polyhomeotic

DEVELOPMENTAL BIOLOGY

Embryonic

Early expression in the cellular blastoderm appears in two domains, an anterior domain extending from 40 to 50% egg length (measured from the posterior) and a posterior domain extending from 30 to 10% egg length. The region anterior to the cephalic furrow is more intensely stained than the posterior (Fauvarque, 1995).

In germ-band extended embryos, polyhomeotic is expressed in most if not all cells of the presumptive neuroectoderm and epidermis. During gastrulation, ph expression evolves rapidly to form a striped pattern, similar to that of Engrailed in germ-band extended embryos. This banded phenotype makes sense in terms of the regulation of polyhomeotic by engrailed. By stage 13 ph is limited to the CNS and disappears from the epidermis. In this case it is no longer under control by engrailed (Serrano, 1995).

The proximal and distal polyhomeotic transcription units are differentially regulated at the mRNA level during development as shown by developmental Northern analysis. The proximal mRNAs do not become abundant until 3-6 hours of development, although maternally deposited mRNA is detectable in young embryos. The 6.1 kb zygotic transcript is initially less abundant than the 6.4 kb transcript, but by 9-12 hours, the 6.1 kb transcript is more abundant. In adult females, the two transcripts are equally abundant, but in males, the smaller transcript predominates. By contrast, the distal mRNA is much more abundant in 0-1.5 hour embryos than is proximal mRNA. Distal mRNA first falls dramatically, but increases again, between 3-6 hours of development. In adult females, a new transcript is detected, just slightly smaller than the transcript found at other developmental stages. No evidence is found for differential binding of proximal and distal products to polytene chromosomes. These results show that the ph locus undergoes complex developmental regulation, and suggest that Polycomb group regulation may be more dynamic than anticipated (Hodgson, 1997).

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins (Polycomb, Polyhomeotic and Posterior sex combs) in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression, there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. Less than 2% of Ph remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression. The findings reported in this paper do not exclude the possibillity that the minor fraction of the PcG proteins, which remains bound to mitotic chromosomes, may be associated with specific nucleation sites within the repressed genes. Repression could then be initiated in telophase from these sites via cooperative binding of previously dispersed PcG protein complexes, insuring that promoters are blocked before reassembly of functional transcription complexes. A similar marking mechanism has been proposed for active genes by residual transcription factors on mitotic chromosomes. In the BX-C, possible candidates for repression nucleation sites are regulatory elements, which are preferentially associated with PC (Buchenau, 1998).

Fluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins Polycomb and Polyhomeotic in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). Translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes were determined in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with the experimental data. Forward and reverse rate constants for complex formation were determined. Complexes exchange within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria (Ficz, 2005).

Most FRAP studies of nuclear proteins have involved components in transcription complexes or transcriptional activators that exchange in less than 2 minutes. The only repressor protein that has previously been investigated is heterochromatin protein 1 (HP1), a protein targeted to heterochromatin in higher eukaryotes. Although HP1 is loaded directly onto the chromatin during replication, it was found by FRAP to bind only transiently to chromatin with a maximum residence time of 60 seconds. Thus, both HP1 and PcG repression complexes appear to function by dynamic competition with other chromatin-binding proteins rather than by formation of a static, higher-order chromatin structure with immobilized bound repressors. FRAP measurements on polytene chromosomes revealed differences in the dissociation rate constants between individual bands -- this implies that a flexible repression system of complexes with various compositions that influence the binding affinity of other members and whose turnover is in the order of a few minutes. It is concluded that: (1) activation and repression can be dynamically controlled by simple chemical equilibria; (2) reduction in PcG levels will facilitate epigenetic change and may explain why non-cycling cells can be reprogrammed more easily than cycling cells, and (3) PcG complexes are exchangeable protein assemblies that maintain repression over many cell cycles by simple chemical equilibria (Fitz, 2005).

Larval

In larvae, Engrailed activates polyhomeotic expression during wing morphogenesis (Serrano, 1995). Larval expression occurs in the eye-antennal disc, the metathoracic leg disc, the brain and anterior midgut (Fauvarque, 1995).

Polycomb group proteins (PcG) repress homeotic genes in cells where these genes must remain inactive during Drosophila and vertebrate development. This repression depends on cis-acting silencer sequences, called Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known sequence-specific DNA-binding PcG protein, binds to PREs, but pho mutants show only mild phenotypes compared with other PcG mutants. pho-like, a gene encoding a protein with high similarity to Pho, has been characterized. Pho-like binds to Pho-binding sites in vitro and pho-like; pho double mutants show more severe misexpression of homeotic genes than do the single mutants. These results suggest that Pho and Pho-like act redundantly to repress homeotic genes. The distribution of five PcG proteins on polytene chromosomes was examined in pho-like, pho double mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins are no longer present in the absence of Pho and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not participate in the anchoring of PcG complexes, but may be necessary for transcriptional repression mediated through PREs. In contrast to Pho and Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other DNA-binding proteins implicated in PRE function, do not cause misexpression of homeotic genes or reporter genes in imaginal discs (Brown, 2003).

The distribution of the PcG proteins Pc, Psc, Polyhomeotic (Ph), Sex combs on midleg (Scm) and Enhancer of zeste [E(z)] on polytene chromosomes was examined. Pc, Ph and Psc are all core components of the PcG protein complex called PRC1. Scm has also been reported to co-purify with PRC1. Scm and Ph may also be present in protein complexes other than PRC1. E(z) is a component of the Esc-E(z) complex, which is distinct from PRC1. The analysis focused on PcG protein binding sites on the X chromosome and on the right arm of chromosome 3, which includes the bithorax and Antennapedia gene complexes (BXC and ANTC) (Brown, 2003).

Pho, Pc, Psc, Ph and Scm all bind the same three sites in wild-type chromosomes. As expected, in phol; pho double mutants, no Pho protein is detected. Binding of Pc, Psc and Scm is lost at polytene subdivision 2D in phol; pho double mutants; binding of these proteins to all other sites on the X chromosome is unaffected. Binding of Ph is completely unaffected in phol; pho double mutants. In particular, the Ph signal at 2D is present, suggesting that Ph can bind at this site even if other PcG proteins are removed. Pc binding to 2D is not lost in either pho or phol single mutants, suggesting that the presence of either of these two proteins is sufficient for Pc to bind to this site (Brown, 2003).

Taken together, the immunolocalization data suggest that binding of PcG proteins to most sites is unaltered in the absence of Pho and Phol protein, but that two proteins are redundantly required for PcG protein binding at a few specific sites. Intriguingly, it appears that all PcG proteins tested in this study are still associated with the BXC and ANTC loci. Nevertheless, the BXC genes Ubx and Abd-B are derepressed in phol; pho double mutant wing discs. Several different explanations for this paradox are proposed. (1) Derepression of homeotic genes and binding of PcG proteins were not assayed in the same tissues. It was not possible to detect derepression of Ubx in salivary gland cells of phol, pho double mutants. (2) Pho and Phol may only be required for anchoring PcG proteins at some PREs in the BXC. Different DNA-binding proteins may provide this function at other PREs. This is supported by the finding that binding of PcG proteins is lost at some sites in phol; pho double mutants. Moreover, several different PREs have been identified in the Ubx gene. The resolution of antibody signals on polytene chromosomes is not refined enough to resolve distinct PREs in a single gene and, hence, loss of only a fraction of PcG protein complexes may not be detectable. Finally, Phol and Pho may not be necessary for the anchoring of PcG protein complexes to the DNA, but may confer the actual transcriptional repression mediated by PREs in imaginal discs, while the PcG protein complexes might function in the propagation and memory of the repression. Thus, PcG protein complexes might serve to recruit Phol and Pho or their co-repressors to the DNA (Brown, 2003).

These results show a strong requirement for the DNA-binding proteins Pho and Pho-like in homeotic gene silencing in imaginal discs. In fact, the strong misexpression of homeotic genes observed in phol; pho double mutant imaginal cells is comparable with that seen in imaginal disc clones mutant for Pc, Scm, Sce or Pcl. The loss of PcG protein binding at only a small number of sites in phol, pho polytene chromosomes is consistent with the idea that Phol and Pho are required to recruit PcG protein complexes at only a subset of PREs. Alternatively, Phol and Pho may be required for transcriptional repression mediated by PREs, but not for anchoring of PcG protein complexes (Brown, 2003).

In vivo Polycomb kinetics and mitotic chromatin binding distinguish stem cells from differentiated cells

Epigenetic memory mediated by Polycomb group (PcG) proteins must be maintained during cell division, but must also be flexible to allow cell fate transitions. This study quantified dynamic chromatin-binding properties of PH::GFP and PC::GFP in living Drosophila in two cell types that undergo defined differentiation and mitosis events. Quantitative fluorescence recovery after photobleaching (FRAP) analysis demonstrates that PcG binding has a higher plasticity in stem cells than in more determined cells and identifies a fraction of PcG proteins that binds mitotic chromatin with up to 300-fold longer residence times than in interphase. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. Phosphorylation of histone H3 at Ser 28 was identified as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages. It is proposed that regulation of the kinetic properties of PcG-chromatin binding is an essential factor in the choice between stability and flexibility in the establishment of cell identities (Fonseca, 2012).

This study used a combination of quantitative live imaging and mathematical modeling to investigate changes in the dynamic behavior of PcG proteins upon mitosis and cell fate transitions in living Drosophila, giving quantitative insight into the properties of the PcG system. For the PH::GFP fusion protein, the use of limited tissue-specific expression strategies was necessary to avoid cell death associated with PH overexpression. This, in turn, precluded the quantification of endogenous PH molecule numbers, since protocols for the isolation of GFP-marked SOPs and neuroblasts are not currently available. A goal of future studies will be to isolate the PH::GFP-expressing cell types of interest in order to enable relative quantification of PH::GFP and endogenous PH. For the PC::GFP fusion protein, the transgene was expressed under the endogenous Pc promoter, enabling quantification of relative amounts of transgenic and endogenous protein from whole tissues. It is important to consider to what extent the partial rescue of Pc mutants by the PC::GFP transgene will affect the quantitative conclusions draw in this study. By quantitative comparison with PH::GFP behavior, it has been proposed that the PC::GFP fusion is less favored by fourfold to fivefold in the PRC1 complex than the endogenous protein. Previous studies have concluded that the population of PRC1 is marked with PC::GFP, but the bound fraction of PC::GFP may be an underestimation of the bound fraction of endogenous PC. This effect may lead to the lower bound fraction that was measure for PC::GFP in comparison with PH::GFP. It also follows from this that second-order kinetic processes (on rates) will be prone to inaccuracies, but first-order processes (off rates and therefore residence times) will be unaffected. It is noted that the accurate determination of the true on rate (k_on) from the pseudo-first-order association rate (k*_on), extracted from FRAP experiments such as these, is also limited by the unknown quantity of free binding sites; thus, at best, one can extract relative k_on values that allow comparisons between different cell types. This in itself allows meaningful comparisons. In summary, it is concluded that the PC::GFP fusion protein is a useful reporter of specific aspects of endogenous protein behavior: It enables the accurate determination of residence times, absolute protein quantities (which do not rely on protein activity), and relative differences between on rates in different cell types and at different cell cycle stages (Fonseca, 2012).

Comparison of PC::GFP and PH::GFP revealed twofold to ninefold longer residence times for PH::GFP than PC::GFP at interphase in all cell types. This result suggests that PC and PH do not solely operate as part of the PRC1 complex. The longer residence times observed for PH::GFP may reflect multimerization of PH via the SAM domain, which has been shown to be required for PH-mediated gene silencing (Robinson, 2012). The cell type-specific differences that were observe in the kinetic behavior of PH::GFP raise the intriguing possibility that some of these may be due to regulation of sterile a motif (SAM) domain polymerization and thus PH silencing properties (Fonseca, 2012).

