Enhancer of Polycomb

Targets of Activity

The genetics of E(Pc) predict that E(Pc) should bind to polytene chromosomes in a pattern that overlaps that of other PcG proteins, and/or bind to the heterochromatin of the chromocenter. Such a binding pattern would support a heterochromatin model of PcG function. It is also possible that E(Pc) is not a chromatin protein, but is somehow required to modify, localize or chaperone chromatin proteins, or is required to construct a boundary between heterochromatin and euchromatin. To test these possibilities, anti-E(Pc) antibodies were used to decorate polytene chromosomes. E(Pc) antibodies bind specifically to about 100 sites on polytene chromosomes, showing that E(Pc) is a chromatin protein. However, none of the three E(Pc) antisera bind to the chromocenter. The E(Pc)-binding sites were mapped and compared to the binding sites of other PcG proteins. E(Pc)-binding sites overlap with 31/96 Asx-binding sites; 28/96 Pc/ph/Pcl-binding sites; 26/96 Psc-binding sites and 28/96 Su(z)2-binding sites. 53 of the 96 E(Pc)-binding sites are shared with at least one other PcG-binding site; 14 are shared with 2 other PcG-binding sites; 8 are shared with 3 other PcG-binding sites, and 7 of the sites are shared with all of Asx, ph, Psc and Su(z)2. E(Pc) binds 84AB, the site of the Antennapedia complex (ANT-C). However, it is not detected at 89EF, the site of the bithorax complex (BX-C). Pc and Ph bind to the sites of 9 PcG genes, including Asx (51A); E(Pc) (48A); esc (33B); Pc (78E); ph (2D); pleiohomeotic (102EF); Psc (49DE); Sex comb on midleg (85E), and super sex combs (41C). However, E(Pc) binds only 3 of these sites: E(Pc) itself, ph and Sex comb on midleg. In view of the observation that E(Pc) mutations are Su(var)s, E(Pc)-binding sites were compared to locations of modifiers of PEV. E(Pc) binds near 6 modifiers of PEV: Su(var)2-4 (23A-D); Su(var)2-8 (24F-25A); Su(var)3-11 (94D); E(var)25F (25F); E(var)33A-D (33A-D); E(var)36A-E (36A-E) and E(var)55 (55A-F) (Stankunas, 1998).

DEVELOPMENTAL BIOLOGY

Embryonic

Developmental Northern analyses in Drosophila show that the 8.5 kb E(Pc) transcript is found at all developmental stages, although it is most abundant just after fertilization, and between 3 and 12 hours of embryogenesis. The smaller 5.2 kb transcript first becomes detectable in late embryogenesis and is most abundant in adult males. There is evidence for additional transcripts in adults, but their structure or function has not been studied further. The E(Pc) protein is ubiquitous throughout Drosophila embryogenesis as shown by immunostaining of embryos with E(Pc) antibody. Later in embryogenesis, E(Pc) appears to be more abundant in the central nervous system. The expression of E(Pc) in embryogenesis appears similar to other characterized PcG genes (Stankunas, 1998).

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).

Effects of Mutation or Deletion

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. Chromosomes containing all alleles of Asx, E(z), Pcl, Psc, and Scm enhance variegation of w^m4, and most also enhance variegation of B^SV, two known variegating chromosomal rearrangements. It is striking that different alleles can modify variegation in different directions. This could either represent allele-specific differences or indicate the presence of modifiers in the background. The esc, l(4)102EFx, Pc, ph, and sxc mutations had no effect on the variegation of w^m4 and B^SV and were not tested further. PcG loci that modified variegation of w^m4 and B^SV were crossed to Sb^V and bw^V, two other variegating chromosomal rearrangements. Because most strong modifiers of PEV modify all variegating rearrangements, it was expected that strong modifiers would affect all four variegating loci tested. Of the alleles tested, only Pcl2, Pcl 12, and Psc1.d20 met this criterion (Sinclair, 1998a).

