and brm²;trx^e2, as do mutations in trithorax group genes (Gilde, 2000).

Mutations in many of the genes that have been classified in the ETP group lead to ectopic expression of homeotic genes in embryos. It has been inferred from such results that the normal function of the products of these genes is to repress transcription. However, a recent study of the consequences of mutations in one of these genes, Enhancer of zeste, demonstrated both ectopic expression and loss of expression of the same homeotic genes. That study was made possible by the availability of a strong temperature-sensitive allele. Without such alleles it would be very difficult to directly assay other members of the group for loss of homeotic gene expression. Nevertheless, the enhancement of the phenotype of mutations in both Polycomb and trithorax group genes by loss-of-function mutations in genes of the ETP group is interpreted as an indication that the products of these genes are required for both activation and repression of transcription. It has been proposed that the product of the zeste gene itself is also involved in both activation and repression of transcription. Little information is available on the biochemical mechanism of action of any of these genes. There is evidence of a multimeric protein complex containing the products of the Polycomb group genes, Polycomb and Polyhomeotic, and of three different complexes containing the products of the trithorax group genes, brahma, ash1, and ash2. One way of rationalizing how mutations in the ETP group of genes could behave as both Polycomb and trithorax group mutations would be to suggest that the products of the ETP genes are components of complexes required for both repression and activation. Perhaps they are responsible for the structure of these complexes or different protein variants encoded by these genes are components of different complexes. Although Polycomb and trithorax group genes were first identified in Drosophila, homologous genes exist in mammals. Until now, most interpretations of the functions of the products of such genes have been based on the idea that the products of Polycomb group genes repress gene transcription and the products of trithorax group genes activate gene transcription. The data presented here together with earlier data suggest that some of the genes previously classified as Polycomb group genes and at least some of the genes identified as suppressors or enhancers of zeste belong to a group of genes whose products play a role in both the repression and activation of gene transcription. These data will require new interpretations of the functions of such genes (Gilde, 2000).

Two antagonistic groups of genes, the trithorax- and the Polycomb-group, are proposed to maintain the appropriate active or inactive state of homeotic genes set up earlier by transiently expressed segmentation genes. Although some details about the mechanism of maintenance are available, it is still unclear how the initially active or inactive chromatin domains are recognized by either the trithorax-group or the Polycomb-group proteins. An unusual dominant allele of a Polycomb-group gene, Enhancer of zeste, is described that mimics the phenotype of loss-of-function mutations in trithorax-group genes. This mutation, named E(z)^{Trithorax mimic} [E(z)^Trm], contains a single-amino-acid substitution in the conserved SET domain. The strong dominant trithorax-like phenotypes elicited by this E(z) allele suggest that the mutated arginine-741 plays a critical role in distinguishing between active and inactive chromatin domains of the homeotic gene complexes. The modification of E(z)^Trm phenotypes have been examined by mutant alleles of PcG and trxG genes and other mutations that alter the phosphorylation of nuclear proteins, covalent modifications of histones, or histone dosage. These data implicate some trxG genes in transcriptional repression as well as activation and provide genetic evidence for involvement of histone modifications in PcG/trxG-dependent transcriptional regulation (Bajusz, 2001).

In a screen for suppressors of Frontabdominal-7¹ (Fab-7¹), a dominant gain-of-function (GOF) mutation was isolated that transforms the sixth abdominal segment (A6) into A7, and exhibits strong dominant trx-like phenotypes. This mutation was termed Trithorax-mimic (Trm), since subsequent experiments proved that it was not allelic to any known members of the trxG. Trm heterozygotes show severe anteriorly directed transformations in the segments that fall under the control of BX-C. These include the partial transformation of the third thoracic (T3) segment into T2, the fifth abdominal segment (A5) into A4, and A6 into A5. Interestingly, A7 is rarely modified. The varying degree of transformation of different segments suggests that the mutation may affect cis-regulatory regions rather than the homeotic genes themselves. Expression of ANT-C is also affected, as shown by the reduced number of sex comb teeth and by the appearance of ectopic apical bristles on the first pair of legs (transformation of T1 toward T2. Homozygous Trm flies die as fully developed pharate adults with an extremely strong trx-like phenotype. For example, not only the haltere (T3) but also the central part of the humerus (T1) is often transformed into wing tissue. A7 is also partially transformed into A6, as indicated by the appearance of a rudimentary seventh tergite. The most extreme transformation is seen in the ventral genitalia of both sexes, which are frequently replaced by leg tissue (Bajusz, 2001). Additionally, clones of anterior wing tissue appear on the posterior wing-blades, indicating that the engrailed gene is inactivated in its normal domain of action (Bajusz, 2001).

It has been suggested that E(z) may be classified as a member of both the Pc-G and the trx-G. One possibility is that E(z) has two distinct functions, one in gene activation and another in gene silencing. In this view, E(z)^Trm could be considered as a dominant antimorphic mutation in the activating function with an essentially wild-type silencing function. On the basis of formal criteria, some observations appear to support this hypothesis. For example, the assumed antimorphic character of E(z)^Trm would be compatible with the findings that E(z)^Trm can be completely reverted by LOF mutations in cis, and its phenotype is enhanced by LOF E(z) alleles in trans and suppressed by extra copies of the E(z) gene. However, other observations are not compatible with this assumption. Thus, while simple LOF and well-characterized antimorphic alleles suppress the phenotype of zeste¹ and nanos, both of these phenotypes are enhanced by E(z)^Trm, suggesting that E(z)^Trm is an excess-of-function allele with respect to silencing. The fact that a single-amino-acid change is responsible for both features makes it unlikely that the E(z)^Trm [and E(z)^TrmTG] mutation affects two distinct and antagonistic functions. Rather, it suggests that the trithorax-like phenotype is the direct consequence of the hyperactivity of the mutant protein in silencing. This is supported by the observation that E(z)^Trm is not only reverted by LOF mutations in cis but it is also suppressed by antimorphic alleles, clearly deficient in silencing, in trans. Therefore, it is conceivable that a subfunction of the E(z) protein is to prevent ectopic or excessive inactivation of target genes by E(z) itself. It is hypothesized that the mutation in E(z)^Trm impairs this subfunction and consequently the mutant protein (partially) inactivates target genes in domains where they should stay active (Bajusz, 2001).

One possible explanation of the phenotype associated with E(z)^Trm could be that the mutant protein binds to ectopic sites. However, the data do not support this explanation: (1) the distribution of TRM protein on polytenic chromosomes suggests a binding specificity for TRM indistinguishable from wild type; (2) increasing the dose of wild-type E(z) gene proportionally suppresses the E(z)^Trm phenotype, indicating that wild-type E(z) competes with TRM for common targets (Bajusz, 2001).

These observations raise the possibility that E(z) may be present in both active and inactive domains of target genes and that it functions differently in the two domains. Indeed, preliminary genetic data suggest that E(z) is required not only for maintaining a silent state of inactive domains but also for setting the appropriate 'strength' of enhancers in active domains of BX-C. Strong reduction of E(z) activity together with a reduction in the number of PREs within a cis-regulatory domain results in a hyperactivation of the affected domain. In contrast to the wild-type protein, E(z)^TRM may be unable to differentiate between active and inactive chromatin domains of the target genes and, therefore, induces inappropriate silencing in active domains. This explanation implies that active or inactive domains are marked by a specific molecular label, which is recognized by the wild-type E(z) protein but not by E(z)^TRM (Bajusz, 2001).

Detailed comparison of E(z)^Trm to another GOF mutation, E(z)¹, supports this hypothesis. Although both Trm and E(z)¹ are dominant enhancers of the z¹-w⁺ interaction, E(z)¹, in sharp contrast to Trm, suppresses z¹ when an insufficient amount of wild-type E(z) protein is produced by the homolog. This suggests that the mutant protein encoded by E(z)¹ exerts its effect on the z¹-w⁺ interaction through the wild-type protein, possibly by forming a heteromeric complex with an altered conformation, which allows the heteromeric complex to generate a more efficient silencing of w. The white gene is not a normal target of E(z); only the binding of the mutant Z¹ protein renders w susceptible to silencing mediated by some PcG proteins, including E(z). Unlike E(z)^Trm, E(z)¹ does not cause an inappropriate inactivation of the homeotic genes or engrailed, suggesting that the E(z)¹-E(z)⁺ heteromeric complex recognizes some specific label present in the active state of its normal target genes (but not in white). In contrast to the E(z)¹-E(z)⁺ complex, the mutant TRM protein inappropriately inactivates target genes in regions where they are normally active, suggesting that it is unable to recognize this label (Bajusz, 2001).

It is noteworthy that the antimorphic E(z)^son1 and E(z)^son3 alleles, which strongly suppress E(z)^Trm, and the GOF E(z)¹ allele all contain point mutations within the SET domain. Although there is no direct biochemical evidence supporting the multimerization of E(z), these data would nevertheless indicate that two or more SET domains of E(z) form an interactive surface (a 'composite' SET domain). Taken together, these data suggest that this composite SET domain carries out two related subfunctions of E(z): It senses signals tethered to the active (or inactive) conformation of target genes and, in response to these signals, modulates PcG silencing (Bajusz, 2001).

How does the E(z) SET domain contribute to the modulation of PcG silencing of target genes? One possibility is suggested by the similarity between the mutant SET domain of TRM (and TRMTG) and the SET domain of wild-type Trx. A recent study has demonstrated that the SET domain of Trx can directly interact with two other trx-group proteins, ASH1 and SNR1, which are also thought to antagonize PcG silencing. TRM may counteract the activating effect of Trx by competing for one or both of these activators. This competition model would be consistent with the finding that the phenotype of E(z)^Trm is strongly alleviated by a duplication that provides an extra wild-type copy of trx. Since duplications of trx magnify the phenotypes of all loss-of-function alleles of E(z), Trx and wild-type E(z) may also compete for common factors in the inactive domains of target genes. It is conceivable that one of the functions of E(z) is to promote PcG-mediated silencing by competing with Trx (Bajusz, 2001).

The partial ectopic inactivation of target genes by E(z)^TRM provides a useful system for testing the effect of factors that are required for, or antagonize, PcG-dependent silencing. For example, all PcG mutations tested, including alleles of the Enhancer of trithorax and Polycomb (ETP) group, modify the E(z)^Trm phenotype. Interestingly, however, using the frequency of transformation of the third leg into the second as an indicator, it was found that none of the ETP alleles enhances the trx-like phenotype E(z)^Trm in T3. In fact, most of the ETP alleles suppress the T3 > T2 transformations as do other 'classical' PcG alleles. However, some of the ETP mutations do enhance the trx-like phenotype of E(z)^Trm in the abdomen. In some cases, even different alleles of the same gene may give opposite results (e.g., Psc¹ and Psc^e22). Moreover, while many of the trxG mutations enhance the E(z)^Trm phenotype as expected, others clearly suppress it in all or in some tissues, which might qualify the genes represented by the latter alleles as members of the ETP group. Two of these genes (sls and skd), originally identified as suppressors of Pc, have not previously been linked to gene silencing. These results show that the assignment of members of the trxG or the PcG to the ETP group greatly depends on the test system used and suggest that in some cases the unexpected or paradoxical phenotypes resulting from the combination of certain trxG and PcG mutations in trans may simply be the consequence of tissue- and allele-specific alterations of a global balance between activators and repressors of homeotic genes. Possible differences in target specificity and complex regulatory interactions among members of the trxG and PcG may be main factors in setting the actual activator/silencer ratio that is reflected in the final level of expression of target genes (Bajusz, 2001).

The phenotype of E(z)^Trm is highly sensitive to the dosage of histone genes, indicating that some components of PcG complexes are able to interact with nucleosomes and that this interaction is necessary to establish efficient silencing. However, as suggested by the effect of Su(var)2-1 mutations and early exposure to Na-butyrate, high levels of histone acetylation appear to be incompatible with the establishment of ectopic PcG-dependent silencing. Involvement of acetylated histones in antagonizing PcG-dependent silencing is supported by the finding that when a transgene containing a PRE is forcibly transcribed early in development, the PRE is unable to silence the reporter gene, concomitant with the appearance of a high level of acetylated histone H4 (but not H3) at the site of the insertion of the transgene (Bajusz, 2001).

A direct link between E(z) and histone deacetylation is suggested by the finding that the Drosophila E(z) binds directly to Esc, and the E(z)-ESC complex is associated with the histone deacetylase RPD3. Moreover, RPD3 is required for silencing mediated by a PRE in vivo. Suppression of E(z)^Trm by Su(var)2-1 mutations and early exposure to Na-butyrate is consistent with conserved inclusion of histone deacetylase activity in Drosophila ESC-E(z) complexes. However, since the interaction of ESC is mediated through an N-terminally located region of E(z), it is unclear how a mutation in the C-terminal SET domain can modify the functioning of HDAC2-ESC-E(z) complexes. Detailed studies of mutations like E(z)^Trm may shed some light on this question (Bajusz, 2001).

It is argued that active domains of PcG target genes should be labeled for recognition by E(z). The putative molecular label in the target domains is unlikely to be the acetylated histones, because Su(var)2-1 mutations suppress not only heterozygous but also homozygous E(z)^Trm, indicating that PcG complexes containing only the mutant E(z)^TRM protein are still able to recognize the difference in the degree of histone acetylation and that E(z)^Trm is not mutant in this respect. In contrast, homozygous E(z)^Trm is not affected by Su(var)3-6 mutations, indicating that the mutant protein is unable to respond to a decreased level of PP1. However, the presence of a wild-type allele renders E(z)^Trm suppressible by Su(var)3-6 mutations, suggesting that wild-type E(z) is able to respond to the level of phosphorylation of some proteins in the active domains. Protein phosphorylation has already been suggested to play a role in PcG silencing by the finding that ESC protein appears to become phosphorylated upon inclusion into the complex formed with E(z). However, this phosphorylation event is likely to be required for the normal ESC function and not for avoiding ectopic silencing, since replacement of the putatively phosphorylated amino acids in ESC results in an esc^- (weakened silencing) phenotype. The results of this study, however, suggest that protein phosphorylation may also play a role in marking active domains of PcG target genes. It is tempting to speculate that binding of a putative moderator phosphoprotein by the SET domain might reduce interactions of the wild-type E(z) with associated PcG proteins or reduce the ability of E(z) to compete with Trx, thus preventing inappropriate formation of the silencing complex in domains of target genes that are designated to be active. The replacement of arginine-741 by lysine in E(z)^Trm [and in E(z)^TrmTG] would prevent binding of the putative moderator, resulting in ectopic inactivation that mimics the consequence of the decreased abundance of Trx. In this view, lysine in the same position of the Trx SET domain would be preserved by selection to avoid its interaction with the moderator (Bajusz, 2001).