The estimation of the number of endogenous PC molecules bound to chromatin in interphase (~2500-7500 depending on cell type) allows comparison with numbers of PcG target genes estimated from profiling studies (between 400 and 2000). It is noted that the interphase residence times for both proteins measured in this study (0.5-10 sec) are shorter than those previously reported for the same fusion proteins in other tissues (2-6 min). These differences may arise from the different cell types examined or from the different FRAP analysis models used. Indeed, the residence times measured in this study are consistent with those measured for several transcription factors using similar FRAP models. These findings suggest that in interphase, several PC molecules are bound to a given target gene and exchange within a matter of seconds on a time scale similar to transcription factor-binding events. The fact that shorter residence times were measured in neuroblasts than in SOPs suggests that the mode of PcG binding, and thus the extent of silencing, may be differently regulated in stem cells and differentiated cells (Fonseca, 2012).

The analysis of different cell lineages and of interphase-to- mitotic transitions led to two key findings. First, a progressive reduction was documented in mobility of both PC::GFP and PH::GFP upon lineage commitment both between cell types and within a single lineage, consistent with and extending previous studies showing reduced mobility of these proteins at later developmental stages (Ficz, 2005) and a general loss of chromatin plasticity upon embryonic stem (ES) cell differentiation. Interestingly, a recent study of TFIIH binding in developing mammalian tissues, performed in living mice, revealed a differentiation-driven reduction in TFIIH mobility, revealing long-lasting but reversible immobilization in post-mitotic cells. It will be of great interest in the future to examine PC, PH, and other PcG and TrxG (Trithorax group) proteins in other cell lineages to determine the extent to which residence times are modulatable upon changes in cell identity. In particular, it will be interesting to examine the kinetics of theDNA-binding proteins that recruit the PcG and TrxG proteins to their sites of action (Fonseca, 2012).

Second, a fraction of PcG molecules was identified that remain strongly bound to mitotic chromatin in both neuroblasts and SOPs. The long residence times (up to several minutes) of this bound fraction raise the important question of whether these molecules are carriers of mitotic memory. Thus, how the mitotic chromatin-binding properties of the PcG are differently regulated in SOPs and neuroblasts will be a key question for future studies. Does a strongly bound subpopulation exist in interphase? In the mathematical model for PC dissociation, all PC molecules are treated as belonging to a single population whose properties change upon entry into mitosis. It is noted that a model in which a subpopulation with long residence time exists during interphase would also be compatible with the observed data, but such a subpopulation was not discernible from the FRAP recovery data (Fonseca, 2012).

The determination of molecule numbers, concentrations, and kinetic constants gives insight into the absolute quantities and mobilities of free and bound PC molecules in specific cell types in the endogenous situation, thus providing in vivo quantitation of an epigenetic system. These in vivo measurements will be essential for interpretation of models based on in vitro findings. Furthermore, this analysis enabled use of quantitative mathematical modeling to examine the predicted behavior of the system over time during an entire cell cycle. The most important insight provided by the model is the requirement for accelerated PC displacement in SOPs and the prediction that this may be provided by a reduction in association rate during prophase. It was demonstrated that H3S28 phosphorylation is a good candidate mechanism for PC displacement during prophase and metaphase, in addition to its documented role in PcG displacement during interphase (Gehani, 2010; Lau, 2011). The increased residence time that was observed for PC::GFP upon RNAi-mediated knockdown of JIL-1 is consistent with a role of H3S28P in ejecting PC from H3K27me3 sites on chromatin. The observation of accumulation of this double mark in prophase and metaphase is consistent with observations of mitotic accumulation of H3K9me3/S10p but is in contrast to a study that report only slight changes in levels of H3K27me3/S28p from interphase to metaphase in human fibroblasts. This discrepancy strongly suggests that the extent of mitotic S28 phosphorylation on K27-methylated H3 tails is cell type-specific, consistent with a potential role for this mark in distinguishing the mitotic behavior of PC in SOPs and neuroblasts (Fonseca, 2012).

Since H3K27me3/S28p is associated with ejection of PC from chromatin, and the double mark is highly enriched on mitotic chromatin, additional mechanisms must contribute to the increased residence times of the small bound fraction of PC::GFP that was observed in mitosis. These may include post-translational modifications of PC and PH proteins themselves, a switch of binding platform (e.g., from histone tails to DNA or RNA), and modification of recruiting or competing molecules. Whether these proposed mechanisms contribute to mitotic PcG displacement and retention and whether they are regulated differently in different lineages will be key questions for future studies (Fonseca, 2012).

In summary, this study demonstrates that the properties of the PcG proteins are not only different in different lineages, but also profoundly altered at mitosis. It is proposed that this regulation of PcG properties may be essential to both the stability of determined cell identities and the flexibility of the stem cell state. The combination of absolute quantification with analysis in living animals that was used in this study offers three key advances to the study of epigenetic regulation: First, single, defined, genetically marked cell lineages were examined as they go through mitosis and differentiation or self-renewal. Only in a living animal can we observe a defined mitotic event and its differentiated or self-renewed daughter cells. Second, only by quantifying absolute numbers of chromatin-bound endogenous molecules in real volumes can the biological meaning of observed differences in terms of cellular concentrations and protein abundance be understood. Third, these quantitative measurements enable not only the comparison of dynamic transitions in different cell types, but also meaningful mathematical models, identifying which parameters of the system can best explain the observed changes in the plasticity of PcG-chromatin binding upon mitosis and differentiation in stem cells and in more determined lineages. In summary, the combined use of live imaging and mathematical modeling in genetically tractable, dynamically changing in vivo experiments provides quantitative insight into how a system whose components are in constant flux can ensure both stability and flexibility (Fonseca, 2012).

Effects of Mutation or Deletion

Homozygous ph flies have homeotic transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes (ANTP-C, BX-C), and in addition show loss of the humerus. ph interacts with three other similar mutations: Polycomb, Polycomb-like, and extra sex comb , and acts as a dominant enhancer of Pc. The expression of ph depends on the ANTP-C and BX-C dosage. ph has no embryonic phenotype, but temperature shift studies on ph2 show that the ph+ product is required during embryogenesis and larval development. ph mutants act to disrupt the normal expression of the ANTP-C and BX-C; therefore, ph+ is needed for maintenance of segmental identity (Dura, 1985).

Viable mutations of polyhomeotic produce transformations similar to those of known gain-of-function mutants in the ANTP-C and the BX-C. These are transformations of anterior segments and structures such as wings into more posterior segments (Dura 1987).

Polyhomeotic is found in immunoprecipitates in a multimeric complex that includes Polycomb. Duplications of ph suppress homeotic transformations of Pc and Polycomb-like, supporting a mass-action model for Pc-G function. ph alleles crossed to all members of the Polycomb group show synergistic effects, suggesting that these gene products might interact directly with ph. Embryonic phenotypes of ph2 embryos that were lethal when heterozygous or homozygous for other mutations suggest that ph may perform different functions in conjunction with differing subsets of Pc-G genes (Cheng, 1994).

Mutations in the proximal and distal proteins have differing effects on regulation of a reporter under the control of a regulatory region from bithoraxoid, suggesting that ph proximal and distal proteins have different functions (Hodgson, 1997).

The ph⁴⁰⁹ mutation disrupts one of the two tandem copies of the polyhomeotic gene located on the X chromosome. Although ph⁴⁰⁹ is a hypomorphic, homozygous viable allele, it is lethal or near-lethal in combination with other PcG mutations, including alleles of Sex combs on midleg . To assess ph interaction, ph⁴⁰⁹ females were crossed to males bearing Scm mutations and viability was scored among the double mutant male progeny. All Scm alleles cause partial lethality among ph⁴⁰⁹; Scm/+ males. However, Su(z)302, R5-13, and ET50 show much higher lethality in this combination than do H1 and M56 (Bornemann, 1998).

The ph^P1 allele of Drosophila polyhomeotic encodes a chimeric P-Ph protein that contains the DNA-binding domain of the P-element transposase and the Ph protein lacking 12 amino-terminal amino acids. It has been shown that the P-Ph protein is responsible for the formation of a repressive complex on P elements inserted at the yellow locus. An enhancer element can suppress the P-Ph-mediated inhibition of yellow transcription. However, an increase of P-element copy number at the yellow locus overcomes the enhancer effect. The mobilization of P-element transposition induces the appearance with a high frequency of Su(y) mutations that partially or completely suppress the inhibitory effect of ph^P1 on yellow expression. The Su(y) mutations are localized at different sites on chromosomes. One strong Su(y) mutation, sn^eP1, was found to be induced by a 1.2-kb P-element insertion into the transcribed noncoding region of the singed locus. The Su(y) mutations result in a high level of transcription of the 1.2-kb P element that contains the sequences encoding one DNA-binding and two protein-protein interaction domains of the transposase. The effect of Su(y) mutations can be explained by the competition between the truncated transposase encoded by a 1.2-kb P element and the P-Ph protein for binding sites on P-element insertions (Biryukova, 1999).

Thus, against the ph^P1 background the P-element sequences function as Polycomb response elements (PREs). PREs are found in the regulatory regions of many homeotic genes and are responsible for transcription repression. PREs are identified by their silencing effect on a reporter gene and by the introduction of variegated or pairing-sensitive expression of the white gene in transgenic flies. DNA fragments with PRE activity are generally from several hundred to several thousand base pairs in length. The y alleles used in this study have been generated by an insertion of the 2.9-kb genomic DNA sequence from the 1A region of X chromosome located more distally with respect to the yellow gene. The genomic duplication is flanked by two identical copies of a deleted 1.2-kb P element. The 1A enhancer located in the 2.9-kb insertion is responsible for yellow activation in the body cuticle and wing blade, compensating the yellow body and wing enhancers blocked by the su(Hw) insulator. The appearance of a strong enhancer disturbs the repressive effect of the P-Ph protein. Addition of the wing and body enhancers by inactivation of the su(Hw) function further suppresses the silencing effect of the ph^P1 mutation. The assembly of a silencing complex depends both on the strength of a PRE site and on the transcriptional activity of the region involved. For example, the formation of a Pc-G complex and the binding of GAL4, which activates transcription, are mutually exclusive and the silencing state can be prevented by a strong activation of transcription (Biryukova, 1999 and references).

The level of repression induced by ph^P1 depends directly on the number of P elements but not on their localization. The ph^P1-mediated repression may act at a distance from the yellow promoter. In the y^+s32 allele the P element is located ~3 kb from the yellow promoter and is separated from the latter by the 1A enhancer. Still, in this case, the ph^P1 mutation inhibits yellow transcription. In this respect, the P elements act like PREs that are responsible in a cooperative manner for the repressive state in the adjacent genome region. The ph^P1 mutation induces only a weak repression in y alleles containing a single P-element copy upstream of the yellow gene. Thus, one copy of P element is not sufficient for the formation of a strong repression complex. Addition to a single copy of the 1.2-kb P element of 153-bp 3'-terminal P-element sequences strongly enhances ph^P1-mediated repression, leading to a complete inactivation of yellow expression. As expected, the deletion of transposase-binding sites or 11-bp inverted repeats reduces the ph^P1-mediated repression. Unexpectedly, the internal region of the 1.2-kb P element is also important for the ph^P1-mediated repression that suggests the presence of a potential site(s) for binding of the chimeric P-Ph protein (Biryukova, 1999 and references therein).