The data above are consistent with the possibility that some PcG mutations modify PEV. However, the data are also consistent with the possibility that the observed modification of PEV results from dominant modifiers in the background, or from recessive modifiers uncovered by deletions, rather than being attributable to PcG mutations themselves. An attempt was made to recombinationally map the enhancement of PEV for the three loci that showed the strongest effects: Asx, Pcl, and Psc, but neither E(z) or Scm were examined. Provided that the Asx mutation was introduced via males into the w^m4 background in females, the enhancement of PEV associated with the Asx1 chromosome to 2 - 71 ± 1.1, could be mapped in reasonable agreement with the published map position of 2 - 72. However, the enhancement of PEV associated with the Pcl12 and Psc1 chromosomes could not be mapped to any defined interval, showing that there are multiple modifiers on the mutant chromosomes. Thus, Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups (Sinclair, 1998a).

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. The phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes have been examined in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. Genetic interactions are shown among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs. The idea that most Pc group genes function independently of esc is reassessed. Most duplications of Pc group genes exhibit neither anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc+ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function (Campbell, 1995).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Mutations in the extra sex combs and Enhancer of Polycomb genes increase homologous recombination in somatic cells of Drosophila melanogaster

Heterozygous mutant alleles of E(Pc) and esc increase homologous recombination from an allelic template in somatic cells in a P-element-induced double-strand break repair assay. Flies heterozygous for mutant alleles of these genes showed increased genome stability and decreased levels of apoptosis in imaginal discs and a concomitant increase in survival following ionizing radiation. It is proposed that this was caused by a genomewide increase in homologous recombination in somatic cells. A double mutant of E(Pc) and esc had no additive effect, showing that these genes act in the same pathway. Finally, it was found that a heterozygous deficiency for the histone deacetylase, Rpd3, masked the radiation-resistant phenotype of both esc and E(Pc) mutants. These findings provide evidence for a gene dosage-dependent interaction between the Esc/E(z) complex and the Tip60 histone acetyltransferase complex. It is proposed that esc and E(Pc) mutants enhance homologous recombination by modulating the histone acetylation status of histone H4 at the double-strand break (Holmes, 2006; full text of article).

Eukaryotes use both homologous recombination and nonhomologous end-joining to repair DSBs; survival is compromised severely when both are inactivated. In a homologous recombination assay, every cell in the adult head had a double-stranded break (DSB) generated at the white locus by excision of the P element. This was true for flies heterozygous for either the esc⁶ or the E(Pc)¹ mutation and for wild-type flies. These breaks were repaired either by homologous recombination using the allelic white gene or by nonhomologous end-joining. Heterozygous mutants of any of three genes, E(Pc), Pcl, or esc, increased the likelihood that homologous recombination was used to repair a DSB made by P-element excision in cells of the developing eye-antennal imaginal disc. Thus, although nonhomologous end-joining was not measured directly, it was possible to conclude that the increase in pigmentation indicated an increase in the repair of DSBs by homologous recombination at the expense of nonhomologous end-joining and that heterozygosity for null alleles of either gene changed the balance between these two pathways. Further studies will be needed to determine if there is a defect in nonhomologous end-joining that is compensated by an increase in homologous recombination, or if nonhomologous end-joining is unaffected and homologous recombination is enhanced (Holmes, 2006).

The repair bias toward homologous recombination was observed when either paired or unpaired allelic templates were used. This suggested that the effects of the esc and E(Pc) mutants were not related to any chromosome-pairing-dependent activities of these genes (Holmes, 2006).

Alterations in DSB repair capacity or DSB pathway choice were not restricted to DSBs made by P-element excision at the white locus. Heterozygous mutants of either esc or E(Pc) showed a significant increase in genome integrity and a significant decrease in apoptosis following exposure to ionizing radiation. Not surprisingly, these animals were somewhat resistant to high doses of ionizing radiation (Holmes, 2006).