Mutations in Aurora kinase, a protein known to be involved in the phosphorylation of H3, enhance the Trm phenotype. Inhibition of PP1 (and PP2A) with okadaic acid increases the level of histone H3 phosphorylated at the amino acid residue Ser 10 in cultured cells, suggesting that PP1 may play a role in the dephosphorylation of phospho-H3. Moreover, biochemical evidence suggests that PP1 and aurora kinases are associated with the chromatin in Xenopus and PP1 regulates the activity of these kinases. These data are compatible with the assumption that the putative moderator of E(z) is the phosphorylated form of histone H3 (Bajusz, 2001 and references therein).

Telomeric associated sequences of Drosophila recruit Polycomb-group proteins in vivo and can induce pairing-sensitive repression

In Drosophila, relocation of a euchromatic gene near centromeric or telomeric heterochromatin often leads to its mosaic silencing. Nevertheless, modifiers of centromeric silencing do not affect telomeric silencing, suggesting that each location requires specific factors. Previous studies suggest that a subset of Polycomb-group (PcG) proteins could be responsible for telomeric silencing. This study presents the effect on telomeric silencing of 50 mutant alleles of the PcG genes and of their counteracting trithorax-group genes. Several combinations of two mutated PcG genes impair telomeric silencing synergistically, revealing that some of these genes are required for telomeric silencing. In situ hybridization and immunostaining experiments on polytene chromosomes reveal a strict correlation between the presence of PcG proteins and that of heterochromatic telomeric associated sequences (TASs), suggesting that TASs and PcG complexes could be associated at telomeres. Furthermore, lines harboring a transgene containing an X-linked TAS subunit and the mini-white reporter gene can exhibit pairing-sensitive repression of the white gene in an orientation-dependent manner. Finally, an additional binding site for PcG proteins was detected at the insertion site of this type of transgene. Taken together, these results demonstrate that PcG proteins bind TASs in vivo and may be major players in Drosophila telomeric position effect (TPE) (Boivin, 2003).

Among the 50 mutant alleles of PcG and trxG genes tested, <10 behave as dominant modifiers of TPE. By contrast, combination analyses reveal that 10 alleles that have no effect alone have synergistic effects on TPE. Interestingly, the subgroup of dominant suppressors that act alone on TPE (Pc, ph, Psc, and Scm) are members of the PRC1 complex that has been purified from embryonic nuclear extracts. Some other PcG mutations, such as Asx, E(z), Pcl, or Sce, act as suppressors in combination, suggesting that the products of these genes participate with a specific telomeric PcG complex. Strikingly, this subgroup of eight PcG genes was already highlighted in a genetic interaction study showing that Pc, Scm, Psc, Pcl, Sce, and Asx are lethal when heterozygous with ph², a temperature-sensitive mutation, all combinations leading to similar phenotypes in the dying embryos (Boivin, 2003).

It has been shown that telomeric inserts are less accessible than euchromatic inserts to restriction enzymes and to DAM methylase. In addition, the accessibility of telomeric inserts to DAM methylase increases in a ph⁴¹⁰ background and this is correlated to derepression of the white gene. This result is similar to that obtained with the ph PRE-mini-white transgenes suggesting that PcG products adopt a similar chromatin-based mechanism to repress their euchromatic and telomeric targets (Boivin, 2003).

PREs were initially identified by their ability to prevent ectopic activation of a Hox reporter gene construct. This capacity depends on the dose of the PcG proteins. Placed in a transgene, PREs can also induce mosaic expression of the flanking reporter gene, a phenotype resembling that of PEV and TPE. Moreover, PRE-mediated repression often exhibits pairing sensitivity, defined as the lower expression of the flanking reporter gene in a homozygous state than in a heterozygous one. This study shows that a 1.2-kb fragment of the 1.8-kb X-chromosome TAS induces variegation or pairing-sensitive repression in an orientation-dependent manner and creates new binding sites for the PcG proteins as detected by immunostaining on polytene chromosomes. These results demonstrate that this TAS fragment mimics some properties of a PRE and thus reinforce the parallels that can be made between telomeric silencing and PcG-mediated euchromatic repression. TASs from the left tip of chromosome 2 (2L-TAS) retain aspects of telomeric silencing in ectopic positions. At this telomere, TASs are composed of repeats of 457 bp that present only limited homology with TASs present at the X, 2R, and 3R telomeres. Analysis of the sequence of one repeat (457 bp) revealed nine GAF-binding sites but no PHO-binding site. Several transgenic lines have been establised carrying different constructs made up of 6 kb of 2L-TAS (~13 repeats) adjacent to the mini-white reporter gene and flanked by Su(Hw) insulator sequences. Depending on the orientation of the TASs inside the transgene, some lines present reduced expression of the mini-white gene when compared to lines carrying a similar transgene without TASs or with TASs in the opposite orientation. Such orientation-dependent silencing has been described for the Fab7 PRE of the Ubx gene, but does not appear to be a general property of PREs since another PRE from Ubx (Mcp) has been shown to function in both orientations . From this study, the more efficient orientation for the 1.2-kb X-TAS-induced repression appears to be the same as that described for the 2L-TASs: repression appears to be stronger from the centromere-proximal side (Boivin, 2003).

Repression induced by the 2L-TAS when inserted within a transgene is weakly sensitive to Su(z)2⁵. Surprisingly, no effect of PcG mutations on the repression induced by the 1.2-kb X-TASs could be detected, except a slight suppressor effect of Su(z)2⁵ on P-CoT-1 in a homozygous state. At the moment, no explanation is available for why the repression induced by the 1.2-kb X-TASs in a euchromatic environment is not sensitive to modification of the dose of PcG proteins that could otherwise affect TPE (Boivin, 2003).

Increasing the distance between the 2L-TAS and the mini-white gene with 2.4 kb of unrelated DNA in another transgene did not change the silencing capacity of 2L-TAS. In this study, the 1.2 kb of X-TAS is located >5 kb away from the mini-white gene, thus showing the silencing capability of TASs over an extended distance. Similar results were obtained with transgenes containing the Fab7 PRE. According to chromatin-immunoprecipitation experiments, PcG products can spread as far as 10-15 kb from PREs and repression could be expected to occur over such a distance (Boivin, 2003).

In fact, what was observed with the 1.2 kb of X-TAS in the pCoT- transgenes resembles what has been observed with PREs from the Bithorax complex. Using Fab7-mini-white transgenes, it has been shown that some insertion sites present pairing sensitivity (as observed with P-CoT-2 and P-CoT-3), while others present variegation with darker spots (as observed with P-CoT-1). The Fab7 PRE has been shown to convey a derepressed state through meiosis after being activated in the embryonic stage by the UAS/GAL4 system. In the case of TPE, the derepressed state observed in a PcG mutant background is not transmitted to the next generation. A fundamental difference between these studies is that the suppressor effect observed in the case of TPE is due to the lack of one PcG partner. It is hyperactivation (forced activation) induced by GAL4 via the UAS sequences that abolishes the repressor capacity of the Fab7 PRE. This activation likely involves fundamental changes in chromatin conformation and/or epigenetic marks (such as hyperacetylation) that may be different from the effect of a decrease in the dosage of a repressor. To compare TPE and the Fab7 PRE it would thus be interesting to test transmission through meiosis of the derepressed state of the UAS-Fab7 transgene induced by a PcG mutation rather than upon activation by GAL4. Different PREs thus share properties but also present particularities that likely depend on their sequence. Indeed, the dissection of Mcp, another PRE from the Bithorax complex, revealed that repression in cis and pairing-sensitive repression could be separated. This shows that PREs may combine several regulatory properties and future dissection of the different TASs will reveal which functions telomeric PREs combine (Boivin, 2003).

A mosaic genetic screen reveals distinct roles for trithorax and Polycomb group genes in Drosophila eye development

The wave of differentiation that traverses the Drosophila eye disc requires rapid transitions in gene expression that are controlled by a number of signaling molecules also required in other developmental processes. A mosaic genetic screen has been used to systematically identify autosomal genes required for the normal pattern of photoreceptor differentiation, independent of their requirements for viability. In addition to genes known to be important for eye development and to known and novel components of the Hedgehog, Decapentaplegic, Wingless, Epidermal growth factor receptor, and Notch signaling pathways, several members of the Polycomb and trithorax classes of genes, encoding general transcriptional regulators, were identified. Mutations in these genes disrupt the transitions between zones along the anterior-posterior axis of the eye disc that express different combinations of transcription factors. Different trithorax group genes have very different mutant phenotypes, indicating that target genes differ in their requirements for chromatin remodeling, histone modification, and coactivation factors (Janody, 2004).

Very similar phenotypes were observed in clones mutant for Pc or E(z), which encode components of two distinct complexes implicated in transcriptional repression. Although likely null alleles for both genes were used, the phenotype of E(z) clones appeared slightly stronger, with a greater likelihood of derepressing hth in posterior regions of the eye disc. The E(z) protein has been shown to act as a histone methyltransferase for H3 K27 within a complex that also includes Extra sex combs (Esc), Suppressor of zeste 12 [Su(z)12], and the histone-binding protein NURF-55. esc appears to act only early in embryonic development, while E(z) and Su(z)12 are continuously required to repress inappropriate homeotic gene expression in wing imaginal discs. The core PRC1 complex contains Pc, as well as Ph, Psc, and dRing1, and can prevent SWI/SNF complexes from binding to a chromatin template. Pc, Psc, and ph are all required to prevent homeotic gene misexpression in wing discs; however, Psc and ph act redundantly with closely related adjacent genes. The two complexes are thought to be linked through binding of the Pc chromodomain to K27-methylated H3. The stronger phenotype of E(z) mutations in the eye disc might suggest that methylation of H3 K27 can recruit other proteins in addition to Pc (Janody, 2004).

In the eye disc, loss of E(z) or Pc leads to misexpression of the homeotic gene Ubx, but this does not seem to account for the entire phenotype. Although Ubx is sufficient to turn on tsh ectopically, misexpression of hth and tsh can occur in E(z) or Pc clones in which Ubx is not misexpressed. This suggests that hth and tsh are either direct targets of Pc/E(z)-mediated repression or targets of a downstream gene other than Ubx, possibly one of the homeotic genes not examined. Tsh misexpression would be sufficient to explain the suppression of photoreceptor differentiation in clones close to the morphogenetic furrow, since it is able to maintain expression of Hth and Ey and, in combination with them, to repress eya. Misexpression of Tsh can also account for overgrowth of Pc or E(z) mutant cells at the posterior margin of the eye disc (Janody, 2004).

trithorax group genes were initially identified as suppressors of Polycomb phenotypes and are therefore thought to contribute to the activation of homeotic gene expression. Some members of the group encode components of the Brahma chromatin-remodeling complex, others encode components of the mediator coactivation complex, and still others encode histone methyltransferases. In addition to their distinct biochemical functions, members of the trithorax group act on different sets of target genes during eye development and can also have different effects on the same target genes. Components of the Brahma complex are strongly required for cell growth and/or survival; brm and mor, but not osa, are also absolutely required for photoreceptor differentiation. However, these three genes do not seem to be required for the restricted expression in anterior-posterior domains of the eye disc of the transcription factors examined. In contrast, the mediator complex subunits encoded by skd and kto are not required for cell proliferation, although they are strongly required for photoreceptor differentiation. trx, which encodes a histone methyltransferase, is required primarily for the normal development of marginal regions of the disc. No significant effect on photoreceptor differentiation were seen in clones mutant for kismet¹, which encodes chromodomain proteins, or ash2¹, which encodes a PHD protein. These differences are unlikely to be due to different expression patterns of the trithorax group genes, since Trx, Skd, Kto, and Osa are ubiquitously expressed in the eye disc (Janody, 2004).

The effects of these genes on the rapid transitions between domains of expression of different transcription factors are of particular interest. In the most anterior region of the eye disc, hth expression is enhanced by skd and kto. The domain just posterior to this also expresses tsh and ey, and activation of both of these genes requires trx. However, skd and kto have opposite effects on the two genes, enhancing tsh expression and preventing the maintenance of ey expression in posterior cells. Since Hth and Tsh can positively regulate each other's expression, it is possible that only one of these genes is directly dependent on skd and kto. Next, dac and h are activated transiently and eya is activated and sustained. The establishment of both dac and eya is delayed in trx mutant clones, and h expression is reduced. This delay in establishing the preproneural domain may be due to the failure to activate ey and tsh earlier in development, since Ey and Tsh combine to activate eya. The effect of Pc or E(z) mutations on eya, dac, and h appears very similar to the effect of trx mutations. However, in Pc or E(z) clones, the delay in eya and dac expression is likely to be caused by the failure to repress tsh and hth, since the combination of these two proteins has been shown to repress genes expressed in the preproneural domain. skd and kto clones also show a reduction in h and anterior eya expression, but an inappropriate maintenance of dac and dpp. These mediator complex components may thus contribute both to the activation of genes such as h in the preproneural domain and to the activation of unknown genes that shut off the expression of ey, dac, and dpp. Alternatively, skd and kto could be directly involved in the repression of these genes. Finally, trx is important to prevent misexpression of hth in cells near the posterior and lateral margins. Although Dpp normally represses hth, in trx mutant clones dpp and hth are both inappropriately expressed in marginal cells. This may reflect a role for trx in the process of morphogenetic furrow initiation, perhaps contributing to the ability of dpp to control gene expression (Janody, 2004).

Further study will be needed to determine which genes are direct targets of each trithorax group protein. However, the results point to a strong specificity of these general transcriptional regulators, suggesting that they may be specialized to mediate the effects of particular signaling pathways or to control specific subsets of downstream genes (Janody, 2004).

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. The expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing were examined by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate the described methods for expression profiling as a way to analyze developmental programs, and they show that modifications to chromatin structure are key to changes in cell fate. These findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells (Klebes, 2005).

When prothoracic (1st) leg discs are fragmented and cultivated in vivo, cells in a proximodorsal region known as the 'weak point' can switch fate and transdetermine. These 'weak point' cells give rise to cuticular wing structures. The leg-to-wing switch is regulated, in part, by the expression of the vestigial (vg) gene, which encodes a transcriptional activator that is a key regulator of wing development. vg is not expressed during normal leg development, but it is expressed during normal wing development and in 'weak point' cells that transdetermine from leg to wing. Activation of vg gene expression marks leg-to-wing transdetermination (Klebes, 2005).