PRE-containing transposons often show a dramatic enhancement of silencing when a fly is homozygous for a transposon insertion, indicating that the homologously paired PREs interact to produce a more stable and more repressive Pc-G complex. No pairing-dependent repression is found when both paired y alleles contain a single P-element copy inducing a weak or no ph^P1-mediated repression. A pairing-dependent repression is found only if one of the y alleles in heterozygous females induces a strong repression complex in the presence of ph^P1 (Biryukova, 1999).

The inhibitory effect of the ph^P1 mutation can be partially suppressed by Su(y) mutations induced by a P-element insertion into regulatory or coding regions of different genes. The inserted 1.2-kb P element is transcribed at a high level and gives rise to a truncated transposase. For example, in the sn^eP1 mutation, the 1.2-kb P element is inserted into the 5' transcribed noncoding region leading to an enhanced P-element transcription. mRNA encoding truncated transposase is also formed as a result of the ph^P1 mutation and this may partially decrease its effect. The 1.2-kb P element has a deletion between 830 and 2560 bp and resembles the previously described KP element. The DNA-binding domain is located within the region of 98 amino-terminal amino acids. The KP protein (a zinc finger transcriptional repressor coded for by the P-element) also contains two protein-protein interaction regions, and dimerization of the KP protein is essential for high-affinity DNA binding. A putative leucine zipper is located between 101 and 122 amino acids of the KP protein . The second protein-protein interaction region is present within a segment of 69 carboxy-terminal amino acids of the KP protein, that is, still in the amino-terminal part of the intact P element . All these sequences are also present in the protein encoded by the 1.2-kb P element (Biryukova, 1999 and references therein).

The KP protein binds to multiple sites on the P-element termini with a higher affinity than the full-length transposase. The binding sites include the high-affinity transposase binding sites, an 11-bp transpositional enhancer, and the terminal 31-bp inverted repeats. Both protein-protein interaction regions are important for this binding. The 1.15-kb P element in sn^e, a derivative of the sn^w mutation, is inserted in almost the same place and has the same orientation as the P element in sn^eP1. The protein product of the 1.15-kb P element contains the DNA-binding domain and a leucine zipper, but not the second protein-protein interaction region. This explains why the sn^eP1 mutation strongly suppresses the ph^P1 inhibitory effect, while sn^e does not. The Delta2-3 construct generating a full length transposase has a very weak suppression effect on the ph^P1 mutation. This may also be explained by its lower affinity to the P-element DNA. The defective transposase with the DNA-binding domain and two protein-protein interaction domains is most efficient for realization of the suppression effect. Thus, the suppression of ph^P1-induced inhibition is a result of the competition for binding sites on the P elements between the KP-like protein and the P-Ph protein. The presence of a strong enhancer in the chimeric element helps the KP-like protein in blocking the assembly of a P-Ph-mediated Pc-G complex on the P element. However, the Su(y) mutations, even expressing the truncated P-element transposase at high levels at all stages of Drosophila development, fail to affect the ph^P1-mediated inhibition of y^2s alleles induced by a double P element in the absence of the 1A enhancer. The formation of a strong repression complex prevents binding of the KP protein to transposase-binding sites on the P elements. Thus, the general low level of transcription activity facilitates P-Ph protein binding to DNA and the formation of a repressive complex (Biryukova, 1999).

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. An investigation was carried out to see whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. Topoisomerase II (TopoII) and Barren (Barr: a subunit of the condensin complex) are shown to interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, the PcG protein Polyhomeotic (Ph) interacts physically with TopoII and Barr and Barr is required for Fab-7-regulated homeotic gene expression. Conversely, defects in chromosome segregation have been found associated with ph mutations. It is proposed that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression (Lupo, 2001).

PcG genes have been proposed to act as chromosomal components maintaining transcriptional repression by 'heterochromatinizing' their target sites. However, the molecular mechanisms underlying chromosomal silencing by the PcG, heterochromatin formation, and the transmission of the silenced state through mitosis are not known. It was reasoned that chromosome condensation machineries could provide an important functional link between the regulation of chromosome domain structure, gene silencing, and mitotic inheritance. Thus, the interaction of the PcG with the machinery involved in orchestrating chromosome dynamics has been investigated and in particular with those machines enabling mitotic chromosome condensation. The in vivo formaldehyde-fixed chromatin immunoprecipitation (X-ChIP) method was used to analyze the distribution in the BX-C locus of two proteins: TopoII, an enzyme involved in the regulation of DNA supercoiling, chromosome condensation, and segregation, and Barr. Barr is the homolog of the Xenopus XCAP-H and C. elegans DPY26 proteins, a TopoII-interacting protein associated with the SMC2/4 condensins complexes, known to be involved in mitotic chromosome condensation (Lupo, 2001).

A striking colocalization of TopoII and Barr with previously mapped PC binding sites was found, suggesting that the two groups of functions are at least acting on the same DNA regions. A clear colocalization was found at major PREs (Fab-7, Mcp, iab-3, bxd, and bx). In particular, the Fab-7 element appears to be a major TopoII/Barr binding site. Strong association of PC to Fab-7 is found. The expression of the major BX-C genes was examined by RT-PCR, and it was found that the AbdB gene is expressed, whereas Ubx and abdA are silent. No Barr/TopoII binding site was found at the Fab-8 PRE, which might define the border between the repressed and active BX-C domains in SL-2 cells (Lupo, 2001).

In iab-2 and iab-3, large fragments (11.0 and 11.5 kb, respectively) have PRE activity. Here specific Barr and TopoII sites are also found. These sites do not match the PC/GAGA peaks previously described. Yet, since these regions show considerable levels of PC, it is suggested that minor PC binding sites adjacent to the reported 'peaks' may also be functionally relevant. Another important aspect of PcG function is the interaction with promoters; major PC binding sites include core promoters, and it is known that PREs perform better when combined with their natural target promoters. Interestingly, a striking colocalization of TopoII and Barr is also found at promoters (AbdB gamma, abdA II, and Ubx) (Lupo, 2001).

Based on the mitotic phenotype and previous immunolocalization data, a direct association of TopoII and Barr with chromosomes mostly at mitosis is expected. In this context, the colocalization of TopoII and Barr in regulatory regions of the BX-C is striking. Although asyncronous tissue culture cells were used, it is believed that the association of Barr and TopoII with the regulatory regions of the BX-C occurs not only at mitosis but also in interphase. In particular, in X-ChIP experiments, the number of mitotic cells at the time of formaldehyde fixation is around 5%, thus, if only mitotic cells contributed to the overall precipitated DNA, this approach would have been below the detectable limit. Hence, it is proposed that TopoII and Barr are associated with their target sites throughout the cell cycle (Lupo, 2001).

The short proximal isoform of Ph (Ph 140p) can be copurified from nuclear extracts with TopoII and Barr. This isoform is not found coimmunoprecipitated with Pc and Psc, and neither Barr nor TopoII copurified with Pc and Psc. The three PcG members Pc, Psc, and the long proximal product Ph 170p have been shown to coimmunopurify from nuclear extracts with antibodies against one of the three. Due to the absence of Ph 140p signals in the Pc/Psc immunoprecipitations, these results might be taken to indicate that there is no functional connection between the presumptive TopoII/Barr/Ph 140p complex and the Pc/Psc/Ph 170p complex. For three reasons this is thought to be unlikely. (1) Both the 170p and the 140p isoforms of Ph are derived from the same transcript by posttranscriptional regulation and differ by a 244 N-terminal stretch of amino acids present only in the 170p isoform. Functional domains of Ph (zinc finger, coiled-coil region, GTP binding site, serine/threonine-rich region, and SAM/SPM domain) are all contained in both isoforms, suggesting that both proteins can fulfill related functions. (2) X-ChIP data, obtained with the same Ph antibodies used in this study, show an extended overlap of Pc and Ph binding regions in the BX-C. Together with the finding of a colocalization of TopoII and Barr with PcG binding sites in regulative regions of the BX-C, this suggests that these proteins act on the same DNA regions. (3) The data show that a reduction of the amount of Barren protein in barren heterozygotes parallels PcG-negative effects on the silencing function of the Fab-7 PRE (Lupo, 2001).

An additional finding supports the conclusion that Ph protein(s) are involved both in PcG function and mitotic chromosome condensation. ph null embryos show defects in chromosome segregation, the same phenotype observed for barren mutant embryos. Conversely, the results of Barren haplo-insufficiency on Fab-7 silencing are suggestive of a role for Barr in early embryogenesis. Since in early embryogenesis Ph 140p is the only Ph product made, these defects are diagnostic of a specific role of Ph 140p in mitosis. These results with regard to Barren protein and Fab-7 silencing are reminiscent of another previously documented role for SMCs in gene regulation. In C. elegans, the DPY27 protein, a homolog of the Xenopus XCAP-C (SMC4), has been shown to bind the X chromosome in females, whereas its absence results in lethality due to abnormally high gene expression levels from the X chromosome. Thus, it is concluded that Ph 140p shares an important role with the Barr/TopoII condensin complexes in mitosis and cell memory processes (Lupo, 2001).

In order to further study interactions between barren and the PcG the null alleles ph⁵⁰² and ph⁶⁰² were used for genetic analysis, and strains heterozygous for ph and barren mutations were crossed. Surprisingly, no effect was found. PcG genes, in contrast, show dosage effects, suggesting that the interaction between PcG and Barr/TopoII may imply a different, more dosage-insensitive regulation. However, it has been shown that barren mutations affect PRE silencing in the same way as mutations in PcG genes do. Taken together, these results may indicate a nonstoichiometric relationship between PcG and Barr/TopoII protein complexes. It is proposed that major PcG and condensin proteins belong to distinct protein complexes, but that they nevertheless cooperate at PREs and promoters to maintain the silenced state of homeotic genes. From the SMC standpoint, these results are intriguing because they show that proteins involved in chromosome condensation and segregation processes bind to regulatory elements in chromosomal domains responsible for the inheritance of transcription states. This would suggest that the 'structural maintenance of chromosome' function could also affect epigenetic control of gene expression (Lupo, 2001).

These data reveal novel molecular aspects of BX-C regulation. The distribution of PC and TopoII/Barr sites in the BX-C appears as a reiterated array suggestive of heterochromatic hallmarks, perhaps providing in cis information for higher-order organization of the BX-C chromosomal domain. In particular, TopoII oligomerizes in a DNA-dependent manner. Similar interactions in trans are proposed to occur between PcG proteins in vivo. According to this ability, spaced molecules at distant sites on the DNA could come into contact, giving rise to more condensed domains. A model has been proposed to explain how condensin proteins and Topoisomerases may act together in condensation. In this model, the size of the condensin complex (perhaps 1000 Å) could introduce (+) supercoils by affecting the global writhe of DNA, thus creating a more condensed state. In this study, Barr is found only at discrete sites, whereas PC and other PcG proteins are associated also with large chromosomal regions. Possibly, one aspect of PcG protein function and binding to chromatin in interphase is to stabilize and expand the condensed state by topological effects (Lupo, 2001 and references therein).