Sustained proliferation appears to be a prerequisite for fate change, and conditions that stimulate growth increase the frequency and enlarge the area of transdetermined tissue. Transdetermination was discovered when fragments of discs were allowed to grow for an extensive period of in vivo culture. More recently, ways to express Wg ectopically have been used to stimulate cell division and cell cycle changes in 'weak point' cells (Sustar, 2005), and have been shown to induce transdetermination very efficiently. Experiments were performed to characterize the genes involved in or responsible for transdetermination that is induced by ectopic Wg. Focus was placed on leg-to-wing transdetermination because it is well characterized, it can be efficiently induced and it can be monitored by the expression of a real-time GFP reporter. These attributes make it possible to isolate transdetermining cells as a group distinct from dorsal leg cells, which regenerate, and ventral leg cells in the same disc, which do not regenerate; and, in this work, to directly define their expression profiles. This analysis identified unique expression properties for each of these cell populations. It also identified a number of genes whose change in expression levels may be significant to understanding transdetermination and the factors that influence developmental plasticity. One is lamina ancestor (lama), whose expression correlates with undifferentiated cells and is shown to control the area of transdetermination. Another has sequence similarity to the mammalian augmenter of liver regeneration (Alr; Gfer -- Mouse Genome Informatics), which controls regenerative capacity in the liver and is upregulated in mammalian stem cells. Fifteen regulators of chromatin structure [e.g. members of the Polycomb group (PcG) and trithorax group (trxG)] are differentially regulated in transdetermining cells, and mutants in seven of these genes have significant effects on transdetermination. These studies identify two types of functions that transdetermination requires -- functions that promote an undifferentiated cell state and functions that re-set chromatin structure (Klebes, 2005).

The importance of chromatin structure to the transcriptional state of determined cells makes it reasonable to assume that re-programming cells to different fates entails reorganization of the Polycomb group (PcG) and trithorax group (trxG) protein complexes that bind to regulatory elements. Although altering the distribution of proteins that mediate chromatin states for transcriptional repression and activation need not involve changes in the levels of expression of the PcG and trxG proteins, the array hybridization data was examined to determine if they do. The PcG Suppressor of zeste 2 [Su(z)2] gene had a median fold repression of 2.1 in eight TD to D_Wg/V_Wg comparisons, but the cut-off settings did not detect significant enrichment or repression of most of the other PcG or trxG protein genes with either clustering analysis or the method of ranking median ratios. Since criteria for assigning biological significance to levels of change are purely subjective, the transdetermination expression data was re-analyzed to identify genes whose median ratio changes within a 95% confidence level. Fourteen percent of the genes satisfied these conditions. Among these genes, 15/32 PcG and trxG genes (47%) had such statistically significant changes. Identification of these 15 genes with differential expression suggests that transdetermination may be correlated with large-scale remodeling of chromatin structure (Klebes, 2005).

To test if the small but statistically significant changes in the expression of PcG and trxG genes are indicative of a functional role in determination, discs from wild-type, Polycomb (Pc), Enhancer of Polycomb [E(Pc)], Sex comb on midleg (Scm), Enhancer of zeste [E(z)], Su(z)2, brahma (brm) and osa (osa) larvae were examined. The level of Wg induction was adjested to reduce the frequency of transdetermination and both frequency of transdetermination and area of transdetermined cells was determined. The frequency of leg discs expressing vg increased significantly in E(z), Pc, E(Pc), brm and osa mutants, and the frequency of leg to wing transdetermination in adult cuticle increased in Scm, E(z), Pc, E(Pc) and osa mutants. Remarkably, Su(z)2 heterozygous discs had no vg expression, suggesting that the loss of Su(z)2 function limits vg expression (Klebes, 2005).

Members of the PcG and trxG are known to act as heteromeric complexes by binding to cellular memory modules (CMMs). The functional tests demonstrate that mutant alleles for members of both groups have the same functional consequence (they increase transdetermination frequency). The findings are consistent with recent observations that the traditional view of PcG members as repressors and trxG factors as activators might be an oversimplification, and that a more complex interplay of a varying composition of PcG and trxG proteins takes place at individual CMMs. Furthermore the opposing effects of Pc and Su(z)2 functions are consistent with the proposal that Su(z)2 is one of a subset of PcG genes that is required to activate as well as to suppress gene expression. In addition to measuring the frequency of transdetermination, the relative area of vg expression was examined in the various PcG and trxG heterozyogous mutant discs. The relative area decreased in E(Pc), brm and osa mutant discs, despite the increased frequency of transdetermination in these mutants. There is no evidence to explain these contrasting effects, but the roles in transdetermination of seven PcG and trxG genes that were identified by these results support the proposition that the transcriptional state of determined cells is implemented through the controls imposed by the regulators of chromatin structure (Klebes, 2005).

The determined states that direct cells to particular fates or lineages can be remarkably stable and can persist after many cell divisions in alien environments, but they are not immune to change. In Drosophila, three experimental systems have provided opportunities to investigate the mechanisms that lead to switches of determined states. These are: (1) the classic homeotic mutants; (2) the PcG and trxG mutants that affect the capacity of cells to maintain homeotic gene expression, and (3) transdetermination. During normal development, the homeotic genes are expressed in spatially restricted regions, and cells that lose (or gain) homeotic gene function presumably change the transcriptional profiles characteristic of the particular body part. In the work reported here, techniques of micro-dissection, RNA amplification and array hybridization were used to monitor the transcription profiles of cells in normal leg and wing imaginal discs, in leg disc cells that regenerate and in cells that transdetermine from leg to wing. The results validate the idea that changing determined states involves global changes in gene expression. They also identify genes whose function may be unrelated to the specific fates of the cells characterized, but instead may correlate with developmental plasticity (Klebes, 2005).

Overlap between the transcriptional profiles in the wing and transdetermination lists (15 genes) and with genes in subcluster IV (high expression in wing discs) is extensive. The overlap is sufficient to indicate that the TD leg disc cells have changed to a wing-like program of development, but interestingly, not all wing-specific genes are activated in the TD cells. The reasons could be related to the incomplete inventory of wing structures produced (only ventral wing) or to the altered state of the TD cells. During normal development, vg expression is activated in the embryo and continues through the 3rd instar. Although the regulatory sequences responsible for activation in the embryo have not been identified, in 2nd instar wing discs, vg expression is dependent upon the vgBE enhancer, and in 3rd instar wing discs expression is dependent upon the vgQE enhancer. Expression of vg in TD cells depends on activation by the vgBE enhancer, indicating that cells that respond to Wg-induction do not revert to an embryonic state. Recent studies of the cell cycle characteristics of TD cells support this conclusion (Sustar, 2005), but the role of the vgBE enhancer in TD cells and the incomplete inventory of 'wing-specific genes' in their expression profile probably indicates as well the stage at which the TD cells were analyzed: they were not equivalent to the cells of late 3rd instar wing discs (Klebes, 2005).

Investigations into the molecular basis of transdetermination have led to a model in which inputs from the Wg, Dpp and Hh signaling pathways alter the chromatin state of key selector genes to activate the transdetermination pathway. The analyses were limited to a period 2-3 days after the cells switched fate, because several cell doublings were necessary to produce sufficient numbers of marked TD cells. As a consequence, these studies did not analyze the initial stages. Despite this technical limitation, this study identified several genes that are interesting novel markers of transdetermination (e.g., ap, CG12534, CG14059 and CG4914), as well as several genes that function in the transdetermination process (e.g., lama and the PcG genes). The results from transcriptional profiling add significant detail to a general model proposed for transdetermination (Klebes, 2005).

(1) It is reported that ectopic wg expression results in statistically significant changes in the expression of 15 PcG and trxG genes. Moreover, although the magnitudes of these changes were very small for most of these genes, functional assays with seven of these genes revealed remarkably large effects on the metrics used to monitor transdetermination -- the fraction of discs with TD cells, the proportion of disc epithelium that TD cells represent, and the fraction of adult legs with wing cuticle. These effects strongly implicate PcG and trxG genes in the process of transdetermination and suggest that the changes in determined states manifested by transdetermination are either driven by or are enabled by changes in chromatin structure. This conclusion is consistent with the demonstrated roles of PcG and trxG genes in the self-renewing capacity of mouse hematopoietic stem cells, in Wg signaling and in the maintenance of determined states. The results now show that the PcG and trxG functions are also crucial to pluripotency in imaginal disc cells, namely that pluripotency by 'weak point' cells is dependent upon precisely regulated levels of PcG and trxG proteins, and is exquisitely sensitive to reductions in gene dose (Klebes, 2005).

The data do not suggest how the PcG and trxG genes affect transdetermination, but several possible mechanisms deserve consideration. A recent study (Sustar, 2005) reported that transdetermination correlates with an extension of the S phase of the cell cycle. Several proteins involved in cell cycle regulation physically associate with PcG and trxG proteins, and Brahma, one of the proteins that affects the metrics of transdetermination, has been shown to dissociate from chromatin in late S-phase and to reassociate in G1. It is possible that changes in the S-phase of TD cells are a consequence of changes in PcG/trxG protein composition (Klebes, 2005).

Another generic explanation is that transdetermination is dependent or sensitive to expression of specific targets of PcG and trxG genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in the Drosophila genome, one is in direct proximity to the vg gene. It is possible that upregulation of vg in TD cells is mediated through this element. Another factor may be the contribution of targets of Wg signaling, since targets of Wg signaling have been shown to be upregulated in osa and brm mutants. These are among a number of likely possible targets, and identifying the sites at which the PcG and trxG proteins function will be necessary if an understand is to be gained of how transdetermination is regulated. Importantly, understanding the roles of such targets and establishing whether these roles are direct will be essential to rationalize how expression levels of individual PcG and trxG genes correlate with the effects of PcG and trxG mutants on transdetermination (Klebes, 2005).

(2) The requirement for lama suggests that proliferation of TD cells involves functions that suppress differentiation. lama expression has been correlated with neural and glial progenitors prior to, but not after, differentiation, and it is observed that lama is expressed in imaginal progenitor cells and in early but not late 3rd instar discs. lama expression is re-activated in leg cells that transdetermine. The upregulation of unpaired in TD cells may be relevant in this context, since the JAK/STAT pathway functions to suppress differentiation and to promote self-renewal of stem cells in the Drosophila testis. It is suggested that it has a similar role in TD cells (Klebes, 2005).

(3) A role for Notch is implied by the expression profiles of several Notch pathway genes. Notch may contribute directly to transdetermination through the activation of the vgBE enhancer [which has a binding site for Su(H)] and of similarly configured sequences that were found to be present in the regulatory regions of 45 other TD genes. It will be important to test whether Notch signaling is required to activate these co-expressed genes, and if it is, to learn what cell-cell interactions and 'community effects' regulate activation of the Notch pathway in TD cells (Klebes, 2005).

(4) The upregulation in TD cells of many genes involved in growth and division, and the identification of DNA replication element (DRE) sites in the regulatory region of many of these genes supports the observation that TD cells become re-programmed after passing through a novel proliferative state (Sustar, 2005), and suggests that this change is in part implemented through DRE-dependent regulation (Klebes, 2005).

There was an interesting correlation between transdetermination induced by Wg mis-expression and the role of Wg/Wnt signaling for stem cells. Wg/Wnt signaling functions as a mitogen and maintains both somatic and germline stem cells in the Drosophila ovary, and mammalian hematopoetic stem cells. Although the 'weak point' cells in the Drosophila leg disc might lack the self-renewing capacity that characterizes stem cells, they respond to Wg mis-expression by manifesting a latent potential for growth and transdetermination. It seems likely that many of the genes are conserved that are involved in regulating stem cells and that lead to disease states when relevant regulatory networks lose their effectiveness (Klebes, 2005).

The prevalence of transcription factors among the genes whose relative expression levels differed most in the tissue comparisons was intriguing. It is commonly assumed that transcription factors function catalytically and that they greatly amplify the production of their targets, so the expectation was that the targets of tissue-specific transcription factors would have the highest degree of tissue-specific expression. In these studies, tissue-specific expression of 15 transcription factors among the 40 top-ranking genes in the wing and leg data sets (38%) is consistent with the large number of differentially expressed genes in these tissues, but these rankings suggest that the targets of these transcription factors are expressed at lower relative levels than the transcription factors that regulate their expression. One possible explanation is that the targets are expressed in both wing and leg disc cells, but the transcription factors that regulate them are not. This would imply that the importance of position-specific regulation lies with the regulator, not the level of expression of the target. Another possibility is that these transcription factors do not act catalytically to amplify the levels of their targets, or do so very inefficiently and require a high concentration of transcription factor to regulate the production of a small number of transcripts. Further analysis will be required to distinguish between these or other explanations, but it is noted that the prevalence of transcription factors in such data sets is neither unique to wing-leg comparisons nor universal (Klebes, 2005).

Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, two Polycomb group epigenetic silencers, Pipsqueak and Lola, have been identified that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis (Ferres-Marco, 2006).

H3K4 methylation is thought to be permissive for maintaining and propagating activated chromatin and is thought to neutralize repressor tags by precluding binding of the HDAC complex and impairing SUV39H1-mediated H3K9 methylation. Thus, H3K4me3 depletion may contribute to tumour formation by permitting aberrant chromatin silencing. It was found that a 50% reduction in dosage of the HDAC gene Rpd3 or of Su(var)3-9 decreased the tumour phenotype dominantly. In contrast, reducing the activity of the H3K4 histone methyltransferase genes Trx (known as ALL1 or MLL in humans) or Ash1, which would be expected to deplete the H3K4me3 tag further, did not visibly enhance the tumours (Ferres-Marco, 2006).

E(z) when complexed with the Extra sex combs (Esc) protein becomes a histone methyltransferase. The E(z)-Esc complex and its mammalian counterpart Ezh2-Eed show specificity for H3K27 but may also target H3K9. The complex also contains the HDAC Rpd3, and this association with Rpd3 is conserved in mammals. H3K27 methylation facilitates binding of the chromodomain protein Pc (HPC in humans), which then creates a repressive chromatin state that is a stable silencer of genes (Ferres-Marco, 2006).

Although loss of E(z) does not cause proliferation defects within discs, halving the E(z) gene dosage dominantly suppressed tumorigenesis, indicating that histone methylation by the E(z)-Esc complex is also a prerequisite for the excessive proliferation of these tumours. Accordingly, Esc^- or Pc^- mutations also notably reduced the tumours (Ferres-Marco, 2006).

Together, these findings suggest that the development of these tumours involves, at least in part, changes in the structure of chromatin brought about by covalent modifications of histones. These changes probably switch the target genes from the active H3K4me3 state to a deacetylated H3K9 and H3K27 methylation silent state (Ferres-Marco, 2006).

Polycomb group mutants exhibit mitotic defects in syncytial cell cycles of Drosophila embryos

The Polycomb Group (PcG) of epigenetic regulators maintains the repressed state of Hox genes during development of Drosophila, thereby maintaining the correct patterning of the anteroposterior axis. PcG-mediated inheritance of gene expression patterns must be stable to mitosis to ensure faithful transmission of repressed Hox states during cell division. Previously, two PcG mutants, polyhomeotic and Enhancer of zeste, were shown to exhibit mitotic segregation defects in embryos, and condensation defects in imaginal discs, respectively. polyhomeotic^proximal but not polyhomeotic^distal is necessary for mitosis. To test if other PcG genes have roles in mitosis, embryos derived from heterozygous PcG mutant females were examined for mitotic defects. Severe defects in sister chromatid segregation and nuclear fallout, but not condensation are exhibited by Polycomb, Posterior sex combs and Additional sex combs. By contrast, mutations in Enhancer of zeste (which encodes the histone methyltransferase subunit of the Polycomb Repressive Complex 2) exhibit condensation but not segregation defects. It is proposed that these mitotic defects in PcG mutants delay cell cycle progression. Possible mitotic roles for PcG proteins are discussed, and suggest that delays in cell cycle progression might lead to failure of maintenance (O'Dor, 2006).

The data for ph mutations confirm the original observation that ph⁵⁰³ mutations exhibit mitotic defects. These original observations have been confirmed in several ways. First, the observation that strains out-crossed to wild-type flies show similar frequencies of defects compared to heterozygous mutants show that the phenotypes arise from ph mutations rather than background effects. Second, embryos derived from homozygous ph⁴⁰⁹ mothers show similar frequencies of mitotic defects to those derived from heterozygous mothers. These results suggest that for ph, the severity of the phenotype reaches a plateau when the amount of Ph is reduced below a threshold which must be greater than 50% of the wild-type amount. Third, only ph^p (ph⁴⁰⁹) is necessary for normal mitosis, because mutations in ph^d (ph⁴⁰¹) have no effect on mitosis. This observation is consistent with data that has shown that only one isoform of Ph-P coimmunoprecipitates with Barren or Topoisomerase II. This observation supports the conclusion that Ph-P and Ph-D have different functions. Fourth, because homozygous ph⁴⁰⁹ flies are viable, the ph phenotypes reported here represent those of maternal germline nulls (O'Dor, 2006).

The results show that early embryos of PcG and Asx mutants exhibit highly penetrant and expressive mitotic phenotypes in syncytial embryos, consistent with problems in cell cycle progression. Two classes of phenotypes are observed: segregation defects and condensation defects, but no mutant exhibits both phenotypes. In these experiments, with the exception of ph, embryos were scored derived from heterozygous mothers, in which 50% of the wild-type product remain. Therefore, the possibility cannot be ruled out that more severe mitotic phenotypes would be observed in embryos derived from homozygous mothers, resulting in less distinct differences between E(z) and other mutants. Consistent with this caveat, when homozygous E(z)⁵ (l(3)1902) mutant imaginal disks were examined, both condensation defects and chromosome breakage consistent with problems in segregation were observed, so E(z) may function in both condensation and segregation. The data show that embryos derived from homozygous ph-proximal mutants do not have condensation defects, so at a minimum, E(z) has at least one role in mitosis different from that of ph. To accurately compare the roles of different PcG and ETP genes in mitosis, it will be necessary to examine mutations derived from homozygous mutant mothers, or from germline clones (O'Dor, 2006).

Mutations in the PcG genes Sex combs extra and Sex combs on midleg reduce proliferation of ovarian follicle cells in Drosophila, suggesting that other PcG members are also required for cell cycle progression. It is predicted that other PcG and ETP group mutants not tested here will also exhibit significant mitotic defects (O'Dor, 2006).

Given the high penetrance and expressivity of the chromatin bridge phenotype in PcG mutant embryos, what becomes of the embryos with severe chromatin bridges, and more specifically, what happens to chromatin bridges themselves? Four observations suggest that most chromatin bridges are resolved in PcG mutants. First, nuclear fallout should remove unresolved nuclei, but relatively few fallout nuclei were observed in any embryo. Second, anaphase and telophase embryos together made up 7–9% of the total developed embryos, a proportion that is consistent with the short duration of those mitotic phases. This low proportion of embryos in anaphase or telophase argues that the embryos that exhibited severe chromatin bridges were not developmentally arrested or dead. Third, only a few embryos out of all mutants tested appeared to have bridged prometaphase nuclei. If chromatin bridges did not resolve, one would expect a higher proportion of these prometaphase bridges. Fourth, unresolved chromatin bridges should break. However, fragmented chromosomes, evidence of chromosome breakage and all low-penetrant mitotic defects accounted for only 4–10% of the total mitotic defects observed in mutant embryos (O'Dor, 2006).

C(2)EN embryos carrying an abnormally long second chromosome exhibited chromatin bridges between some nuclei since the extra-long chromosomes were not able to fully segregate during anaphase. These bridged nuclei lagged behind neighboring nuclei and were subsequently removed from the cortex by the fallout mechanism once they reached telophase. Therefore, the fallout nuclei observed in PcG mutants are likely previously-bridged nuclei not able to resolve in time to maintain overall mitotic synchrony. Interestingly, the fallout nuclei were never joined by chromatin bridges. This may be because the delay is only detected once the bridges are resolved, or the bridged nuclei “snap-back” and fuse with each other, as has been observed for bridged nuclei in embryos mutant for grapes, a checkpoint gene required at several cell cycle stages (O'Dor, 2006).

Occasionally, the fallout mechanism may be unable to detect or remove delayed nuclei. If resolved, these nuclei may appear as asynchronous to neighboring nuclei, or, if bridged, they appear as prometaphase bridges, polyploid, giant nuclei or chromosome breaks. The embryos of polyhomeotic mutants develop at a slower rate than those of wild-type flies as judged by timed embryo collections. The slower developmental rate may reflect delays in the mitotic cycles due to segregation defects. In other cases, the most extreme segregation defects overwhelm the fallout mechanisms and continue with the mitotic program until the segregation failures reach a critical point and the embryo dies. In some embryos, the cortex is completely disorganized with very large amorphous nuclei and extensive chromosome breakage. These embryos are probably dead and appear to be the result of cumulative effects of several rounds of segregation defects (O'Dor, 2006).

It remains to be determined whether the length of cell cycle stages in PcG mutants is altered by a checkpoint pathway. In syncytial embryos, the metaphase to anaphase transition is delayed in response to damaged DNA, improper spindle assembly, or faulty centrosome activation. Activation of the spindle checkpoint also delays mitotic progression. It is possible that the mitotic defects of PcG embryos also delay the mitotic cycle by activating a pre-mitotic checkpoint (O'Dor, 2006).

PcG proteins could have a direct structural or enzymatic role in mitosis, separate from their role in silencing. PcG proteins associate with chromatin in a cell cycle-dependent manner. In Drosophila embryos, Polyhomeotic (PH), Polycomb (PC), and Posterior sex combs (PSC) proteins associate with chromatin at S phase, almost completely dissociate by metaphase and reassociate at telophase. BMI1, the human homologue of PSC, shows a similar pattern of association and dissociation in primary and tumor cell lines. Therefore, PcG proteins are present during the key events of mitosis that occur prior to metaphase. An interesting recent report shows that Set1, the yeast homolog of the MP Trithorax, methylates a component of the kinetochore, consistent with the possibility that the methyltransferase activity of E(z) could directly modify proteins needed for mitosis (O'Dor, 2006).

The presence of anaphase bridges in ph, Pc, Psc, and Asx mutants does not necessarily imply that PcG proteins act at anaphase. Mitotic defects may arise at other cell cycle stages but carry forward to manifest as a segregation phenotype. Many different Drosophila cell cycle genes regulating every stage of the cell cycle also have chromatin bridge phenotypes. Some examples include kinesin-like enzymes, a variety of regulatory kinases such as polo kinase and aurora-like kinases, replication checkpoint regulators such as grapes, chk2, and mei-41, and genes involved in sister chromatid segregation such as pimples and three rows (O'Dor, 2006).

PcG proteins could be required at DNA synthesis. Cramped colocalizes with PCNA, which is required for DNA synthesis. Therefore, Cramped, and by extension, other PcG proteins could have a role in elongation of replication forks. RAE28, the homolog of PH, interacts and colocalizes with GEMININ, a replication licensing factor. There have been suggestions in the literature that silenced genes are late-replicating, and this observation has been supported in Drosophila. Therefore, PcG mutations, by interfering with silencing or chromatin structure, could affect replication timing in mitosis. Consistent with this idea, PC tethered near an origin interferes with origin activity. However, it has been reported that an E(z) mutation does not affect replication timing in polytene chromosomes. This may be a reflection of the differences in possible mitotic roles between E(z) and other PcG members. Interestingly, in mammals, heritable gene silencing delays chromatid resolution without affecting timing of DNA replication (O'Dor, 2006).

PcG proteins could also be required for association of sister chromatids. Interaction between PcG Response Elements (PREs), presumably mediated by PcG proteins, is important for repression of PcG targets. Interaction between PcG proteins has been proposed to account for the high likelihood of insertion of PRE-containing transgenes in genomic regions that already contain a PRE. By analogy, PcG proteins could have roles in sister chromatid adhesion or resolution (O'Dor, 2006).

Finally, PcG proteins could be required for chromatin condensation prior to metaphase. This hypothesis is consistent with the E(z) phenotype, in which mitotic chromosomes fail to condense. E(z) is a histone methyltransferase. Histone modifications, notably hypoacetylation and methylation of histone H3 lysine 9 and 27, have been associated with heterochromatin and silencing, consistent with the idea that E(z) might have a role in chromosome condensation. In support of this idea, PH-P coimmunoprecipitates with Barren and Topoisomerase II. Though Barren is a member of the condensin complex, it is not essential for condensation, but is required for sister chromatid resolution. It is speculated that E(z) has a specific role in condensation separate from the role of other PcG proteins, perhaps because its role as a methyltransferase might be required for targets other than histones. It will be interesting to determine if all PRC2 members exhibit condensation defects (O'Dor, 2006).

PcG genes could have indirect effects on mitosis if they are required for regulation of genes that are themselves important for mitosis, or to prevent expression of genes that disrupt mitosis. There are two clear precedents for this possibility. Bmi1, the mammalian Psc homolog originally identified as an oncogene, is also required for regulating lymphoid cell proliferation via repression of the ink4a tumor suppressor locus. Mel18, another mammalian Psc homologue, was originally identified as a tumor suppressor and inhibits cell cycle progression likely via repression of c-myc, leading to downregulation of cyclins and CDKs. If PcG-mediated regulation of proteins important for the cell cycle accounts for the mitotic phenotypes observed in embryos, then this challenges the assumption that maintenance proteins are required only to propagate expression states of genes between cell cycles (O'Dor, 2006).

Steroid hormone-dependent transformation of polyhomeotic mutant neurons in the Drosophila brain

Polyhomeotic (Ph), which forms complexes with other Polycomb-group (PcG) proteins, is widely required for maintenance of cell identity by ensuring differential gene expression patterns in distinct types of cells. Genetic mosaic screens in adult fly brains allow for recovery of a mutation that simultaneously disrupts the tandemly duplicated Drosophila ph transcriptional units. Distinct clones of neurons normally acquire different characteristic projection patterns and can be differentially labeled using various subtype-specific drivers in mosaic brains. Such neuronal diversity is lost without Ph. In response to ecdysone, ph mutant neurons are transformed into cells with unidentifiable projection patterns and indistinguishable gene expression profiles during early metamorphosis. Some subtype-specific neuronal drivers become constitutively activated, while others are constantly suppressed. By contrast, loss of other PcG proteins, including Pc and E(z), causes different neuronal developmental defects; and, consistent with these phenomena, distinct Hox genes are differentially misexpressed in different PcG mutant clones. Taken together, Drosophila Ph is essential for governing neuronal diversity, especially during steroid hormone signaling (Wang, 2006).

Ph is well implicated in maintaining cell fates via controlling transcription of genes in distinct cell type-characteristic manners. Deregulation of multiple genes aberrantly occurs in ph mutant tissues. A similar mechanism probably underlies most of the abnormalities in ph mutant neurons. In particular, there are multiple lines of evidence suggesting mal-expression of various subtype-specific GAL4 drivers in ph mutant clones. First, with respect to GAL4-OK107, GAL4-NP225 and elav-GAL4, the use of various GAL4 drivers results in labeling of similar numbers of clones. Second, clones were induced in the central brain versus the optic lobe, depending on when mitotic recombination was induced; the result is the same as in wild-type mosaic brains. Third, ato-GAL4 and GAL4-EB1 fail to label any clone, arguing against constitutive expression of UAS-transgenes in mutant clones. Finally, examining clones through development reveals no evidence for derivation of some clones from other clones; and, instead, sudden labeling of full-sized clones was constantly observed shortly after a big ecdysone pulse. Apparently, loss of Ph function alone is short of causing the full spectrum of abnormalities. Mass ecdysone is required for the pathological transformation of ph mutant neurons in the Drosophila brain, raising several interesting possibilities about mutual involvement between the epigenetic function of PcG and the global nuclear signaling of steroid hormones (Wang, 2006).

Distinct wild-type cells respond differentially to ecdysone, but ph mutant neurons of distinct origins become no longer distinguishable after ecdysone signaling. Ecdysone mediates diverse biological activities partially via binding to different heterodimeric receptors. Its conventional receptors consist of the nuclear receptor superfamily members ecdysone receptor (EcR) and Ultraspiracle (USP; the Drosophila RXR). There are three documented EcR isoforms; and cells that express different EcR isoforms have been shown to undergo different changes in response to the prepupal ecdysone peak. For example, abundant EcR-B1 exists selectively in the neurons that remodel projections during early metamorphosis. Since no change was observed in EcR expression patterns in ph mutant neurons, it is unlikely that the aberrant responses of ph mutant neurons to the prepupal ecdysone peak occur as a result of derepression of specific EcR isoforms. In addition, derepression of multiple Hox genes appears not to be involved either. Nevertheless, given the involvement of Ph in silencing transcription, it remains possible that derepression of other unidentified genes directly re-programs ecdysone-induced transcriptional hierarchies, leading to transformation of ph mutant neurons. Alternatively, it is possible that loss of the epigenetic function of Ph may permit diffuse activation of prohibited genes by normal transcriptional hierarchies. Moreover, massive steroid hormone signaling might directly modify genomic imprinting when PcG functions are compromised (Wang, 2006).

Ecdysone-dependent transformation of ph mutant neurons provides a possible model system for characterizing the epigenetic functions of steroid hormones. In addition, the demonstration of the unusual potent epigenetic effects of ecdysone in ph mutant neurons suggests complex mechanisms may underlie pathogenesis of other documented PcG loss-of-function phenotypes (Wang, 2006).