The positioning of TopoII at complex regulatory regions (e.g., abx/bx and iab-3-iab-8) may indicate the existence of minidomains providing tight control on the chromatin structure of intervening regulatory DNA sequences by localized changes of DNA superhelicity. The activity of TopoII could be locally regulated by the association with other proteins like Barr and perhaps some PcG and trxG members [e.g., Ph 140p, CCF, E(z), and Gaga]. Interestingly, Barr has been found to stimulate TopoII activity. It has to be pointed out that these data show, in a direct way, where in vivo TopoII binds to single-copy genes but they cannot tell if these sites correspond to TopoII cutting sites. However, it is likely that a tight association with DNA corresponds to enzymatic activity. Thus, it is proposed that in vivo TopoII activity may be enhanced at specific sites, whereas at others it could be reduced, resulting in local differences in chromatin condensation states controlled by DNA topology (Lupo, 2001).

The presence of multiple Barr and TopoII sites within the BX-C could thus provide a powerful way to fine-tune the structure of each of the parasegment-specific chromosomal subdomains. As a direct consequence of controlled condensation of specific parts of the BX-C, determined states could be fixed by enabling or not enabling specific interactions between cis elements. The mechanism by which Fab-7 regulates the AbdB promoters is, in fact, not known. It has been proposed that a combination of 'chromatin effects' and insulating activity may regulate enhancer-promoter interactions. It is proposed that the homeotic loss-of-function phenotypes observed in Fab-7 or Mcp deletions could be due to a change in local DNA topology altering the communication of segment-specific enhancers with the AbdB promoters. In this way, local differences in chromosome domain topology may contribute to stabilize or interfere with correct phasing between regulatory elements and promoters. If topological effects are at least part of Fab-7 function, this may also help to explain distance-dependent effects on enhancer-promoter interactions. Interestingly, in Drosophila, mutations in the Nipped-B gene facilitate enhancer-promoter interactions by overcoming the action of ectopic insulator elements in the Ubx domain. Nipped-B is the homolog of the yeast SMC-associated protein Scc2 (sister chromatid cohesion 2), suggesting that adherins may have a broader role in chromosomal domain organization and gene regulation. It is proposed that chromatin condensation proteins may be involved in a pathway acting also in interphase that regulates chromosome domain structure by DNA topology and is essential for maintenance of gene expression (Lupo, 2001).

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, since bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repress white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC is stronger than ZnF-SCM, which is stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression is more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produce silencing, indicating that these effects are long range. Repression by ZnF-SCM requires a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo (Roseman, 2001).

Biochemical studies indicate that PcG repression involves multiple, distinct PcG complexes. Thus, an underlying assumption of the assay system that was used is that gene silencing by the tethered ZnF-PcG protein involves assembly with endogenous PcG proteins at the reporter site. This hypothesis leads to the prediction that repression by a tethered ZnF-PcG protein should be compromised by loss of function for an endogenous PcG partner. It was difficult to test this prediction for the comprehensive set of endogenous PcG proteins because the basic assay system involved generating a very complex genotype. Nevertheless, the requirement was tested for endogenous PH protein, which is encoded by an X-linked gene and for which a hemizygous viable allele is available (Roseman, 2001).

A reporter integration site was identified that normally lacks PH binding, as scored on polytene chromosomes: it was repressed by all three ZnF-PcG proteins. Genetic tests show that reduction in PH dosage relieves tether-based repression by PC and SCM at this site. These results can be reconciled with the known PC-PH and SCM-PH molecular interactions. Surprisingly, ZnF-ESC repression is sensitive to PH dosage. This result was not expected since ESC-PH interactions have not been reported and there is evidence that ESC and PH are in separate complexes in embryos (Roseman, 2001).

Several explanations may account for the effect of PH dosage upon ZnF-ESC repression. (1) Since only a single reporter site was investigated, the PH dependency at this site may not be a general property at other genomic sites. It is noted, however, that this reporter site was chosen for analysis because polytene chromosome immunostaining studies indicate that it is not pre-equipped with endogenous PH. (2) Alternatively, it is possible that the functions of biochemically separable PcG complexes are interdependent in vivo, at least at certain loci. This could also explain the basic observation that PC and ESC are both required for repression at homeotic loci even though they sort into distinct complexes. An excellent example of the interplay between distinct chromatin complexes at a single locus is provided by regulation of the HO gene in yeast. Both the SWI/SNF nucleosome remodeling complex and the SAGA histone acetyltransferase complex are required for HO activation in vivo. These complexes cooperate in an ordered series of events, wherein SWI/SNF action is a prerequisite for SAGA activity upon HO chromatin. (3) Similarly, loci that require multiple PcG complexes for transcriptional repression may use a multistep mechanism where one PcG complex alters the chromatin template to 'pave the way' for binding or action of another PcG complex. Indeed, this type of interplay could explain the observation that E(Z) function is required for association of the PRC1 components PSC and PH at many chromosomal sites (Roseman, 2001).

The carboxyl-terminal SPM domain of SCM is highly conserved in mammalian SCM homologs. Analyses of Scm mutant alleles that remove the SPM domain, together with site-directed mutational analysis, have shown that the SPM domain is required for SCM function in vivo. Although in vitro studies indicate that the SPM domain is a protein interaction module, the functional contribution of this domain to SCM repression in vivo is not known. One possible role for the SPM domain would be to recruit SCM to target sites in chromatin, by analogy to the role that the conserved chromodomain plays in chromatin targeting of PC. Deletion of the SPM domain in ZnF-SCM^DeltaSPM abolishes silencing in the tethering system. Although ZnF-SCM^DeltaSPM accumulates to a level similar to that of wild-type ZnF-SCM, its repression activity is indistinguishable from ZnF alone. These results imply that the SPM domain does not solely provide interactions that target SCM to chromosomes, since the Su(Hw)-binding domain circumvents this targeting function. Instead, it is suggested that the SPM domain is more directly involved in the repression mechanism or in maintaining integrity of SCM complexes (Roseman, 2001).

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 tell which functions telomeric PREs combine (Boivin, 2003).

polyhomeotic is required for somatic cell proliferation and differentiation during ovarian follicle formation

The polyhomeotic (ph) gene of Drosophila is a member of the Polycomb group (Pc-G) genes, which are required for maintenance of a repressed state of homeotic gene transcription, which stabilizes cell identity throughout development. The ph gene was recovered in the course of a gain-of-function screen aimed at identifying genes with a role during ovarian follicle formation in Drosophila, a process that involves coordinated proliferation and differentiation of two cell lineages, somatic and germline. Subsequent analysis revealed that ph loss-of-function mutations led to production of follicles with greater or fewer than the normal number of germ cells associated with reduced proliferation of somatic prefollicular cells, abnormal prefollicular cell encapsulation of germline cysts and an excess of both interfollicular stalk cells and polar cells. Clonal analysis showed that ph function for follicle formation resides specifically in somatic cells and not in the germline. This is thus the first time that a role has been shown for a Pc-G gene during Drosophila folliculogenesis. In addition, mutations in a number of other Pc-G genes were tested, and two of them, Sex combs extra (Sce) and Sex comb on midleg (Scm), also display ovarian defects similar to those observed for ph. These results provide a new model system, the Drosophila ovary, in which the function of Pc-G genes, distinct from that of control of homeotic gene expression, can be explored (Narbonne, 2004).

A new role for ph was first revealed by the reduced fecundity and associated ovarian anomalies observed upon analysis of a P{y+}UAS insertion in the first exon of the ph-p locus (4061 line). That the ovarian phenotypes characterized for this line are due to overexpression of ph is supported by three lines of evidence: (1) the ovarian phenotypes produced depend on the presence of a GAL4 driver (da-GAL4); (2) the 4061/w; da-GAL4/+ flies also present a ph gain-of-function haltere-to-wing transformation; and (3) flip-out clone overexpression in ovarian somatic cells of several UAS-ph cDNA transgenes gave very similar ovarian phenotypes. In particular, overexpression of ph is associated with production of multicyst follicles in which several (two to four) germline cysts develop within a single follicular epithelium. Importantly, each multicyst follicle contains several pairs of polar cells corresponding to the number of cysts present in the follicle. Here, therefore, unlike other mutants (notch and hedgehog) for which inclusion of several cysts in one follicle has been attributed to a problem in polar cell specification, this does not seem to be the case. Interestingly, multicyst follicles produced by ph overexpression are covered by a follicular epithelium that is not completely regular, showing indentations that appear to mark boundaries between cysts as evidenced by the presence of polar cells at the level of the indentations. This suggests that earlier, cyst individualization may have begun and been subsequently aborted. In support of this, analysis of the associated germaria shows an abnormally long region 3, with adjacent mature germline cysts between which prefollicular cells fail to complete centripetal migration (visualized by specific anti-Fas III antibody staining of prefollicular cells). Overexpression of ph may thus specifically affect the expression of proteins necessary for recognition and/or adhesion between prefollicular cells and germline cysts for encapsulation. These effects seem to be specific to this stage since later interactions between these two cell lineages, for instance between follicular epithelial cells and the nurse cells and oocyte, are not perturbed by overexpression of ph (Narbonne, 2004).

Surprisingly, a similar phenotype as that observed in the ph overexpression study, multicyst follicles (several cysts within one follicle), was observed with loss-of-function ph alleles. Importantly, in contrast to ph overexpression, multicyst follicles in ph loss-of-function mutant ovaries always have only two groups of polar cells, one at each pole. Therefore, it seems that, unlike for overexpression of ph, delayed or deficient polar cell specification in ph mutants contributes to inclusion of several cysts within a single follicle. Thus, ph overexpression and loss-of-function phenotypes are distinguishable, indicating that the origin of the phenotypes is probably different (Narbonne, 2004).

The implication of the ph gene in ovarian somatic cells was also studied using two different loss-of-function mutations: the hypomorphic ph^lac mutation, which consists of a PlacW transposon inserted in the first intron of ph-p; and via clonal analysis of the amorphic ph⁵⁰⁴ (noted ph⁰) allele, which eliminates the functions of both ph-p and phd. The origin of the multicyst phenotype caused by ph loss-of-function mutations was characterized more precisely by analysis of the process of follicle formation in the germarium. This study showed that several early aspects of the somatic cell developmental program (including proliferation, morphogenesis and differentiation) are perturbed by these ph mutations (Narbonne, 2004).

In contrast, the rate of division of germarial somatic cells is reduced in a ph hypomorphic mutant background, as assayed by immunohistochemical analysis of the mitosis-specific PH3. This probably contributes to delayed follicle encapsulation and budding, evidenced by the accumulation of mature germline cysts in germarial region 3 of ph mutant ovarioles, and, consequently, by the formation of multicyst follicles. Although the same type of analysis was not possible upon induction of clones of the ph⁰ amorphic mutation in somatic ovarian cells, the fact that these clones are very small or absent compared with control clones suggests that a proliferation defect may also be associated with this ph mutation (Narbonne, 2004).

In contrast, the morphogenetic properties of prefollicular cells and their differentiation into polar cells, interfollicular stalks and follicular epithelia are also specifically perturbed in ph mutants, which also probably contributes to formation of multicyst follicles. ph⁰ clonal analysis shows that ph function is necessary specifically in somatic cells, and not in the germline, for proper follicle formation. Almost all the phenotypes observed in ph mutant ovaries were reproduced upon induction of ph⁰ clones in prefollicular cells and their descendants (Narbonne, 2004).

In one observed phenotype, prefollicular cell individualization of germline cysts was shown to be compromised in germarial regions 2a and 2b of ph mutant ovarioles. Fas III, which is specifically upregulated in prefollicular cells in wild-type germaria, is expressed normally in ph mutant prefollicular cells (ph^lac and ph⁰), but these cells remain at the periphery of the germarium and fail to undergo normal morphological changes necessary for germline cyst encapsulation, allowing multiple cysts, not individualized by somatic cells, to accumulate in region 3. In other ph mutant germaria in which prefollicular cells have migrated between germline cysts, encapsulation is disorganized, and germline cysts can be split and follicle budding significantly delayed. Therefore, in the germarium, interaction between prefollicular cells and the germline is defective (Narbonne, 2004).