Both derepression and inactivation of genes occur in transformed ph mutant neurons, characterization of which offers some molecular insights into this status of transformation. First, the fine-tuning of gene expression in transformed cells was no longer detected; and all the examined drivers appeared either fully on or completely off. Second, on or off could not be simply attributed to the genomic locations of drivers, as evidenced by constitutive silencing of the multiple independently inserted atonal-GAL4 transgenes. Third, transformed cells retained neuron-type morphologies and remained positive for the neuron-specific gene elav; and ph mutant neurons had been earlier reported to acquire normal-looking neurites in culture. Taken together, the transformation leads to loss of subtype identity without affecting basic neuronal fates, abolishes the genomic imprints governing fine controls over gene expression, and locks gene expression in 'on' or 'off' possibly in a promoter-autonomous manner (largely independent of its chromatin environment) (Wang, 2006).

Finally, loss of Ph, Pc, versus E(z) results in distinct phenotypes in the developing fly brain. Differences in their underlying pathological mechanisms are well exemplified by differential derepression of distinct Hox genes in different PcG clones. In addition, for a given PcG mutation, patterns of Hox gene derepression vary from neural clones to wing disc clones and visceral mesoderm. It remains to be elucidated how distinct PcG functions are governed in diverse cell type-characteristic manners (Wang, 2006).

E(z) was identified in a genome-wide analyses for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4²²¹) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4²²¹-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

RNAi of the Polycomb group (PcG) genes Su(z)12, E(z), esc, or Caf1 similarly caused an increase in branch number and an expansion of the receptive field of class I neurons. Consistent with the similar RNAi phenotypes for these genes, Su(z)12, E(z), esc, and Caf1 are components of the multiprotein esc/E(z) polycomb repressor complex. One critical role for PcG-mediated gene silencing is the regulation of hox gene expression. Therefore, Polycomb-mediated regulation of hox gene expression likely contributes to arborization of class I neurons (Parrish, 2006).

Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins

Polycomb group (PcG) and trithorax group (trxG) proteins act as antagonistic regulators to maintain transcriptional OFF and ON states of HOX and other target genes. To study the molecular basis of PcG/trxG control, the chromatin of the HOX gene Ultrabithorax (Ubx) was analyzed in Ubx^OFF and Ubx^ONcells purified from developing Drosophila. PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all constitutively bound to Polycomb response elements (PREs) in the OFF and ON state. In contrast, the trxG protein Ash1 is only bound in the ON state; not at PREs but downstream of the transcription start site. In the OFF state, extensive trimethylation was found at H3-K27, H3-K9, and H4-K20 across the entire Ubx gene; i.e., throughout the upstream control, promoter, and coding region. In the ON state, the upstream control region is also trimethylated at H3-K27, H3-K9, and H4-K20, but all three modifications are absent in the promoter and 5' coding region. These analyses of mutants that lack the PcG histone methyltransferase (HMTase) E(z) or the trxG HMTase Ash1 provide strong evidence that differential histone lysine trimethylation at the promoter and in the coding region confers transcriptional ON and OFF states of Ubx. In particular, these results suggest that PRE-tethered PcG protein complexes act over long distances to generate Pc-repressed chromatin that is trimethylated at H3-K27, H3-K9, and H4-K20, but that the trxG HMTase Ash1 selectively prevents this trimethylation in the promoter and coding region in the ON state (Papp, 2006; Full text of article).

Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and dSfmbt, a novel PcG protein that selectively binds to histone H3 and H4 tail peptides that are mono- or dimethylated at H3-K9 or H4-K20 (H3-K9me1/2 and H4-K20me1/2, respectively). PRC1 contains the PcG proteins Ph, Psc, Sce/Ring, and Pc. PRC1 inhibits nucleosome remodeling and transcription in in-vitro assays and its subunit Pc specifically binds to trimethylated K27 in histone H3 (H3-K27me3). PRC2 contains the PcG proteins E(z), Su(z)12, and Esc as well as Nurf55, and this complex functions as a histone methyltransferase (HMTase) that specifically methylates K27 in histone H3 (H3-K27) in nucleosomes (Papp, 2006).

This study used quantitative X-ChIP analysis to examine the chromatin of the HOX gene Ubx in its ON and OFF state in developing Drosophila larvae. Previous genetic studies had established that all of the PcG and trxG proteins analyzed in this study are critically needed to maintain Ubx OFF and ON states in the very same imaginal disc cells in which their binding to Ubx was analyzed in this study. The following conclusions can be drawn from the analyses reported in this study. (1) The PcG protein complexes PhoRC, PRC1, and PRC2 (recognized using antibodies against Su(z)12 and Pcl) and the Trx protein are all highly localized at PREs, but they are all constitutively bound at comparable levels in the OFF and ON state. (2) The trxG protein Ash1 is bound only in the ON state, where it is specifically localized ~1 kb downstream of the transcription start site. (3) In the OFF state, PRC2 and other unknown HMTases trimethylate H3-K27, H3-K9, and H4-K20 over an extended 100-kb domain that spans the whole Ubx gene. (4) In the ON state, comparable H3-K27, H3-K9, and H4-K20 trimethylation is restricted to the upstream control regions and Ash1 selectively prevents this trimethylation in the promoter and coding region. (5) Repressed Ubx chromatin is extensively tri- but not di- or monomethylated at H3-K27, H3-K9, and H4-K20. (6) Trimethylation of H3-K27, H3-K9, and H4-K20 at imaginal disc enhancers in the upstream control region does not impair the function of these enhancers in the ON state. (7) TBP and Spt5 are bound at the Ubx transcription start site in the ON and OFF state, but Kis is only bound in the ON state. This suggests that in the OFF state, transcription is blocked at a late step of transcriptional initiation, prior to the transition to elongation. A schematic representation of PcG and trxG protein complex binding and histone methylation at the Ubx gene in the OFF and ON state is presented (Papp, 2006).

Unexpectedly, ChIP analysis by qPCR used in this study and in a similar study by the laboratory of Vincent Pirrotta (V. Pirrotta, pers. comm. to Papp, 2006) reveals that the relationship between PcG and trxG proteins and histone methylation is quite different from the currently held views. Specifically, X-ChIP studies have reported that H3-K27 trimethylation is localized at PREs and this led to the model that recruitment of PRC1 to PREs occurs through H3-K27me3 (i.e., via the Pc chromodomain). In contrast, the current study and that by Vincent Pirrotta found H3-K27 trimethylation to be present across the whole inactive Ubx gene, both in wing discs and in S2 cells (V. Pirrotta, pers. comm. to Papp, 2006). No specific enrichment of H3-K27 trimethylation at PREs has been detected; rather, a reduction of H3-K27me3 signals is observed at PREs, consistent with the reduced signals of H3 that are detected at these sites. Consistent with these results, genome-wide analyses of PcG protein binding and H3-K27me3 profiles in S2 cells revealed that, at most PcG-binding sites in the genome, PcG proteins are tightly localized, whereas H3-K27 trimethylation is typically present across an extended domain that often spans the whole coding region. How could the differences between this study and the earlier studies be explained? It should be noted that in contrast to the qPCR analysis used in the current study, previous studies all relied on nonquantitative end-point PCR after 36 or more cycles to assess the X-ChIP results. It is possible that these experimental differences account for the discrepancies (Papp, 2006).

PhoRC, PRC1, and PRC2 are all tightly localized at PREs but they are all constitutively bound at the inactive and active Ubx gene. This suggests that recruitment of PcG complexes to PREs occurs by default. Although all three complexes are bound at comparable levels to the bxd PRE in the inactive and active state and PhoRC is also bound at comparable levels at the bx PRE, it should be pointed out that the levels of PRC1 and PRC2 binding at the bx PRE are about twofold reduced in the active Ubx gene compared with the inactive Ubx gene. Even though there is still high-level binding of PRC1 and PRC2 at the bx PRE, it cannot be excluded that the observed reduction in binding helps to prevent default PcG repression of the active Ubx gene. It is possible that transcription through the bx PRE reduces PRC1 and PRC2 binding at this PRE. Transcription through PREs has been proposed to serve as an 'anti-silencing' mechanism that prevents default silencing of active genes by PREs (Papp, 2006),

The highly localized binding of all three PcG protein complexes at PREs, together with earlier studies on PRE targeting of PcG protein complexes supports the idea that not only PhoRC but also PRC1 and PRC2 are targeted to PRE DNA through interactions with Pho and/or other sequence-specific DNA-binding proteins. In the case of trxG proteins, the binding modes are more diverse. In particular, recruitment of Trx protein to PREs and to the promoter is also constitutive in both states but recruitment of Ash1 to the coding region is clearly observed only at the active Ubx gene. At present, it is not known how Trx or Ash1 are targeted to these sites. It is possible that a transcription-coupled process recruits Ash1 to the position 1 kb downstream of the transcription start site (Papp, 2006).

In contrast to the localized and constitutive binding of PcG protein complexes and the Trx protein, it was found that the patterns of histone trimethylation are very distinct in the active and inactive Ubx gene. The results also suggest that the locally bound PcG and trxG HMTases act across different distances to methylate chromatin. For example, H3-K4 trimethylation is confined to the first kilobase of the Ubx coding region where Ash1 and Trx are bound, whereas H3-K27 trimethylation is present across an extended 100-kb domain of chromatin that spans the whole Ubx gene. This suggests that PRE-tethered PRC2 is able to trimethylate H3-K27 in nucleosomes that are as far as 30 kb away from the bxd or bx PREs. Unexpectedly, it was found that the H3-K9me3 and H4-K20me3 profiles closely match the H3-K27me3 profile. At present it is not known which HMTases are responsible for H3-K9 and H4-K20 trimethylation, but analysis of E(z) mutants indicate that these modifications may be generated in a sequential manner, following H3-K27 trimethylation by PRC2. The molecular mechanisms that permit locally tethered HMTases such as PRE-bound PRC2 to maintain such extended chromatin stretches in a trimethylated state are only poorly understood. However, a recent study showed that the PhoRC subunit dSfmbt selectively binds to mono- and dimethylated H3-K9 and H4-K20 in peptide-binding assays (Klymenko. 2006). One possibility would be that dSfmbt participates in the process that ensures that repressed Ubx chromatin is trimethylated at H3-K27, H3-K9, and H4-K20. For example, dSfmbt, tethered to PREs by Pho, may interact with nucleosomes of lower methylated states (i.e., H3-K9me1/2 or H4-K20me1/2) in the flanking chromatin and thereby bring them into the vicinity of PRE-anchored HMTases that will hypermethylate them to the trimethylated state (Papp, 2006).

These analyses suggest that H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region is critical for Polycomb repression. (1) Although H3-K27, H3-K9, and H4-K20 trimethylation is present at the inactive and active Ubx gene, it is specifically depleted in the promoter and coding region in the active Ubx gene. (2) Misexpression of Ubx in wing discs with impaired E(z) activity correlates well with loss of H3-K27 and H3-K9 trimethylation at the promoter and 5' coding region. It is possible that the persisting H3-K27 and H3-K9 trimethylation in the 3' coding region is responsible for maintenance of repression in those E(z) mutant wing discs cells that do not show misexpression of Ubx. (3) In haltere and third-leg discs of ash1 mutants, the promoter and coding region become extensively trimethylated at H3-K27 and H3-K9, and this correlates with loss of Ubx expression. Previous studies showed that Ubx expression is restored in ash1 mutants cells that also lack E(z) function. Together, these findings therefore provide strong evidence that Ash1 is required to prevent PRC2 and other HMTases from trimethylating the promoter and coding region at H3-K27 and H3-K9. The loss of H3-K4 trimethylation in ash1 mutants is formally consistent with the idea that Ash1 exerts its antirepressor function by trimethylating H3-K4 in nucleosomes in the promoter and 5' coding region, but other explanations are possible (Papp, 2006).

But how might H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region repress transcription? The observation that TBP and Spt5 are also bound to the promoter in the OFF state suggests that these methylation marks do not prevent assembly of the basic transcription apparatus at the promoter. However, the nucleosome remodeling factor Kis is not recruited in the OFF state, and transcription thus appears to be blocked at a late step of transcriptional intiation prior to elongation. It was found that the low-level binding of Pc in the coding region correlates with the presence of H3-K27 trimethylation; i.e., Pc and H3-K27me3 are both present in the OFF state, but are absent in the ON state. One possible scenario would thus be that H3-K27 trimethylation in the promoter and coding region permits direct recruitment of PRC1. According to this view, locally recruited PRC1 would then repress transcription; e.g., by inhibiting nucleosome remodeling in the promoter region. However, several observations are not easily reconciled with such a simple 'recruitment-by-methylation' model. First, peak levels of all PRC1 components are present at PREs and, apart from Pc, very little binding is observed outside of PREs. Second, excision of PRE sequences from a PRE reporter gene during development leads to a rapid loss of silencing, suggesting that transcriptional repression requires the continuous presence of PREs and the proteins that are bound to them. A second, more plausible scenario would therefore be that DNA-binding factors first target PcG protein complexes to PREs, and that these PRE-tethered complexes then interact with trimethylated nucleosomes in the flanking chromatin in order to repress transcription. For example, it is possible that bridging interactions between the Pc chromodomain in PRE-tethered PRC1 and H3-K27me3-marked chromatin in the promoter or coding region permit other PRE-tethered PcG proteins to recognize the chromatin interval across which they should act, e.g., to inhibit nucleosome remodeling in the case of PRC1 or to trimethylate H3-K27 at hypomethylated nucleosomes in the case of PRC2 (Papp, 2006).

The analysis of a HOX gene in developing Drosophila suggests that histone trimethylation at H3-K27, H3-K9, and H4-K20 in the promoter and coding region plays a central role in generating and maintaining of a PcG-repressed state. Contrary to previous reports, the current findings provide no evidence that H3-K27 trimethylation is specifically localized at PREs and could thus recruit PRC1 to PREs; widespread H3-K27 trimethylation is found across the whole transcription unit. The data presented in this study provide evidence that PREs serve as assembly platforms for PcG protein complexes such as PRC2 that act over considerable distances to trimethylate H3-K27 across long stretches of chromatin. The presence of this trimethylation mark in the chromatin that flanks PREs may in turn serve as a signal to define the chromatin interval that is targeted by other PRE-tethered PcG protein complexes such as PRC1. The results reported here also provide a molecular explanation for the previously reported antirepressor function of trxG HMTases; selective binding of Ash1 to the active HOX gene blocks PcG repression by preventing PRC2 from trimethylating the promoter and coding region. It is possible that the extended domain of combined H3-K27, H3-K9, and H4-K20 trimethylation creates not only the necessary stability for transcriptional repression, but that it also provides the molecular marks that permits PcG repression to be heritably maintained through cell division (Papp, 2006).

Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila

Genes of the Polycomb group (PcG) are part of a cellular memory system that maintains appropriate inactive states of Hox gene expression in Drosophila. This study investigates the role of PcG genes in postembryonic development of the Drosophila CNS. Mosaic-based MARCM techniques were used to analyze the role of these genes in the persistent larval neuroblasts and progeny of the central brain and thoracic ganglia. Proliferation in postembryonic neuroblast clones is dramatically reduced in the absence of Polycomb, Sex combs extra, Sex combs on midleg, Enhancer of zeste or Suppressor of zeste 12. The proliferation defects in these PcG mutants are due to the loss of neuroblasts by apoptosis in the mutant clones. Mutation of PcG genes in postembryonic lineages results in the ectopic expression of posterior Hox genes, and experimentally induced misexpression of posterior Hox genes, which in the wild type causes neuroblast death, mimics the PcG loss-of-function phenotype. Significantly, full restoration of wild-type-like properties in the PcG mutant lineages is achieved by blocking apoptosis in the neuroblast clones. These findings indicate that loss of PcG genes leads to aberrant derepression of posterior Hox gene expression in postembryonic neuroblasts, which causes neuroblast death and termination of proliferation in the mutant clones. These findings demonstrate that PcG genes are essential for normal neuroblast survival in the postembryonic CNS of Drosophila. Moreover, together with data on mammalian PcG genes, they imply that repression of aberrant reactivation of Hox genes may be a general and evolutionarily conserved role for PcG genes in CNS development (Bello, 2007).

Genetic analysis indicates that the PcG genes Sce, Scm, Pc, E(z) and Su(z)12 are required for postembryonic neurogenesis in the central brain and thoracic ganglia of Drosophila. In the absence of any one of these genes, several mutant phenotypes are observed in the third-instar CNS: (1) neural proliferation is dramatically reduced and only small numbers of cells are found in neuroblast clones; (2) proliferating postembryonic neuroblasts are absent in most of the mutant clones due to apoptosis; (3) posterior Hox genes are ectopically expressed in the postembryonic neuroblast lineages. It is hypothesized that these phenotypes are causally related, in that loss of PcG genes leads to ectopic Hox gene expression in postembryonic neuroblasts resulting in their premature cell death and, thereby, in drastically reduced neuroblast lineage size. Strong support for this hypothesis is provided by the fact that the mutant lineages proliferate normally if apoptosis is blocked. Corollary support for this notion is provided by the fact that Psc-Su(z)2 mutant clones, which do not show ectopic Hox gene expression, are consistently wild-type-like in size and presence of neuroblast (Bello, 2007).

Numerous genes are required for the continued mitotic activity of neuroblasts during postembryonic life. These findings provide the first demonstration that PcG genes are essential for neuroblast survival and proliferation in the postembryonic CNS. Previous work on PcG gene action during embryonic neurogenesis has demonstrated that the derepression of posterior Hox genes in PcG mutants leads to a change in the segmental determination of neuroblasts and their lineage, but not to their mitotic arrest and death. Thus, the effects of PcG gene loss on neurogenesis are context-dependent and differ during embryonic development as compared with postembryonic development. This is underscored in recent work which indicates that the PcG gene ph is essential for maintaining neuronal identity and diversity during metamorphosis (Bello, 2007).

In postembryonic development of the Drosophila CNS, a remarkable link exists between neuroblast survival and Hox gene expression. In the ventral ganglia, a neuroblast-specific pulse of abd-A during the third instar provides the cue for cell death, which limits the number of progeny produced per neuroblast. These data indicate that this mechanism, which in the wild type relates Hox gene expression to the clone size of neural stem cells, also operates in PcG mutants and is responsible for the PcG mutant phenotypes. Indeed, a general function of PcG genes in postembryonic neurogenesis may be to prevent the premature and widespread operation of this mechanism for temporal regulation of neurogenesis through termination of neuroblast life. It is noteworthy that the Hox gene-dependent activation of apoptosis within the CNS is selective for the neuroblast and does not occur when Hox genes are derepressed in neurons, either during normal development or in misexpression experiments. This explains why the neurons in PcG mutant clones, which were generated before the induction of neuroblast cell death, continue to survive despite the presence of ectopic Hox gene derepression (Bello, 2007).

These data indicate that loss of specific PcG genes in larval neuroblasts leads to ectopic Hox gene expression that is sufficient to cause neuroblast cell death. However, the PcG proteins may also contribute to neuroblast survival by repressing other unidentified target genes which, when derepressed, might result in premature death of postembryonic neuroblasts. Indeed, although deregulation of Hox gene expression is one of the hallmarks of PcG phenotypes in Drosophila, a diverse set of other target genes, including genes involved in cell cycle regulation, are controlled by PcG genes (Bello, 2007).

Interesting parallels to these findings on the role of PcG genes in neural proliferation come from studies of mammalian PcG genes, specifically of the Bmi1 gene. Bmi1 mutant mice develop ataxia, seizures and tremors in early postnatal life, and display a significant reduction in overall brain size, which is particularly severe in the granular and molecular layers of the cerebellum. Strikingly, Bmi1-deficent mice become depleted of cerebellar neural stem cells postnataly, indicating an in vivo requirement for Bmi1 in neural stem cell renewal. Bmi1 deficiency leads to increased expression of the cell cycle regulators p16Ink-4a and p19Arf (both now known as Cdkn2a - Mouse Genome Informatics), and the neurogenesis defect in the mutant mice can be partially rescued by further deleting p16Ink4a. This suggests that one way in which Bmi1 promotes the maintenance of adult stem cells is by repressing the p16Ink4a pathway. However, it is also likely that Hox gene repression through Bmi1 is involved in this process, given that loss of Bmi1 has been shown to cause a deregulation of posterior Hox gene expression in neural stem cells in vitro. Moreover, a direct molecular link between Bmi1 and Hox gene regulation has recently been discovered in mammalian development, in that the promyelocytic leukemia zinc finger (Plzf; Zbtb16 -- Mouse Genome Informatics) protein directly binds Bmi1 and recruits PcG proteins in the HoxD cluster (Bello, 2007 and references therein).

In Drosophila, the homologs of the mammalian Bmi1 gene are the PcG genes Psc and Su(z)2. Psc and Su(z)2 encode very similar proteins and are partially redundant in function, but both genes are eliminated in a deletion in the Psc-Su(z)2 line. Rather surprisingly, mutational loss of Psc-Su(z)2 does not lead to ectopic Hox gene derepression and, in consequence, does not appear to affect neuronal proliferation in the postembryonic CNS of Drosophila. This is in stark contrast to the other five PcG genes investigated, which do play important roles in proliferation control by preventing ectopic Hox gene expression and cell death in postembryonic neuroblasts. The discrepancy between murine Bmi1 and Drosophila Psc-Su(z)2 function in neuronal proliferation suggests that although a general role of PcG genes in neuronal proliferation control may be conserved between mammals and flies, conservation of gene action may not always be retained at the level of individual PcG homologs (Bello, 2007).

In terms of overall development, it is clear that one and the same PcG gene can have very different functions depending on the developmental context in which it acts. For example, as mentioned above, during embryonic neurogenesis the Drosophila Pc gene acts in tagmata-specific differentiation of neuroblasts, in contrast to its role in postembryonic neurogenesis. Moreover, in postembryonic development of imaginal discs, deletions in the Drosophila Psc-Su(z)2 genes have been shown to result in cellular hyperproliferation, which contrasts with the lack-of-proliferation phenotype of Psc-Su(z)2 mutants in postembryonic development of the CNS. Similarly, in the mouse, the Bmi1 gene has been implicated in tumor progression in mantle cell lymphoma, colorectal cancer, liver carcinomas and non-small cell lung cancer, in addition to its role in nervous system development. Nevertheless, in all of the Drosophila and mammalian phenotypes mentioned, deregulation of Hox gene expression appears to be one of the conserved and thus unifying features of PcG gene functional loss (Bello, 2007).

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. This study shows that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. It is proposed that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing (Tie, 2009).

The major findings of this work are: (1) that Drosophila CBP acetylates H3K27; (2) that this acetylation requires TRX; and (3) that it prevents H3K27 trimethylation by E(Z) at Polycomb target genes and antagonizes Polycomb silencing. The remarkably complementary developmental profiles of H3K27ac and H3K27me3 (but not H3K27me2) during embryogenesis suggest that the deposition of H3K27me3, which increases steadily after ~4 hours with the onset of Polycomb silencing, occurs at the expense of a substantial fraction of the H3K27ac already present. This suggests that the establishment of Polycomb silencing might require active deacetylation of this pre-existing H3K27ac. The reciprocal effects of knockdown and overexpression of CBP and E(Z) on H3K27 trimethylation and acetylation in bulk chromatin further suggest that the two modifications constitute alternative chromatin states associated with active and inactive genes. Consistent with this, ChIP-chip experiments revealed that H3K27me3 and H3K27ac are mutually exclusive genome wide. Moreover, in S2 cells, the inactive abd-A gene does not have the H3K27ac modification in its promoter region, but acquires it upon RNAi knockdown of E(Z). It will be important to determine whether such a modification switch occurs genome wide after loss of E(Z) (Tie, 2009).

The ability of E(Z) overexpression to suppress the small rough eye phenotype of CBP overexpressers further supports the conclusion that H3K27 trimethylation by E(Z) antagonizes H3K27 acetylation by CBP and suggests that deacetylation of H3K27 by RPD3, and possibly other deacetylases, might be a prerequisite for subsequent methylation by E(Z) and therefore important for reversal of an active state. Conversely, the ability of CBP and TRX overexpression to increase the global H3K27ac level at the expense of H3K27me3 suggests that either active demethylation of H3K27me3 by the H3K27-specific demethylase UTX (Agge, 2007; Lee, 2007; Smith, 2008), or histone replacement (Ahmad, 2002), might be a prerequisite to acetylation by CBP. Indeed, depletion of Drosophila UTX in vivo using a GAL4-inducible UTX RNAi transgene line results in an increase in H3K27me3, as previously reported (Smith, 2008), and in a marked decrease in H3K27ac. These data, together with the evidence of developmentally programmed reversal of Polycomb silencing, now suggest that the widely accepted stability of Polycomb silencing during development might be more dynamically regulated than previously appreciated (Tie, 2009).

This is the first report that CBP/p300 acetylates H3K27. Recombinant Drosophila CBP acetylates H3K27 and K18 in vivo and in vitro. The greatly reduced H3K27ac levels in CBP-depleted S2 cells also strongly suggest that CBP is the major H3K27 acetylase in Drosophila. The conservation of H3K27 acetylation by human p300, together with the reported association of CBP with the TRX homolog MLL in humans (Ernst, 2001), suggest that it is likely to play a similar role in antagonizing Polycomb silencing in mammals (Tie, 2009).

The genome-wide distribution of H3K27ac, as estimated from human ChIP-chip experiments, appears very similar to that of H3K4me3. This suggests that H3K27ac is much more widely distributed than just at Polycomb target genes, which are estimated to number several thousand in mammalian cells and hundreds in Drosophila. Although these numbers could grow with the identification of additional Polycomb-silenced genes in additional cell types, the recently reported strong correlation of H3K27ac with active genes suggests that it plays an additional role(s) in promoting the transcription of active genes, including those that are never targets of Polycomb silencing. (Note that the H3K27ac at non-Polycomb target genes will not be directly affected by global changes in H3K27me3.) Interestingly, like H3K27me3, H3K27ac appears on the transcribed regions of Polycomb target genes, which might reflect a role for H3K27ac in facilitating transcriptional elongation, and, conversely, a role for H3K27me3 in inhibiting elongation. In addition to its anti-silencing role in preventing H3K27 trimethylation, H3K27ac may also serve as a signal for recruitment of other proteins with additional enzyme activities that alter local chromatin structure further to facilitate or promote transcription. Prime candidates are those containing a bromodomain, a conserved acetyl-lysine-binding module present in several dozen chromatin-associated proteins, including a number of TrxG proteins that also antagonize Polycomb silencing (Tie, 2009).

The results presented in this study provide new insight into how TRX and CBP function together to antagonize Polycomb silencing. Robust H3K27 acetylation by CBP is dependent on TRX, suggesting that H3K27ac plays a crucial role in the anti-silencing activity of TRX. Consistent with this, preliminary genetic evidence suggests that the Polycomb phenotypes caused by TRX overexpression are dependent on CBP, as they are suppressed by RNAi knockdown of CBP. The nature of this dependence is currently unknown, but could involve targeting of CBP by TRX or regulation of the H3K27 acetylation activity of CBP by TRX (Tie, 2009).

The physical association of TRX and CBP and the widespread coincidence of H3K27ac and H3K4me3 sites in the human ChIP-chip data further suggest that the two modifications might be coordinately executed by TRX and CBP. However, the results also raise the possibility that H3K4 trimethylation by TRX itself might be less important for antagonizing Polycomb silencing than H3K27 acetylation. This possibility is also suggested by the discovery of Polycomb-silenced genes in ES and human T cells that contain 'bivalent' marks (both H3K4me3 and H3K27me3) in their promoter regions (although the H3K4me3 levels at these inactive genes are typically lower, on average, than they are at active genes, hinting at the possible importance of quantitative effects of the two marks) (Tie, 2009).

A speculative model is proposed for the regulation of Polycomb silencing that incorporates the activities of TRX, CBP, E(Z), RPD3 and UTX. Repressed genes are marked with H3K27me3. H3K27 trimethylation by PRC2 (which can also control DNA methylation in mammals) requires RPD3 (and possibly other histone deacetylases) to deacetylate any pre-existing H3K27ac. H3K27me3 promotes binding of PC-containing PRC1 complexes, which may inhibit H3K27 acetylation and maintain silencing through 'downstream' events, including those promoted by the H2AK119 mono-ubiquitylation mediated by its RING subunit. Conversely, active genes are marked with H3K4me3 and H3K27ac. H3K27 acetylation by CBP is dependent on TRX and possibly other TrxG proteins, as suggested by the observation that H3K27me3 levels are significantly increased on salivary gland polytene chromosomes from trx, ash1 and kis mutants. The current results predict that this increase will be accompanied by a decrease in H3K27ac. Interestingly, ash1 encodes another HMTase that also interacts with CBP and antagonizes Polycomb silencing. Acetylation of H3K27 is likely to also require the K27-specific demethylase UTX when removal of pre-existing H3K27me3 is a prerequisite for acetylation, e.g. for developmentally regulated reversal of Polycomb silencing at the onset of differentiation. H3K27ac prevents H3K27 trimethylation and might also serve as a signal for recruitment of other TrxG proteins with additional chromatin-modifying activities that may protect the H3K27ac modification and also alter local chromatin structure to promote transcription and further inhibit Polycomb silencing (Tie, 2009).

Polycomb repressive complex 2 and Trithorax modulate Drosophila longevity and stress resistance

Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are key epigenetic regulators of global transcription programs. Their antagonistic chromatin-modifying activities modulate the expression of many genes and affect many biological processes. This study reports that heterozygous mutations in two core subunits of Polycomb Repressive Complex 2 (PRC2), the histone H3 lysine 27 (H3K27)-specific methyltransferase E(Z) and its partner, the H3 binding protein ESC, increase longevity and reduce adult levels of trimethylated H3K27 (H3K27me3). Mutations in trithorax (trx), a well known antagonist of Polycomb silencing, elevate the H3K27me3 level of E(z) mutants and suppress their increased longevity. Like many long-lived mutants, E(z) and esc mutants exhibit increased resistance to oxidative stress and starvation, and these phenotypes are also suppressed by trx mutations. This suppression strongly suggests that both the longevity and stress resistance phenotypes of PRC2 mutants are specifically due to their reduced levels of H3K27me3 and the consequent perturbation of Polycomb silencing. Consistent with this, long-lived E(z) mutants exhibit derepression of Abd-B, a well-characterized direct target of Polycomb silencing, and Odc1, a putative direct target implicated in stress resistance. These findings establish a role for PRC2 and TRX in the modulation of organismal longevity and stress resistance and indicate that moderate perturbation of Polycomb silencing can increase longevity (Siebold, 2009).

The evidence presented in this study establishes a role for PRC2 and TRX in the modulation of life span and stress resistance. Using multiple alleles of several PRC2 subunits, evidence is provided that heterozygous mutations in the PRC2 subunits E(z) and esc extend life span and increase resistance to oxidative stress and starvation in Drosophila. Consistent with the enzymatic function of PRC2 in the methylation of H3K27, long-lived E(z) and esc mutants have reduced H3K27me3 levels. Furthermore, mutations in trx suppress the increased longevity and stress resistance phenotypes of E(z) mutants, while concomitantly increasing their reduced H3K27me3. The moderate reduction of H3K27me3 in long-lived E(z) mutants is sufficient to partially derepress some direct targets of Polycomb silencing, and this is also counteracted by mutations in trx. These results provide strong evidence that derepression of one or more Polycomb target genes is likely to be responsible for their increased longevity. Interestingly, E(z) was also recently identified as one of a number genes whose mRNA expression levels were significantly associated with variation in longevity in a large set of wild-type derived inbred lines (Siebold, 2009).

The counterbalancing effects of PRC2 and TRX on H3K27me3 levels suggest a simple model for their modulation of longevity. Although complete loss of PRC2 activity results in preadult lethality, moderately reducing H3K27me3 destabilizes Polycomb silencing sufficiently to cause partial derepression of some Polycomb target genes that can increase life span and stress resistance. Simultaneously reducing TRX and E(Z) exerts a compensatory effect, reestablishing more normal levels of H3K27me3 and Polycomb target gene expression. Based on this model, it is expected that heterozygous trx mutations would decrease longevity. However, the modestly elevated H3K27me3 level (13%) of the heterozygous trxB11 null mutant may simply be insufficient to cause this effect in a wild-type background. It will be interesting to see whether increased TRX levels, which decrease H3K27me3 levels (much like PRC2 mutants) by elevating CBP-mediated H3K27 acetylation, will promote increased longevity, as the model would predict. The evolutionary conservation of PRC2 components in metazoans and their conserved function in epigenetic silencing raises the possibility that they may play a conserved role in modulating life span in other organisms. Although histone methyl-transferases have not been previously implicated in modulating organismal longevity, several other highly conserved chromatin- modifying enzymes have been. In addition to SIR2 and RPD3, the histone H3K4 demethylase LSD-1 has also recently been implicated in modulating longevity in C. elegans. Given the roles of these enzymes in the epigenetic maintenance of transcriptional states, it seems likely that additional chromatin modifying enzymes will be found to modulate longevity. The most well-characterized targets of Polycomb silencing are the homeotic genes of the Bithorax and Antennapedia complexes (Siebold, 2009).

Although heterozygous PRC2 mutants exhibit no overt homeotic phenotypes, the elevated level of Abd-B expression in E(z) heterozygotes demonstrates that their moderately reduced H3K27me3 level is sufficient to partially derepress Polycomb target genes. Could modest derepression of one or more of the homeotic genes be responsible for the increased longevity? Given that they encode transcription factors, their potential for regulating expression of many other genes leaves this possibility open. PRC2 mutants exhibit increased resistance to oxidative stress and starvation. The elevated expression level of Odc1, a putative direct target of Polycomb silencing may contribute to this as it has been shown to mediate resistance to oxidative stress and a variety of other chemical and environmental stresses. Dietary supplementation with the polyamine spermidine was also recently shown to increase longevity in yeast, C. elegans, Drosophila, and mice, consistent with the possibility that Odc1 overexpression may contribute to the increased longevity of PRC2 mutants. Recent evidence suggests that other changes in metabolism and adult physiology might also contribute to the increased longevity of PRC2 mutants. YY1, the mammalian homolog of Drosophila PHO (a DNA-binding PcG protein involved in recruiting PRC2 to chromatin), directly regulates many genes required for mitochondrial oxidative metabolism. It will be interesting to determine whether transcriptional regulation of metabolic genes is a broader theme in the adult function of PcG proteins. PRC2 and TRX play key roles in promoting epigenetically stable transcriptional states through their mutually antagonistic effects on H3K27me3 levels. Recent work has revealed a growing number of biological processes in which they play an important role. The results presented here now point to a role for these epigenetic transcriptional regulators in modulating life span (Siebold, 2009).

A mutation in the E(Z) methyltransferase that increases trimethylation of histone H3 lysine 27 and causes inappropriate silencing of active Polycomb target genes

Drosophila Polycomb Repressive Complex 2 (PRC2) is a lysine methyltransferase that trimethylates histone H3 lysine 27 (H3K27me3), a modification essential for Polycomb silencing. Mutations in its catalytic subunit, E(Z), that abolish its methyltransferase activity disrupt Polycomb silencing, causing derepression of Polycomb target genes in cells where they are normally silenced. In contrast, the unusual E(z) mutant allele Trithorax mimic (E(z)^Trm) causes dominant homeotic phenotypes similar to those caused by mutations in trithorax (trx), an antagonist of Polycomb silencing. This suggests that E(z)^Trm causes inappropriate silencing of Polycomb target genes in cells where they are normally active. This study shows that E(z)^Trm mutants have an elevated level of H3K27me3 and reduced levels of H3K27me1 and H3K27me2, modifications also carried out by E(Z). This suggests that the E(z)^Trm mutation increases the H3K27 trimethylation efficiency of E(Z). Acetylated H3K27 (H3K27ac), a mark of transcriptionally active genes that directly antagonizes H3K27 methylation by E(Z), is also reduced in E(z)^Trm mutants, consistent with their elevated H3K27me3 level causing inappropriate silencing. In 0–4 h E(z)^Trm embryos, H3K27me3 accumulates prematurely and to high levels and does so at the expense of H3K27ac, which is normally present at high levels in early embryos. Despite their high level of H3K27me3, expression of Abd-B initiates normally in homozygous E(z)^Trm embryos, but is substantially lower than in wild type embryos by completion of germ band retraction. These results suggest that increased H3K27 trimethylation activity of E(Z)^Trm causes the premature accumulation of H3K27me3 in early embryogenesis, 'predestining' initially active Polycomb target genes to silencing once Polycomb silencing is initiated (Stepanik, 2012).

Evidence presented in this study suggests that the trithorax-like phenotypes of E(z)^Trm mutants are due to the substantial bulk increase in H3K27me3, which leads to inappropriate silencing of Polycomb target genes in cells where they are normally active. How does this come about? The suppression of the high H3K27me3 levels of E(z)^Trm mutants by Su(z)12 and esc mutations as well as increased E(z)⁺ gene dosage indicates that, like wild type E(Z), the E(Z)^Trm mutant protein must be assembled into PRC2 complexes and that its activity depends on other PRC2 subunits. This means that E(Z)^Trm mutant protein is likely to be targeted to the same regions of the genome as wild type E(Z) and not mistargeted to novel sites. Developmental analysis indicates that inappropriate silencing of Abd-B in E(z)^Trm mutants begins in early embryogenesis, and is presaged by premature accumulation of H3K27me3 in early (0–4 h) embryos to much higher levels than in wild type embryos. Surprisingly, this does not prevent normal initiation of Abd-B expression on schedule, but appears to potentiate its subsequent attenuation around the time that Polycomb silencing normally becomes required (Stepanik, 2012).

A recent analysis of the in vitro enzymatic activities of recombinant human PRC2 complexes demonstrated that wild type EZH2 displays the greatest catalytic efficiency for the H3K27 monomethylation reaction and lower efficiency for the dimethylation (mono- to di-) and the trimethylation (di- to tri-) reactions (Sneeringer, 2010). The increase in H3K27me3 at the expense of H3K27me1 and H3K27me2 in E(z)^Trm mutants suggests that the R741K substitution alters E(Z) catalytic activities to preferentially increase its H3K27 trimethylation efficiency. The mono- and di-methylation activities might also be affected, but the reduced steady state levels of H3K27me1 and H3K27me2 levels must be interpreted cautiously, since they are also influenced by presence of the H3K27 demethylase UTX and since some H3K27me1 may be produced by another methyltransferase besides E(Z) (Stepanik, 2012).

The decrease in H3K27ac also indicates that not all of the increase in H3K27me3 occurs at the expense of H3K27me1/2. The reduced level of H3K27ac in E(z)^Trm mutants, a modification that directly antagonizes H3K27 methylation, strongly suggests that at least some of the increase in H3K27me3 occurs at the expense of H3K27ac, i.e., occurs at H3K27 sites that are normally acetylated and associated with transcriptionally active Polycomb target genes. Indeed, in the absence of direct evidence that the excess H3K27me3 in E(z)^Trm mutants occupies genomic sites of normally active Polycomb target genes, the lower level of bulk H3K27ac is perhaps the strongest evidence that the elevated H3K27me3 level in E(z)^Trm mutants is the cause of the inappropriate silencing of normally active Polycomb target genes (Stepanik, 2012).

The R741K substitution occurs in a region of the catalytic SET domain of E(Z) that plays an important role in the methyltransferase activity of other SET domain proteins. The adjacent Y740 residue is invariant in all SET domain proteins. Mutating the corresponding tyrosine to alanine in SET7/9 or to phenylalanine in DIM-5 all but eliminates the methyltransferase activity of these proteins. A structural study showed that this conserved tyrosine is positioned in close proximity to the substrate lysine targeted for methylation. Residue Y742 is also invariant in E(Z) orthologs, though it varies among other SET domain proteins. The residue present at the corresponding position in the H3K9 trimethylase DIM-5 is involved in recognition of the substrate peptide (Stepanik, 2012).

Of particular relevance to the current work are several structure-based in vitro mutagenesis studies of other SET domain proteins that identified a 'phenylalanine/tyrosine switch' that affects the number of methyl groups the enzymes can add to the substrate lysine. This 'switch' affects the residue corresponding to F738 in E(Z). Mutating the phenylanine present at this position to the bulkier tyrosine in the H3K9 trimethylases DIM-5 and G9a results in enzymes that can only catalyze mono- and dimethylation. Conversely, mutating the tyrosine present at this position to phenylalanine in the H4K20 monomethylase SET8 allows the enzyme to add a second methyl group to H4K20 (Stepanik, 2012).

The results of these studies suggest that the E(Z)^Trm R741K substitution may increase trimethylation efficiency by altering the dimensions of the catalytic pocket either directly, via its more compact side chain, or by altering the positioning of the adjacent Y740, Y742, F738 or other residues that affect the catalytic pocket of E(Z). This could occur by altering the rate of the reaction, the affinity of E(Z) for the substrates of the methyltransferase reaction, enhancing the release of products of the reaction, or by enabling the enzyme to better accommodate the bulkier products of the trimethylation reaction (Stepanik, 2012).

Recently, a number of other dominant mutations in human EZH2 that cause elevated H3K27me3 levels have been detected in follicular lymphomas and diffuse-large B-cell lymphomas, always in heterozygous condition (Morin, 2010). These recurring mutations, single amino acid substitutions for Y641 (Y655 in E(Z)), a conserved residue in the SET domain, were found to have increased H3K27 di- and trimethylation activity but no monomethylation activity, explaining their obligate heterozygosity for manifestation of their elevated H3K27me3 phenotype. In contrast, the E(Z)^Trm mutant enzyme produces a higher H3K27me3 level in the absence of wild type protein than when heterozygous, indicating that the R741K substitution does not abolish monomethylation activity (Stepanik, 2012).

Remarkably, 0–4 h E(z)^Trm embryos exhibit an 8-fold increase in bulk H3K27me3 and a greater than 55% reduction in the level of H3K27ac. This suggests that if the H3K27ac present in early embryos normally plays a role in preventing the premature onset of H3K27 trimethylation, the increased trimethylation efficiency of E(Z)^Trm is sufficient to overcome it. It further suggests that despite the very low level of H3K27me3 in 0–4 h wild type embryos, the PRC2 subunits that are already present in the nuclei of early cleavage stage embryos are likely to be assembled into enzymatically active PRC2 complexes (Stepanik, 2012).

In spite of the much higher H3K27me3 level in 0–4 hour E(z)^Trm embryos, the levels of ABD-B in E(z)^Trm and wild type embryos are indistinguishable until after completion of germ band elongation, i.e., after the time when Polycomb silencing of the homeotic genes is normally established. This indicates that while H3K27me3 is required for Polycomb silencing, the elevated H3K27me3 level in 0–4 hour E(z)^Trm embryos does not, by itself, exert a 'repressive' effect on early Abd-B expression or advance the time of onset of Polycomb silencing. Thus, some other factor or signal that is required for Polycomb silencing must be absent or inactive during this early stage. This might include, for example, a developmentally programmed delay in the maturation of functionally active PRC1 complexes, which bind to H3K27me3-containing nucleosomes produced by PRC2 and carry out additional activities required for Polycomb silencing. Whatever the nature of this limiting factor/signal, once Polycomb silencing begins, the premature accumulation of H3K27me3 appears to 'predestine' or potentiate the inappropriate silencing of already activated Polycomb target genes (Stepanik, 2012).