In a second observed phenotype, prefollicular cell differentiation into polar cells, interfollicular stalk cells and follicular epithelial cells was delayed and/or incomplete in the presence of ph mutations. Using a polar cell specific marker (A101/neu-lacZ), specification of polar cells, normally appearing by stage 2 in wild-type follicles, was shown to be delayed in ph mutant ovarioles until stage 3. In addition, multicyst follicles produced in ph mutant ovarioles contained two pairs of polar cells, one at each extremity of the anterior/posterior axis. Therefore, polar cell specification, which is necessary for individualization of germline cysts, is perturbed by ph mutations. Interfollicular stalk cell differentiation was assayed by expression of the stalk-specific marker, alpha-Spectrin, and by loss of Fas III, which is normally expressed at high levels in precursor prefollicular cells. In stalks of both ph mutant and ph⁰/ph⁺ mosaic ovarioles, although alpha-Spectrin expression was normally upregulated, Fas III was also present at high levels, indicating an ambiguous state of differentiation. Poor differentiation of stalk cells was further substantiated by the abnormally long and disorganized interfollicular stalks that showed intercalation defects. Abnormal perdurance of the early prefollicular cell marker Fas III was also observed at the level of the follicular epithelium in very affected ph mutant ovarioles, as well as in ph⁰ epithelial cell clones. Taken together, these results suggest that ph mutations result in the prolongation of a precursor state for polar, stalk and follicular epithelial cells (Narbonne, 2004).

In a third observed phenotype, ph mutant ovarioles exhibited an excess of polar cells (up to 11) at both the anterior and posterior poles of follicles, which persisted beyond stage 9, accompanied by an excess of adjacent interfollicular stalk cells (from 10 to 50). ph⁰ clones induced in the polar cell lineage also produced an excess of polar cells after stage 5, and the presence of ph⁰ cells was also associated with abnormally long stalks. Overproliferation of the pool of precursor cells common to both polar and stalk cells would result in an excess of these cells. However, the mitotic activity of germarial somatic cells was assayed by PH3 staining and a reduction compared to wild-type was observed in regions 2a, 2b and 3. It has recently been shown that an excess of polar cells is normally present in early stage follicles in wild-type females, and that the final pair of polar cells is selected from this group via apoptosis-induced cell death. However, in that study, a maximum of six such pre-polar cells was observed when apoptosis was specifically blocked. Since ph mutant ovarioles exhibit up to 11 polar cells, it would seem that apoptosis of pre-polar cells is probably not the only aspect of polar cell development affected. Finally, it is possible that the process by which the pool of polar and stalk cell precursors, distinct from the progenitors of the epithelial follicle cells, is set aside may be affected by ph mutations, leading to both problems in their number and differentiation. Determination of this pool probably involves several cell-cell signaling pathways in region 2b of the germarium, implicating Delta/Notch and EGFR signaling initiating from the germline and Hedgehog signaling from anterior terminal filament somatic cells. ph may participate, in parallel or within one (or several) of these signaling pathways, to the regulation of the somatic cell differentiation program in the germarium. So far, attempts to uncover genetic or molecular interactions between ph and genes of these signaling pathways have proven unfruitful (Narbonne, 2004).

The ph gene was first characterized as one of the Drosophila Pc-G genes, which encode transcriptional repressors required for maintaining the spatial pattern of homeotic gene expression during embryonic and larval development. ph has additional functions during development, since it has also been implicated in restriction of anterior compartment expression of engrailed and hedgehog in the wing imaginal disc. The present results, which implicate ph function and that of two other Pc-G genes (Sce and Scm) in somatic cell development during early oogenesis, thus suggest that Pc-G function may be more generalized than previously thought. One other study reported ovarian defects associated with two temperature-sensitive alleles (pco^ox736hs and pco^my939hs) of the E(z) gene, but the defects observed do not resemble those of ph (degeneration of nurse cells and little growth in the size of the follicle beyond stage 3 or 4) (Narbonne, 2004).

Ovarian phenotypes associated with mutations in several other Pc-G genes were also examined in this study. The product of the Scm gene interacts directly with Ph, and, with Ph, forms part of the same complex, PRC1, in Drosophila embryos. The presence of large somatic cell clones of Scm^D1 (amorphic mutation) leads to similar, though not completely overlapping, phenotypes than those observed for mutations in the ph gene. In particular, the results suggest that somatic cells are poorly differentiated and this leads to formation of multicyst follicles and abnormal interfollicular stalks with an excess number of cells. Other Pc-G members were examined; some also belong to the PRC1 complex (Sce and Psc), while others do not [Asx, Su(z)2 and Pcl]. Ovarian defects were only observed with a mutation in the Sce gene, and these defects closely resembled those obtained with Scm^D1. Since follicle cell clones mutant for Scm^D1 and Sce¹ covered large areas of the follicular epithelium, like wild-type clones, it is concluded that (unlike ph⁰, Scm^D1 and Sce¹) somatic cells are not affected in their proliferative property and/or viability. In addition, Scm^D1 and Sce¹ mutations did not affect polar cell number or differentiation. These results indicate that these anomalies are specific to mutations in the ph gene. Thus, several components of the PRC1 complex, but not all, seem to be implicated in follicle formation and their functions do not seem to overlap fully. In addition, none of the genetic interactions between Pc-G genes known to exist for embryonic segment identity were reproduced in the ovary system. Two hypotheses can be made concerning the role of these Pc-G genes in ovarian folliculogenesis: (1) either each Pc-G gene acts specifically on its own specific subset of target genes in somatic cells of the ovary, possibly regulating the transcriptional machinery directly rather than forming particular Pc-G complexes that alter chromatin structure, or (2) repression of target genes in somatic cells of the ovary occurs via Pc-G complexes in a chromatin-dependent manner, but the complexes involved differ markedly in composition from those identified for embryonic cell identity. Further experiments will be needed to distinguish between these two possibilities (Narbonne, 2004).

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).

Drosophila ptip is essential for anterior/posterior patterning in development and interacts with the PcG and trxG pathways

Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. This study describes an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation (Fang, 2009).

The establishment and maintenance of gene expression patterns in development is regulated in part at the level of chromatin modification through the concerted actions of the Polycomb and trithorax family of genes (PcG/trxG). In Drosophila, Polycomb and Trithorax response elements (PRE/TREs) are cis-acting DNA sequences that bind to Trithorax or Polycomb protein complexes and maintain active or silent states, presumably in a heritable manner. In mammalian cells however, such PRE/TREs have not been conclusively identified. Polycomb and Trithorax gene products function by methylating specific histone lysine residues, yet how these complexes recognize individual loci in a temporal and tissue specific manner during development is unclear. Recently, a novel protein, PTIP (also known as PAXIP1), was identified that is part of a histone H3K4 methyltransferase complex and binds to the Pax family of DNA-binding proteins (Patel, 2007). PTIP is essential for assembly of the histone methyltransferase (HMT) complex at a Pax DNA-binding site. These data suggest that Pax proteins, and other similar DNA-binding proteins, can provide the locus and tissue specificity for HMT complexes during mammalian development (Fang, 2009).

In mammals, the PTIP protein is found within an HMT complex that includes the SET domain proteins ALR (GFER) and MLL3, and the accessory proteins WDR5, RBBP5 and ASH2. This PTIP containing complex can methylate lysine 4 (K4) of histone H3, a modification implicated in epigenetic activation and maintenance of gene expression patterns. Furthermore, conventional Ptip^-/- mouse embryos and conditionally inactivated Ptip^-/- neural stem cell derivatives show a marked decrease in the levels of global H3K4 methylation, suggesting that PTIP is required for some subset of H3K4 methylation events (Patel, 2007). The PTIP protein contains six BRCT (BRCA1 carboxy terminal) domains that can bind to phosphorylated serine residues. This is consistent with the observation that PAX2 is serine-phosphorylated in response to inductive signals. In mammals, PAX2 specifies a region of mesoderm fated to become urogenital epithelia at a time when the mesoderm becomes compartmentalized into axial, intermediate and lateral plate. These data suggest that PTIP provides a link between tissue specific DNA-binding proteins that specify cell lineages and the H3K4 methylation machinery (Fang, 2009).

To extend these finding to a non-mammalian organism and address the evolutionary conservation of Ptip, it was asked whether a Drosophila ptip homolog could be identified and if so, whether it is also an essential developmental regulator and part of the epigenetic machinery. The mammalian Ptip gene encodes a novel nuclear protein with two amino-terminal and four carboxy-terminal BRCT domains, flanking a glutamine-rich sequence. Based on the number and position of the BRCT domains and the glutamine-rich domain, the Drosophila genome contains a single ptip homolog. To understand the function of Drosophila ptip in development, a ptip mutant allele was characterized that contained a piggyBac transposon insertion between BRCT domains three and four. Maternal and zygotic ptip mutant embryos exhibited severe patterning defects and developmental arrest, whereas zygotic null mutants developed to the third instar larval stage but also exhibited anterior/posterior (A/P) patterning defects. In cell culture, depletion of Polycomb-mediated repression activates developmental regulatory genes, such as the homeotic gene Ultrabithorax (Ubx). This derepression is dependent on trxG activity and also requires PTIP. Microarray analyses in cell culture of Polycomb and polyhomeotic target genes indicate that many, but not all, require PTIP for activation once repression is removed. The activation of PcG target genes is coincident with PTIP binding to promoter sequences and increased H3K4 trimethylation. These data argue for a conserved role for PTIP in Trithorax-mediated epigenetic imprinting during development (Fang, 2009).

Embryonic development requires epigenetic imprinting of active and inactive chromatin in a spatially and temporally regulated manner, such that correct gene expression patterns are established and maintained. This study shows that Drosophila ptip is essential for early embryonic development. In larval development, ptip coordinately regulates the methylation of histone H3K4 and demethylation of H3K27, consistent with the reports that mammalian PTIP complexes with HMT proteins ALR and MLL3, and the histone demethylase UTX. In wing discs, ptip is required for appropriate A/P patterning by affecting morphogenesis determinant genes, such as en and ci. These data demonstrate in vivo that dynamic histone modifications play crucial roles in animal development and PTIP might be necessary for coherent histone coding. In addition, ptip is required for the activation of a broad array of PcG target genes in response to derepression in cultured fly cells. These data are consistent with a role for ptip in trxG-mediated activation of gene expression patterns (Fang, 2009).

Early development requires ptip for the appropriate expression of the pair rule genes eve and ftz. The characteristic seven-stripe eve expression pattern is regulated by separate enhancer sequences, which are not all equally affected by the loss of ptip. The complete absence of en expression at the extended germband stage also indicates the dramatic effect of ptip mutations on transcription. The characteristic 14 stripes of en expression depends on the correct expression of pair rule genes, which are clearly affected in ptip mutants. However, the maintenance of en expression at later stages and in imaginal discs is regulated by PREs and PcG proteins. If ptip functions as a trxG cofactor, then expression of en along the entire A/P axis in the imaginal discs of ptip mutants might be due to the absence of a repressor. This might explain the surprising presence of ectopic en in the anterior halves of imaginal discs from zygotic ptip mutants. This ectopic en expression is likely to result in suppression of ci through a PcG-mediated mechanism. Yet, it is not clear how en is normally repressed in the anterior half, nor which genes are responsible for derepression of en in the ptip mutant wing and leg discs (Fang, 2009).

The direct interaction of PTIP protein with developmental regulatory genes is supported by ChIP studies in cell culture. Given the structural and functional conservation of mouse and fly PTIP, mPTIP was expressed in fly cells; it can localize to the 5' regulatory regions of many PcG target genes that are activated upon loss of PC and PH activity. Consistent with the interpretation that a PTIP trxG complex is necessary for activation of repressed genes, mPTIP only bound to DNA upon loss of Pc and ph function. In the Kc cells, suppression of both Pc and ph results in the activation of many important developmental regulators, including homeotic genes. A recent report details the genome-wide binding of PcG complexes at different developmental stages in Drosophila and reveals hundreds of PREs located near transcription start sites. Strikingly, most of the genes found to be activated in the Kc cells after PcG knockdown also contain PRE elements near the transcription start site (Fang, 2009).

In vertebrates, PTIP interacts with the Trithorax homologs ALR/MLL3 to promote assembly of an H3K4 methyltransferase complex. The tissue and locus specificity for assembly may be mediated by DNA-binding proteins such as PAX2 (Patel, 2007) or SMAD2 (Shimizu, 2001), which regulate cell fate and cell lineages in response to positional information in the embryo. In flies, recruitment of PcG or trxG complexes to specific sites also can require DNA-binding proteins such as Zeste, DSP1, Pleiohomeotic and Pipsqueak. Whereas PcG complexes have been purified and described in detail, much less is known about the Drosophila trxG complexes. Purification of a trxG complex capable of histone acetylation (TAC1) revealed the proteins CBP and SBF1 in addition to TRX. By contrast, the mammalian MLL/ALL proteins are components of large multi-protein complexes capable of histone H3K4 methylation. Although the mutant analysis, the reduction of H3K4 methylation and the dsRNA knockdowns in Kc cells all suggest that Drosophila ptip has trxG-like activity and hence might be a suppressor of PcG proteins, a more definitve biochemical analysis awaits the generation of antibodies and the delineation of in vivo DNA-binding sites for PTIP and its associated proteins at specific target genes (Fang, 2009).

Mammalian PTIP is also thought to play a role in the DNA damage response based on its ability to bind to phosphorylated p53BP1. PTIP also binds preferentially to the P-SQ motif, which is a good substrate for the ATR/ATM cell cycle checkpoint regulating kinases. Several reports demonstrate that PTIP is part of a RAD50/p53BP1 DNA damage response complex, which can be separated from the MLL2 histone H3K4 methyltransferase complex. Both budding and fission yeast contain multiple BRCT domain proteins that are involved in the DNA damage response, including Esc4, Crb2, Rad9 and Cut5. All of these yeast proteins have mammalian counterparts. However, neither the fission nor budding yeast genomes encodes a protein with six BRCT domains and a glutamine-rich region between domains two and three, whereas such characteristic PTIP proteins are found in Drosophila, the honey bee, C. elegans and all vertebrate genomes. These comparative genome analyses suggest that ptip evolved in metazoans, consistent with an important role in development and differentiation (Fang, 2009).

In summary, Drosophila ptip is an essential gene for early embryonic development and pattern formation. Maternal ptip null embryos show early patterning defects including altered and reduced levels of pair rule gene expression prior to gastrulation. In cultured cells PTIP activity is required for the activation of Polycomb target genes upon derepression, suggesting an important role for the PTIP protein in trxG-mediated activation of developmental regulatory genes. The conservation of gene structure and function, from flies to mammals, suggests an essential epigenetic role for ptip in metazoans that has remained unchanged (Fang, 2009).

Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation

Reactive oxygen species (ROS), produced during various electron transfer reactions in vivo, are generally considered to be deleterious to cells. In the mammalian haematopoietic system, haematopoietic stem cells contain low levels of ROS. However, unexpectedly, the common myeloid progenitors (CMPs) produce significantly increased levels of ROS. The functional significance of this difference in ROS level in the two progenitor types remains unresolved. This study shows that Drosophila multipotent haematopoietic progenitors, which are largely akin to the mammalian myeloid progenitors, display increased levels of ROS under in vivo physiological conditions, which are downregulated on differentiation. Scavenging the ROS from these haematopoietic progenitors by using in vivo genetic tools retards their differentiation into mature blood cells. Conversely, increasing the haematopoietic progenitor ROS beyond their basal level triggers precocious differentiation into all three mature blood cell types found in Drosophila, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb downregulation. It is concluded that the developmentally regulated, moderately high ROS level in the progenitor population sensitizes them to differentiation, and establishes a signalling role for ROS in the regulation of haematopoietic cell fate. These results lead to a model that could be extended to reveal a probable signalling role for ROS in the differentiation of CMPs in mammalian haematopoietic development and oxidative stress response (Owusu-Ansah, 2009).

The Drosophila lymph gland is a specialized haematopoietic organ which produces three blood cell types -- plasmatocytes, crystal cells and lamellocytes -- with functions reminiscent of the vertebrate myeloid lineage. During the first and early second larval instars, the lymph gland comprises only the progenitor population. However, by late third instar, multipotent stem-like progenitor cells become restricted to the medial region of the primary lymph gland lobe, in an area referred to as the medullary zone; whereas a peripheral zone, referred to as the cortical zone, contains differentiated blood cells. By late third instar, the progenitors within the medullary zone are essentially quiescent, whereas the mature, differentiated population in the cortical zone proliferates extensively. The posterior signalling centre is a group of about 30 cells that secretes several signalling molecules and serves as a stem-cell niche regulating the balance between cells that maintain 'stemness' and those that differentiate (Owusu-Ansah, 2009).

Although several studies have identified factors that regulate the differentiation and maintenance of Drosophila blood cells and the stem-like progenitor population that generates them, intrinsic factors within the stem-like progenitors are less explored. Interrogation of these intrinsic factors is the central theme of this investigation. It was observed that by the third instar, the progenitor population in the normal wild-type lymph gland medullary zone contains significantly increased ROS levels compared with their neighbouring differentiated progeny that express mature blood cell markers in the cortical zone. ROS are not increased during the earlier larval instars but increase as the progenitor cells become quiescent and subside as they differentiate. This first suggested that the rise in ROS primes the relatively quiescent stem-like progenitor cells for differentiation. ROS was reduced by expressing antioxidant scavenger proteins GTPx-1 or catalase, specifically in the progenitor cell compartment using the GAL4/UAS system, and it was found that suppressing increased ROS levels in haematopoietic progenitors significantly retards their differentiation into plasmatocytes. As a corollary, mutating the gene encoding the antioxidant scavenger protein superoxide dismutase (Sod2) led to a significant increase in differentiated cells and decrease in progenitors (Owusu-Ansah, 2009).

ROS levels in cells can be increased by the genetic disruption of complex I proteins of the mitochondrial electron transport chain, such as ND75 and ND42. Unlike in wild type, where early second-instar lymph glands exclusively comprise undifferentiated cells, mitochondrial complex I depletion triggers premature differentiation of the progenitor population. This defect is even more evident in the third instar, where a complete depletion of the progenitors is seen as primary lobes are populated with differentiated plasmatocytes and crystal cells. The third differentiated cell type, the lamellocyte, defined by the expression of the antigen L1, is rarely observed in the wild-type lymph gland but is abundantly seen in the mutant. Finally, the secondary and tertiary lobes, largely undifferentiated in wild type, also embark on a robust program of differentiation upon complex I depletion. Importantly, the phenotype resulting from ND75 disruption can be suppressed by the co-expression of the ROS scavenger protein GTPx-1, which provides a causal link between increased ROS and the premature differentiation phenotype. It is concluded that the normally increased ROS levels in the stem-like progenitors serve as an intrinsic factor that sensitizes the progenitors to differentiation into all three mature cell types. Any further increase or decrease in the level of ROS away from the wild-type level enhances or suppresses differentiation respectively (Owusu-Ansah, 2009).

In unrelated systems, increased ROS levels have been demonstrated to activate the JNK signal transduction pathway. Consequently, it was tested whether the mechanism by which the progenitors in the medullary zone differentiate when ROS levels increase could involve this pathway. The gene puckered (puc) is a downstream target of JNK signalling and its expression has been used extensively to monitor JNK activity. Although puc transcripts are detectable by reverse transcriptase PCR (RT- PCR), the puc-lacZ reporter is very weakly expressed in wild type. After disruption of ND75, however, a robust transcriptional upregulation of puc-lacZ expression can be seen, indicating that JNK signalling is induced in these cells in response to high ROS levels. The precocious progenitor cell differentiation caused by mitochondrial disruption is suppressed upon expressing a dominant negative version of basket (bsk), the sole Drosophila homologue of JNK. This suppression is associated with a decrease in the level of expression of the stress response gene encoding phosphoenol pyruvate carboxykinase; quantitatively a 68% suppression of the ND75 crystal cell phenotype was observed when JNK function was removed as well. Although disrupting JNK signalling suppressed differentiation, ROS levels remain increased in the mutant cells, as would be expected from JNK functioning downstream of ROS (Owusu-Ansah, 2009).

In several systems and organisms, JNK function can be mediated by activation of FoxO as well as through repression of Polycomb activity. FoxO activation can be monitored by the expression of its downstream target Thor, using Thor-lacZ as a transcriptional read-out. This reporter is undetectable in wild-type lymph glands although Thor transcripts are detectable by RT-PCR; however, the reporter is robustly induced when complex I is disrupted, suggesting that the increase in ROS that is mediated by loss of complex I activates FoxO. To monitor Polycomb de-repression, a Polycomb reporter was used that expresses lacZ when Polycomb proteins are downregulated. Although undetectable in wild-type lymph glands, disrupting ND75 leads to lacZ expression suggesting that Polycomb activity is downregulated by the altered ROS and resulting JNK activation. Direct FoxO overexpression causes a remarkable advancement in differentiation to a time as early as the second instar, never seen in wild type. By early third instar, the entire primary and secondary lobes stained for plasmatocyte and crystal cell markers when FoxO is expressed in the progenitor population. Unlike with ROS increase, no a significant increase in lamellocytes was found upon FoxO overexpression. However, downregulating the expression of two polycomb proteins, Polyhomeotic Proximal (Php-x) and Enhancer of Polycomb [E(Pc)], that function downstream of JNK, markedly increased lamellocyte number without affecting plasmatocytes and crystal cells. When FoxO and a transgenic RNA interference (RNAi) construct against E(Pc) are expressed together in the progenitor cell population, differentiation to all three cell types is evident. It is concluded that FoxO activation and Polycomb downregulation act combinatorially downstream of JNK to trigger the full differentiation phenotype: an increase in plasmatocytes and crystal cells due to FoxO activation, and an increase in lamellocytes primarily due to Polycomb downregulation (Owusu-Ansah, 2009).

This analysis of ROS in the wild-type lymph gland highlights a previously unappreciated role for ROS as an intrinsic factor that regulates the differentiation of multipotent haematopoietic progenitors in Drosophila. Any further increase in ROS beyond the developmentally regulated levels, owing to oxidative stress, will cause the progenitors to differentiate into one of three myeloid cell types. It has been reported that the ROS levels in mammalian haematopoietic stem cells is low but that in the CMPs is relatively high. The Drosophila haematopoietic progenitors give rise entirely to a myeloid lineage and therefore are functionally more similar to CMPs than they are to haematopoietic stem cells. It is therefore a remarkable example of conservation to find that they too have high ROS levels. The genetic analysis makes it clear that the high ROS in Drosophila haematopoietic progenitors primes them towards differentiation. It will be interesting to determine whether such a mechanism operates in mammalian CMPs. In mice, as in flies, a function of FoxO is to activate antioxidant scavenger proteins. Consequently, deletion of FoxO increases ROS levels in the mouse haematopoietic stem cell and drives myeloid differentiation. However, even in the mouse haematopoietic system, FoxO function is dose and context dependent, as ROS levels in CMPs are independent of FoxO. Thus, although the basic logic of increased ROS in myeloid progenitors is conserved between flies and mice, the exact function of FoxO in this context may have diverged (Owusu-Ansah, 2009).