Interestingly, it has been observed that feeding the histone deacetylase inhibitor sodium butyrate to E(z)^Trm/ + females partially suppressed the trx-like phenotypes of their E(z)^Trm/ + progeny, suggesting that a deacetylase activity may be required during early embryogenesis for these phenotypes to develop. The observation that during embryogenesis, H3K27ac levels are highest in 0–4 h wild type embryos, but are more than 55% lower in 0–4 h E(z)^Trm embryos is consistent with the possibility that it is specifically the inhibition of H3K27ac deacetylation by maternal butyrate feeding that suppresses the development of trx-like phenotypes in E(z)^Trm mutants, indirectly inhibiting the premature accumulation of high levels of H3K27me3 by helping to maintain H3K27ac. The histone deacetylase RPD3/HDAC1 has been implicated in H3K27ac deacetylation in vivo by RNAi knockdown in Drosophila S2 cells. RPD3 is physically associated with PRC2 and PRC1 complexes in both Drosophila and mammals and is required in vivo for robust Polycomb silencing. RPD3 of maternal origin is already present in the early embryo, and is presumably available to deacetylate H3K27. The results thus suggest that the initial establishment of Polycomb silencing during normal development may require active deacetylation of preexisting H3K27ac as a prerequisite to at least some H3K27 trimethylation (Stepanik, 2012).

If the H3K27ac already present in 0–4 h embryos normally plays a role in inhibiting the premature accumulation of H3K27me3, then the effect of the E(z)^Trm mutation on the H3K27ac level indicates that the mutant enzyme can override this inhibition. How could it do this? It seems unlikely that simply altering the H3K27 trimethylation efficiency of E(Z) would lead to increased H3K27ac deacetylation activity. Instead, it is speculated that the higher trimethylation efficiency of the E(Z)^Trm mutant enzyme alters an ongoing dynamic balance between H3K27 acetylation/deacetylation and methylation/demethylation activities in the early embryo. It was recently shown that the CBP-mediated acetylation of H3K4me3-containing nucleosomes is highly dynamic and its dynamic nature is critical for transcriptional activation (Crump, 2011). The physical association of E(Z) and RPD3 likely increases the efficiency of the sequential deacetylation and methylation reactions required for replacement of an H3K27ac with H3K27me3, a possible advantage for efficient developmentally programmed switching from a transcriptionally active to silent state. This suggests that with no change in an already dynamic acetylation–deacetylation cycle, the presence of E(Z)^Trm would further increase the probability that an un/deacetylated H3K27 will be methylated before it can be (re)acetylated, and would have the cumulative effect of increasing the bulk H3K27me3 level at the expense of H3K27ac, as observed (Stepanik, 2012).

To gain further insight into how E(z)^Trm promotes inappropriate silencing of normally active Polycomb target genes, it will be important to determine the effect of E(z)^Trm on the genome-wide distributions of H3K27me3/2/1 and H3K27ac, as well as PcG and TrxG proteins. Understanding the effects of E(z)^Trm in further detail could provide new insights into the role of aberrant Polycomb silencing in cancer. In addition to the above EZH2 mutations with increased H3K27 trimethylation activity in B cell lymphomas, EZH2 and other PRC2 subunits have been found to be overexpressed in many solid tumors, leading to aberrant H3K27 trimethylation and silencing of tumor suppressor genes. The level of overexpression is positively correlated with poor prognosis and RNAi knockdown of elevated EZH2 levels in can halt tumor cell proliferation. While the mechanisms underlying inappropriate silencing due to EZH2 overexpression and due to E(Z)^Trm and other hyper-trimethylating mutants are likely to differ, they underscore the importance of understanding how TRX, CBP, UTX and other TrxG factors antagonize H3K27 methylation by PRC2 to prevent inappropriate silencing of normally active Polycomb target genes and maintain their active states. Further understanding of how E(z)^Trm overrides their antagonistic activities should provide additional insights into how they do so (Stepanik, 2012).

PRC2 Controls Drosophila Oocyte Cell Fate by Repressing Cell Cycle Genes

The oocyte is a unique cell type that undergoes extensive chromosome changes on its way to fertilization, but the chromatin determinants of its fate are unknown. This study shows that Polycomb group (PcG) proteins of the Polycomb repressive complex 2 (PRC2) determine the fate of the oocyte in Drosophila. Mutation of the enzymatic PRC2 subunit Enhancer of zeste [E(z)] in the germline abolishes spatial and temporal control of the cell cycle and induces sterility via transdetermination of the oocyte into a nurse-like cell. This fate switch depends on loss of silencing of two PRC2 target genes, Cyclin E and the cyclin-dependent kinase inhibitor dacapo. By contrast, the PRC1 component Polycomb (Pc) plays no role in this process. These results demonstrate that PRC2 plays an exquisite role in the determination of the oocyte fate by preventing its switching into an endoreplicative program (Iovino, 2013).

Together, the data show that PRC2 controls oogenesis by direct corepression of CycE and dap in a time window that stretches from region 2b to region 3. This prevents improper endocycling, before the oocyte becomes fully silenced and determined by stage 3. Interestingly, E(z) levels rapidly drop in the germline at stage 4, suggesting that the continuous presence of PRC2 is no longer required after oocyte determination. This observation is consistent with the fact that depletion of E(z) after stage 3 using a late Gal4 driver does not affect oocyte fate (Iovino, 2013).

Although differences between PRC1 and PRC2 have been previously reported in mammals (Lessard, 1999; Sauvageau, 2010) and Drosophila (Richter, 2011), it is crucial to understand whether the molecular differences reflect specific biological roles for each of the two complexes. This study has shown that PRC2 controls oocyte cell fate determination, whereas PRC1 components, such as Pc, had no obvious function in the oocyte, consistent with absence of Pc from the germline. Intriguingly, a different situation is seen in the fly male testis, where PRC1 components are required in the germline (Chen, 2011) while PRC2 is dispensable. Therefore, the production of gametes is a critical biological function that separates the function of these two complexes (Iovino, 2013).

Although a few genes that are responsible for oocyte determination have been identified previously, none is known to act on chromatin. The identification of PRC2 as a chromatin effector complex that is required to fix the oocyte fate offers the possibility of starting to dissect the molecular mechanisms that transduce the early asymmetry between the preoocyte and the surrounding cells into a terminally determined fate (Iovino, 2013).

Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling

Histone H3 lysine²⁷-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. This study established a Drosophila animal model for the pathogenic histone H3K27M mutation and showed that its overexpression resembles polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing derepression of PRC2 target genes and developmental perturbations. Similarly, an H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M-containing nucleosomes, and its misregulation in Drosophila results in changes of H3K9 methylation levels and heterochromatic silencing defects. This study has established histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin signaling pathways (Herz, 2014).

Histone proteins constitute the core of eukaryotic chromatin. SET domain-containing histone methyltransferase complexes such as complex of proteins associated with Set1 (COMPASS) and polycomb repressive complex 2 (PRC2) methylate lysine residues within the histone H3 amino-terminal tail and are essential for normal development. Establishing direct functions for modified lysine residues in histones is difficult because there are multiple histone gene copies in metazoans. Moreover, histone methyltransferase enzymes occur in multimember families with potential redundant activities and histone methylation-independent functions. In Drosophila, replacing all copies of histone H3 with H3 Lys²⁷-to-Arg²⁷ (H3K27R) in a clonal substitution recapitulates the phenotype of mutating E(z), the PRC2 H3 Lys²⁷ methyltransferase gene, suggesting this mark is indeed required for PRC2-mediated repression. Single-allele mutations of histone H3.3 Lys²⁷-to-Met²⁷ (H3.3K27M) occur in a subtype of aggressive pediatric brain cancers and act in a dominant manner to deplete H3K27 methylation by inhibiting PRC2 methyltransferase activity. Other histone H3 lysine-to-methionine mutants also possess dominant gain-of-function activities, making them attractive tools for in vivo functional studies of histone lysine modifications. Trimethylation of histone H3 Lys²⁷ (H3K27me3) and Lys⁹ (H3K9me3) are associated with distinct forms of transcriptionally silenced chromatin. Histone H3K27me3 catalyzed by PRC2 is enriched at so-called facultative heterochromatin and is implicated in the silencing of key developmental genes, in particular the homoeotic gene clusters. By contrast, H3K9me3 is associated with 'constitutive' heterochromatin at telomeres and centromeres (Herz, 2014).

This study established wild-type histone H3.3, H3.3K27M, and H3.3K9M constructs with a C-terminal FLAG-hemagglutinin (HA) tag for tissue-specific overexpression in Drosophila. Overexpression of H3.3K27M in the posterior compartment of wing imaginal discs driven by engrailed-GAL4 caused a strong reduction in all three H3K27 methylation states and derepression of the PRC2 target gene Ultrabithorax (Ubx), thus phenocopying knockdown of the catalytic PRC2 subunit E(z). Also, increased H3K27 acetylation was observed for H3.3K27M overexpression in Drosophila and mammalian cells and E(z)-RNAi in Drosophila. Genome-wide RNA sequencing (RNA-seq) analysis of H3.3K27M-overexpressing wing imaginal discs revealed up-regulated RNA transcripts for known polycomb target genes, including Ubx, wingless (wg), and the PRC1 subunits Posterior sex combs (Psc) and Suppressor of zeste 2 ([Su(z)2]). Other Homeobox (Hox)-containing genes such as engrailed (en) and invected (in), and signaling pathway components such as cubitus interruptus (ci), were down-regulated upon H3.3K27M overexpression. Moreover, flies expressing H3.3K27M under a tissue-specific Distal-less-GAL4 driver exhibit gross morphological defects-such as severe leg malformations and fusion phenotypes and malformed, reduced, or missing proboscis-and die around eclosion, phenocopying E(z)-RNAi under the same conditions (Herz, 2014).

Trimethylation of histone H3K9me3 by suppressor of variegation 3-9 [Su(var)3-9] proteins is a hallmark of constitutive heterochromatin. Histone H3K9me3 serves as a binding substrate for heterochromatin protein 1 α (HP1α, also known as CBX5) and establishes a transcriptionally repressed state. Euchromatic genes that become abnormally juxtaposed to heterochromatic regions are subject to transcriptional silencing through position-effect variegation (PEV). Less is known about the direct role of H3K9 methylation in the regulation of gene expression. Indeed, studies in fission yeast point to H3K9 methylation-independent functions for the Su(var)3-9 homolog Clr4 in chromatin silencing. To test a direct role for H3K9 methylation in the regulation of gene expression in metazoans, H3.3K9M was overexpressed in Drosophila wing imaginal discs and mammalian cells, and a global depletion of H3K9 methylation levels was observed but no effect was seen on H3K27 methylation. In contrast, H3K9 mono- and dimethylation were slightly reduced when H3.3K27M was overexpressed. Mononucleosomes were purified from wild-type H3.3-, H3.3K9M-, and H3.3K27M-overexpressing human embryonic kidney (HEK) 293 cells, and these samples were subjected to multidimensional protein identification technology (MudPIT) mass spectrometry). The bindings of HP1α (CBX5), HP1β (CBX1), and HP1γ (CBX3) were substantially reduced for H3.3K9M-containing mononucleosomes, as were the interactions of the HP1-associated proteins chromatin assembly factor 1a (CHAF1A/p150) and CHAF1B/p60. Substantially increased association of the H3K9 demethylase KDM3B and the H3K9/K56 deacetylase SIRT6 were found with H3K9M-containing mononucleosomes (Herz, 2014).

Reduced dosage of Drosophila HP1 α [also known as Su(var)205] and Su(var)3-9 results in suppression of PEV. By using a heat shock-inducible lacZ construct inserted within Y-chromosomal heterochromatin, this study found that overexpression of H3.3K9M results in suppression of PEV in both Drosophila salivary glands and eye-antenna imaginal discs. Bulk histone H3K9 methylation levels were decreased in H3.3K9M-overexpressing salivary glands. The effects of H3.3K9M overexpression on heterochromatic silencing were assessed in Drosophila ovaries. The gypsy-lacZ construct is normally silenced in almost all follicle cells but is up-regulated upon loss of heterochromatin function. Overexpression of H3.3K9M results in derepression of lacZ. Thus, the H3.3K9M mutation disrupts heterochromatic silencing of retroelements (Herz, 2014).

KDM3B is a JumonjiC domain-containing histone demethylase that shows specificity toward H3K9 and is involved in gene activation in leukemia cells. Because KDM3B specifically interacts with H3.3K9M-containing nucleosomes, it was of interest to test whether changes in KDM3B levels would alter H3K9 methylation by knocking down or overexpressing its Drosophila homolog, JHDM2, in wing imaginal discs. Depletion of JHDM2 results in increased H3K9 mono- and dimethylation. Conversely, the overexpression of JHDM2 in wing imaginal discs results in depletion in H3K9 dimethylation levels and, to a lesser extent, H3K9 trimethylation and suppresses PEV in both Drosophila salivary glands and eye-antenna imaginal discs. JHDM2 and SIRT6 also globally affect H3K9 acetylation to a similar degree as H3.3K9M overexpression. Sirt6 is not a major regulator of PEV in eye-antenna imaginal discs and salivary glands, but Sirt6-RNAi results in a somewhat modest derepression of the gypsy-lacZ reporter (Herz, 2014).

Histone lysine-to-methionine mutants were used to globally modulate histone methylation in vivo. A Drosophila animal model of the H3K27M mutation was established, that may help elucidate the molecular pathogenesis of pediatric gliomas. To gain mechanistic insight into the molecular function of these mutants, an unbiased proteomic strategy was used to identify histone lysine-to-methionine-interacting partners. Biochemical studies do not identify PRC2 components, such as EZH2, SUZ12, and EED, as significantly enriched on H3.3K27M-containing nucleosomes as previously suggested. However, an increase in H3K27 acetylation levels was detected, along with association of bromodomain-containing protein 1 (BRD1) and BRD4 to H3.3K27M-containing nucleosomes. These findings suggest that inhibitors of H3K27 acetylation or BRD4 inhibitors, such as JQ1 and iBET, could be useful for the treatment of the H3.3K27M-mutated subtype of aggressive pediatric glioblastomas (Herz, 2014).

It was also demonstrated that H3K9M globally depletes H3K9 methylation levels in vivo, disrupts interaction of HP1 proteins, and thus suppresses PEV. Via an unbiased proteomic strategy, KDM3B/JHDM2 and Sirt6 were identified as regulators of H3K9 methylation-dependent heterochromatic silencing. Indeed, JHDM2 acts as a suppressor of variegation in multiple tissues in these assays, whereas Sirt6 function seems to be restricted to retroelement silencing. Mutations of histone H3.3K36M were recently discovered in a subtype of bone cancer. Thus, histone lysine-to-methionine mutations are associated with highly tissue-specific cancer types. Given the importance of heterochromatin in maintaining genomic stability, it is plausible that as-yet-uncharacterized H3K9M mutations might occur in some cancers. The system that was established will provide a powerful tool to inhibit histone lysine modifications at specific residues in vivo and allow to biochemically capture the molecular players involved in chromatin signaling pathways (Herz, 2014).

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