Past work has hinted that ROS can function as signalling molecules at physiologically moderate levels. This work supports and further extends this notion. Although excessive ROS is damaging to cells, developmentally regulated ROS production can be beneficial. The finding that ROS levels are moderately high in normal Drosophila haematopoietic progenitors and mammalian CMPs raises the possibility that wanton overdose of antioxidant products may in fact inhibit the formation of cells participating in the innate immune response (Owusu-Ansah, 2009).

Loss of the Polycomb group gene polyhomeotic induces non-autonomous cell overproliferation

Polycomb group (PcG) proteins are conserved epigenetic regulators that are linked to cancer in humans. However, little is known about how they control cell proliferation. This study reports that mutant clones of the PcG gene polyhomeotic (ph) form unique single-cell-layer cavities that secrete three JAK/STAT pathway ligands, which in turn act redundantly to stimulate overproliferation of surrounding wild-type cells. Notably, different ph alleles cause different phenotypes at the cellular level. Although the ph-null allele induces non-autonomous overgrowth, an allele encoding truncated Ph induces both autonomous and non-autonomous overgrowth. It is proposed that PcG misregulation promotes tumorigenesis through several cellular mechanisms (Feng, 2011). <>In summary, mosaic clones homozygous for the ph-null allele induce overproliferation of surrounding wild-type cells through Notch-Upd-JAK/STAT signalling, whereas mosaic clones homozygous for a ph hypomorphic allele that encodes truncated Ph proteins induce both autonomous and non-autonomous cell overproliferation. These results highlight an important but largely overlooked phenomenon: different mutations in the same gene might induce tumours and cancers through distinct cellular mechanisms, depending on the nature of the mutations and/or genetic backgrounds. This fact adds another layer of complexity to cancer pathology (Feng, 2011).

Diverse tumor pathology due to distinctive patterns of JAK/STAT pathway activation caused by different Drosophila polyhomeotic alleles

Drosophila polyhomeotic (ph) is one of the important polycomb group genes that is linked to human cancer. In the mosaic eye imaginal discs, while ph^del, a null allele, causes only non-autonomous overgrowth, ph⁵⁰⁵, a hypomorphic allele, causes both autonomous and non-autonomous overgrowth. These allele-specific phenotypes stem from the different sensitivities of ph mutant cells to the Upd homologs that they secrete (Feng, 2012).

Different ph alleles cause tissue overgrowth in different ways. While a ph null allele, ph^del , causes only non-autonomous cell over-proliferation, a ph hypomorphic allele, ph⁵⁰⁵ , causes both autonomous and non-autonomous cell overproliferation. In mosaic tissues, overproliferation of mutant cells was defined as autonomous, whereas over-proliferation of genotypically wild type cells induced by mutant cells was defined as non-autonomous. The signaling pathway involved in ph^del induced non-autonomous cell over-proliferation. In summary, elevated Notch activity in ph cells up-regulates the expression of JAK/STAT pathway ligands Upd homologs, which in turn activate the JAK/STAT pathway in neighboring wild type cells and cause their over-proliferation. This study addressed why a ph null allele and a ph hypomorphic allele both cause tumors but in such different ways (Feng, 2012).

First whether the same signaling pathway underlay non-autonomous overproliferation induced by both ph^del and ph⁵⁰⁵ was tested. The functions of Notch and Upd homologs in the ph⁵⁰⁵ mosaic eyes were examined with the same strategy used for ph^del. A ph⁵⁰⁵ -Notch double mutant line was generated, and eyes mosaic for this line were essentially of the same size as wild type eyes. The mosaic eye discs had normal size and normal cell proliferation level, as shown by PH3 staining, which marks mitotic cells. Moreover, the size of ph⁵⁰⁵ -Notch clones was significantly reduced when compared to that of ph⁵⁰⁵ clones. These results indicated that Notch was required for both autonomous and non-autonomous overproliferation induced by ph⁵⁰⁵ (Feng, 2012).

Next ph⁵⁰⁵ was recombined with upd^Δ1-3, a deficiency line that lacks all three upd homologs in the Drosophila genome Mosaic analyses were then performed using this double mutant line. ph⁵⁰⁵ -upd^Δ1-3 mosaic eyes were significantly smaller than ph⁵⁰⁵ mosaic eyes and were comparable to wild type eyes, indicating that tissue overgrowth was largely suppressed. PH3 staining of the double mutant mosaic eye discs showed that these discs had relatively normal size and cell proliferation level. Importantly, ph⁵⁰⁵ -updd1-3 clones were also drastically reduced in size compared to ph⁵⁰⁵ clones. These results indicated that Upd homologs are required for not only non-autonomous but also autonomous cell over-proliferations induced by ph⁵⁰⁵ (Feng, 2012).

It is not surprising that the same signaling pathway is responsible for non-autonomous over-proliferation induced by both ph^del and ph⁵⁰⁵ , and it is not completely unexpected that Notch is also required for ph⁵⁰⁵ induced autonomous over-proliferation, as Notch is a transcription factor that has been shown to autonomously regulate cell proliferation. However, the three Upd proteins are secreted and are not expected to have any direct effect on autonomous cell proliferation. To interpret these observations, it was hypothesized that ph⁵⁰⁵ cells still respond to Upd ligands secreted by themselves in an autocrine or paracrine manner, and therefore over-proliferate. However, ph^del cells were thought to be no longer responsive to Upd ligands (Feng, 2012).

To functionally test this hypothesis, the double mutant strategy was applied, taking advantage of the fact that the genes domeless (dome, encoding the only transmembrane receptor of the Drosophila JAK/STAT pathway) and hopscotch (hop, encoding the only Drosophila JAK kinase) are also on X chromosome as is ph. First ph⁵⁰⁵ was recombined with two dome alleles to generate ph⁵⁰⁵ -dome double mutant lines. Eye discs mosaic for these lines were still significantly larger than wild type, but the size of double mutant clones was dramatically reduced, so that only a tiny portion of the disc was composed of mutant cells. PH3 staining indicated that non-autonomous proliferation level was still high, but autonomous proliferation largely disappeared. The adult eyes mosaic for such double mutant lines were further examinedm and these eyes were found to be still much larger than wild type and similar to ph⁵⁰⁵ mosaic eyes in size, but they generally were not folded as seen in ph⁵⁰⁵ mosaic eyes (Feng, 2012).

Next a ph⁵⁰⁵ -hop double mutant line was generated. Autonomous proliferation was found in mosaic eye discs of this double mutant that was also significantly suppressed, with mutant cells only accounted for a small portion of the whole disc. In contrast, non-autonomous cell over-proliferation was not affected and the overall size of these discs was still significantly larger than wild type. Adult eyes mosaic for this double mutant showed similar phenotypes as those of ph⁵⁰⁵ -dome mosaic eyes. These eyes were still significantly larger than wild type but they were generally not folded. Therefore, the removal of either dome or hop from ph⁵⁰⁵ cells only suppressed autonomous over-proliferation but did not affect non-autonomous overproliferation, making such double mutant mosaic discs phenotypically similar to ph^del mosaic discs (Feng, 2012).

As controls, ph^del -dome and ph^del -hop double mutant lines were also generated using the same dome and hop alleles. Mosaic analyses on eye discs showed that the removal of dome or hop from ph^del cells did not affect non-autonomous cell over proliferation. It did, however, mildly reduce the mutant clone size, suggesting that ph^del cells might still have a weak response to Upd ligands. Adult eyes mosaic for these double mutant lines were phenotypically indistinguishable from ph^del mosaic eyes, consistent with the above observations in mosaic eye discs (Feng, 2012).

Finally it was asked why ph^del and ph⁵⁰⁵ cells responds differently to the Upd ligands secreted by themselves. It was hypothesized that some of the JAK/STAT pathway modulators might be differentially expressed in ph^del and ph⁵⁰⁵ cells. To test this hypothesis, TU-Tagging, a technique that enables the purification of RNA from mutant cells without having to physically isolate such cells, was chosen. Briefly, Drosophila is unable to synthesize uridine from uracil due to the lack of phosphoribosyltransferase (UPRT). When exogenous UPRT is expressed in mutant cells by MARCM, such cells would acquire the ability to utilize uracil. If these larvae are fed with 4-thiouracil (4-TU), a uracil derivative that contains a thio group, only mutant cells would be able to use 4-TU and eventually incorporate thio- containing uridine into newly synthesized RNA. This treatment has little toxicity, and the thio-labeled RNA can be purified from total RNA using conventional biochemical methods (Feng, 2012).

TU-tagging was performed to isolate RNA from ph^del cells and ph⁵⁰⁵ cells, and qRTPCR was used to examine candidate gene expression. The expression of the JAK/STAT pathway receptor dome was significantly higher in ph⁵⁰⁵ cells than in ph^del cells. A higher receptor expression might sensitize ph⁵⁰⁵ cells to the Upd ligands. The levels of enok and socs42a, both negative regulators of the JAK/STAT pathway, were also significantly higher in ph⁵⁰⁵ cells compared to ph^del cells. This might represent feedback loops that negatively regulate the pathway activity. In fact, several such negative feedback loops, in which elevated pathway activity upregulates a negative pathway regulator, have been reported in JAK/STAT pathway (Feng, 2012).

Together, it is concluded that ph^del and ph⁵⁰⁵ both cause autonomous over-expression of Upd homologs in mutant cells, which represents the only driving force of cell overproliferation in ph^del and ph⁵⁰⁵ mosaic tissues and in essence acts non-autonomously to activate JAK/STAT pathway. The different phenotypes of these two types of mosaics are due to different sensitivity of mutant cells to Upd homologs. ph⁵⁰⁵ mutant cells robustly respond to Upd ligands that they secreted. Therefore, Upd ligands secreted by ph⁵⁰⁵ cells simultaneously induce over-proliferation in both mutant and wild type cells. In contrast, ph^del cells are largely insensitive to Upd ligands, so that Upd ligands secreted by ph^del cells only induce over-proliferation in wild type but not mutant cells. Furthermore, differential expression of the JAK/STAT pathway receptor dome might underlie the different sensitivity of ph^del and ph⁵⁰⁵ cells to Upd ligands (Feng, 2012).

The role of the histone H2A ubiquitinase Sce in Polycomb repression

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRing) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. This study created an Sce knockout allele and showed that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression (Gutiérrez, 2012).

This study analyzed how PRC1 regulates target genes in Drosophila to investigate how the distinct chromatin-modifying activities of this complex repress transcription in vivo. Because H2A monoubiquitylation is thought to be central to the repression mechanism of PRC1-type complexes, focus was placed on the role of Sce. The following main conclusions can be drawn from the work reported in this study. First, in the absence of Sce, bulk levels of H2A-K118ub1 are drastically reduced but the levels of the PRC1 subunits Psc and Ph are undiminished. Sce is therefore the major E3 ligase for H2A monoubiquitylation in developing Drosophila but is not required for the stability of other PRC1 subunits. Second, PRC1-bound genes fall into two classes. Class I target genes are misexpressed if any of the PRC1 subunits is removed. Class II target genes are misexpressed in the absence of Ph or Psc-Su(z)2 but remain stably repressed in the absence of Sce or Pc. At class II target genes, Ph and the Psc-Su(z)2 proteins work together to repress transcription by a mechanism that does not require Sce and Pc and is therefore independent of H2A monoubiquitylation. Third, removal of the Ph, Psc-Su(z)2 or Pc proteins results in imaginal disc tumors that are characterized by unrestricted cell proliferation. However, removal of Sce does not cause this phenotype, suggesting that this tumor suppressor activity by the PcG system does not require H2A monoubiquitylation. Finally, these analyses reveal that PRC1 subunits are essential for repressing the elB, noc, dac and pros genes outside of their normal expression domains in developing Drosophila. This expands the inventory of developmental regulator genes in Drosophila for which PcG repression has been demonstrated in a functional assay (Gutiérrez, 2012).

In the Sce^33M2 allele Arg65 is mutated to Cys, but this mutant Sce protein is undetectable and therefore does not appear to be stable in vivo. The crystal structure of the Ring1B-Bmi1 complex provides a molecular explanation for this observation: the Arg70 residue in Ring1B that corresponds to Arg65 in Sce is thought to be critical for interaction with Bmi1. A likely scenario therefore is that the Sce^Arg65Cys protein in Drosophila is unstable and is degraded because it is unable to associate with Psc or its paralog Su(z)2. Interestingly, removal of Sce protein has no detectable effect on the levels of the Psc and Ph proteins. Psc is therefore stable in the absence of its binding partner Sce. This is in contrast to the situation in mice in which Ring1B mutant ES cells show a drastic reduction in the levels of the Ring1B partner protein Bmi1 and its paralog Mel18 (Pcgf62) and also a reduction in the levels of Mph2 (Phc2) and Mpc2 (Cbx4) (Leeb, 2007). The interdependence between PRC1 subunits for protein stability is therefore different in mammals and Drosophila (Gutiérrez, 2012).

Reconstitution of the Drosophila PRC1 core complex in a baculovirus expression system suggests that Sce is important for complex stability. At present, it is not know whether the Psc, Ph and Pc proteins still form a complex in vivo in the absence of Sce. It is currently unknown whether Psc, Ph and Pc are still bound to all PRC1 target genes in the absence of Sce. However, the finding that class II genes remain repressed in the absence of Sce, even though their repression depends on Psc-Su(z)2 and Ph, argues against a crucial role of Sce in the targeting of these other PRC1 subunits to these genes. Interestingly, the repression of all class II target genes analyzed in this study always requires both the Ph and the Psc-Su(z)2 proteins. A possible explanation for this observation is that Ph and Psc-Su(2) still form a PRC1 subcomplex in the absence of Sce and that this complex is fully functional to repress class II target genes. Alternatively, it is possible that Ph and Psc-Su(z)2 repress class II target genes as components of as yet uncharacterized complexes that are distinct from PRC1 and Drosophila dRing-associated factors (dRAF) complex (Gutiérrez, 2012).

In vitro, Psc and Su(z)2 proteins compact nucleosome templates, inhibit nucleosome remodeling by SWI/SNF complexes and repress transcription on chromatin templates. The observation that repression of class II target genes requires Psc-Su(z)2 and Ph but not Pc and Sce supports the idea that the chromatin-modifying activities of Psc-Su(z)2 identified in vitro are the main mechanism by which PRC1 represses these genes. Previous structure/function analyses in Drosophila showed that the same domains of the Psc protein responsible for chromatin compaction and remodeling inhibition in vitro are crucial for HOX gene repression in vivo. Chromatin modification by Psc and Su(z)2 is therefore also crucial for repression of class I target genes. Regulation of the class I target gene en further illustrates this point. In some tissues (e.g. in the dorsal hinge region of the wing imaginal disc) repression of en requires all PRC1 core subunits, but in other tissues (e.g. in the pouch of the wing imaginal disc) en remains repressed in the absence of Sce and Pc, and only Psc-Su(z)2 and Ph seem to be crucial to keep the gene inactive. At present, the molecular mechanism of Ph is not well understood. In vitro, Ph protein has the capacity to inhibit chromatin remodeling and transcription but it does so less effectively than Psc. At the target genes analyzed in this study, Ph is required for transcriptional repression wherever Psc-Su(z)2 is required, suggesting that Ph and Psc-Su(z)2 act in concert in this repression. Nevertheless, it is possible that repression of other PRC1 target genes requires a different subset of PRC1 subunits, or that, as in the case of en, the subunit requirement for repression changes depending on the cell type (Gutiérrez, 2012).

In mammals, Ring1B and Ring1A are responsible for the bulk of H2A-K119 monoubiquitylation. Similarly, Sce generates the bulk of H2A-K118 monoubiquitylation in Drosophila, both in tissue culture cells (Lagarou, 2008) and in the developing organism (this study). The requirement for Sce at class I target genes is consistent with the idea that H2A monoubiquitylation of their chromatin is part of the repression mechanism. Repression of a subset of class I genes, namely the HOX genes, also requires the H2A deubiquitinase PR-DUB (Gaytán de Ayala Alonso, 2007; Scheuermann, 2010). Moreover, PR-DUB and Sce strongly synergize to repress HOX genes. Specifically, the phenotype of Sce PR-DUB double mutants suggests that H2A monoubiquitylation becomes ineffective for HOX gene repression if PR-DUB is absent. However, embryos that lack PR-DUB alone show a 10-fold increase in the bulk levels of H2A-K118ub1 and it is estimated that ~10% of all H2A molecules become monoubiquitylated in these animals. How could this conundrum be explained? One possibility is that H2A monoubiquitylation and deubiquitylation at HOX gene chromatin need to be regulated in a precisely balanced manner. However, an alternative explanation considers H2A-K118ub1 levels in the context of ubiquitin homeostasis. In particular, the high H2A-K118ub1 levels in PR-DUB mutants suggest that Sce generates widespread H2A monoubiquitylation at most Sce-bound genes and possibly also elsewhere in the genome, but that in wild-type animals PR-DUB continuously deubiquitylates H2A-K118ub1 at these locations and thereby recycles ubiquitin. The observation that PR-DUB is widely co-bound with Sce, not only at HOX but also at many other class I and class II target genes, is consistent with this idea. It is tempting to speculate that the widespread H2A monoubiquitylation in PR-DUB mutants sequesters a substantial fraction of the pool of free ubiquitin. It is therefore possible that removal of PR-DUB effectively depletes the pool of free ubiquitin in the nucleus to an extent that H2A monoubiquitylation at HOX target genes becomes inefficient and, consequently, their repression can no longer be maintained. According to this model, the crucial function of PR-DUB would not be the deubiquitylation of H2A-K118ub1 at HOX genes but rather at class II target genes and elsewhere in the genome where Sce 'wastefully' monoubiquitylates H2A (Gutiérrez, 2012).

A Deterministic Analysis of Genome Integrity during Neoplastic Growth in Drosophila

The development of cancer has been associated with the gradual acquisition of genetic alterations leading to a progressive increase in malignancy. In various cancer types this process is enabled and accelerated by genome instability. While genome sequencing-based analysis of tumor genomes becomes increasingly a standard procedure in human cancer research, the potential necessity of genome instability for tumorigenesis in Drosophila melanogaster has never been determined at the DNA sequence level. Therefore, this study induced formation of tumors by depletion of the Drosophila tumor suppressor Polyhomeotic and subjected them to genome sequencing. To achieve a highly resolved delineation of the genome structure the Deterministic Structural Variation Detection (DSVD) algorithm was developed; the algorithm identifies structural variations (SVs) with high accuracy and at single base resolution. The employment of long overlapping paired-end reads enables DSVD to perform a deterministic, i.e. fragment size distribution independent, identification of a large size spectrum of SVs. Application of DSVD and other algorithms to sequencing data reveals substantial genetic variation with respect to the reference genome reflecting temporal separation of the reference and laboratory strains. The majority of SVs, constituted by small insertions/deletions, is potentially caused by erroneous replication or transposition of mobile elements. Nevertheless, the tumor did not depict a loss of genome integrity compared to the control. Altogether, the results demonstrate that genome stability is not affected inevitably during sustained tumor growth in Drosophila implying that tumorigenesis, in this model organism, can occur irrespective of genome instability and the accumulation of specific genetic alterations (Sievers, 2014).

An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling

Polycomb group proteins are epigenetic regulators maintaining transcriptional memory during cellular proliferation. In Drosophila larvae, malfunction of Polyhomeotic (Ph), a member of the PRC1 silencing complex, results in neoplastic growth. This study reports an intrinsic tumour suppression mechanism mediated by the steroid hormone ecdysone during metamorphosis. Ecdysone alters neoplastic growth into a nontumorigenic state of the mutant ph cells which then become eliminated during adult stage. This study demonstrates that ecdysone exerts this function by inducing a heterochronic network encompassing the activation of the microRNA lethal-7, which suppresses its target gene chronologically inappropriate morphogenesis (chinmo). This pathway can also promote remission of brain tumours formed in brain tumour mutants, revealing a restraining of neoplastic growth in different tumour types. Given the conserved role of let-7, the identification and molecular characterization of this innate tumour eviction mechanism in flies might provide important clues towards the exploitation of related pathways for human tumour therapy (Jiang, 2018).

Deregulation of PcG gene expression has been associated with various types of human cancer. For example, loss of expression of the human ph homologue has been linked to the formation of osteosarcomas. As the molecular mechanisms of PcG proteins in human cancers are largely unknown, understanding the tumour-suppressor function of PcG genes in Drosophila therefore could provide insights in human cancer biology. During the past decades imaginal discs have been used as a powerful paradigm to investigate mechanisms underlying the formation and progression of several types of tumour, including Ras-, PcG-, or Hippo pathway-induced tumours. In addition, it is worth noting that the let-7 consensus sequence is identical from Caenorhabditis elegans to humans, suggesting that let-7 may control functionally conserved targets in regulating proliferation and differentiation during development. In various types of human cancer, downregulation of one or more let-7 members has been observed. Moreover, induced expression of let-7 in cancer cell lines can suppress cell proliferation and tumour growth. In the human genome, the let-7 family consists of more than ten members. However, the transcriptional regulation, spatial and temporal expression, and their tissue-specific and/or redundant functions of the let-7 family in human are far more complicated and still remain elusive (Jiang, 2018).

This study identified an intrinsic mechanism reprogramming tumorigenic to nontumorigenic cells of at least two different tumour types, by marking the cells for destruction in adult Drosophila. The steroid hormone-induced miRNA let-7 is a key mediator of this mechanism. Interestingly, let-7 and its target genes, including chinmo have been shown to act as heterochronic genes that regulate developmental transitions. Other findings from this lab indicate that ph⁵⁰⁵ tumour cells are reprogrammed from the original larval imaginal disc identity to an early embryonic state. By artificially overexpressing a differentiation factor, these cancer cells can be induced to lose their neoplastic state, however. It appears as if these tumour cells are trapped in an immature condition, unable to differentiate. Pulses of ecdysone are a major timer of developmental transitions in flies and their target, let-7, is for example required for enforcing the terminal cell cycle arrest in pupal stage wing discs. These findings suggest that flies have evolved tumour suppressive mechanisms by inducing let-7-controlled heterochronic gene networks to enforce cellular differentiation in epigenetically derailed tumours. Indeed, differentiation therapy is considered a promising approach for curing human cancers. However, the strategy has been applied only in limited cases. As such, identification of an innate tumour eviction mechanism in flies based on these principles may provide new ideas how such cancer treatments could be further improved in human patients (Jiang, 2018).

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