The increase in homologous recombination and the increase in genome integrity seen in esc⁶ heterozygous animals was suppressed by a P{esc⁺} transgene. This demonstrated that the effects were specific to the esc mutations and were not caused by genetic background effects. While the effect of the E(Pc)¹ mutant was not suppressed with a transgene, it was observed that a large deficiency that included the E(Pc) gene had a similar effect to that of E(Pc)¹ in the homologous recombination assay (Holmes, 2006).

Notably, the increase in homologous recombination was not seen in animals heterozygous for either single mutant of Psc. Furthermore, Psc, Pc, or Scm heterozygous animals are not resistant to ionizing radiation. The esc, Pcl, E(Pc), and Psc genes produce proteins that are localized to three different complexes. The proteins produced from the esc and Pcl genes are members of the esc/E(z) histone H3 methyltransferase complex, the E(Pc) protein is found in the Tip60/NuA4 histone acetyltransferase complex, and the Psc, Pc, and Scm proteins are components of the PRC1 complex. Interestingly, the esc and E(Pc) mutants alter the choice of repair pathway by a common mechanism since the esc⁶/E(Pc)¹ double mutant lacked an additive effect in the homologous recombination assay over either single mutant. This suggests that the effects that were observed on the choice of repair pathway are not linked to the methylation activity of the Esc/E(z) complex or to the methyl-H3-binding activity of PCR1, but rather are linked to some other function that connects the Esc/E(z) and Tip60/NuA4 complexes (Holmes, 2006).

Since the Esc/E(z) complex often associates with a histone chaperone and the Rpd3 histone deacetylase and the E(Pc) protein is a component of a histone acetyl transferase known to be required for DSB repair in yeast, the hypothesis was tested that histone acetylation forms a link between these two complexes, and it was found that a heterozygous mutation for Rpd3 blocked the ability of heterozygous esc⁶ or E(Pc)¹ alleles to confer resistance to ionizing radiation. This suggests that the Rpd3 gene product acts upstream of the esc and E(Pc) proteins in a pathway that influences the choice of which mechanism is used to repair DSBs (Holmes, 2006).

In yeast, the acetylation status of histone H4 plays a crucial role in determining whether a DSB is repaired by nonhomologous end-joining or by homologous recombination, and this role is distinct from its role in regulation of gene expression. Recent work in yeast and Drosophila shows that the NuA4/Tip60 histone acetyltransferase complex [which includes the yeast E(Pc) ortholog] is recruited to DSB sites and acetylates the tail lysines of histone H2A and H4. Histone acetylation is thought to neutralize the positively charged histone tail, thereby reducing the affinity between DNA and histones and loosening the compaction of the chromatin. The homologous recombination or nonhomologous end-joining repair machinery can then gain access to the damage and facilitate repair (Holmes, 2006).

Furthermore, nonhomologous end-joining requires the acetylation of all four lysine residues on the H4 histone tail, whereas homologous recombination requires only partial acetylation of these lysines. Likewise, mutations in the nonessential NuA4 subunit, Yng2, result in global hypoacetylation of histone H4 and are synthetic lethal with the YKU70 gene, further supporting the argument that nonhomologous end-joining requires complete acetylation of histone H4. Finally, yeast with hypoacetylated histone H4 not only are proficient in homologous recombination but also show enhanced recombination in a sister-chromatid exchange assay. These data suggest a compensatory relationship between nonhomologous end-joining and homologous recombination in Saccharomyces cerevisiae (Holmes, 2006).

The yeast data, together with the current results, suggest a model for the gene dose-dependent interaction among Rpd3, Esc, and E(Pc) in Drosophila. It is proposed that, similar to S. cerevisiae, nonhomologous end-joining in Drosophila somatic cells is dependent upon full acetylation of histone H4 tails, but that homologous recombination is not. In this model, a heterozygous E(Pc) mutation would cause a decrease in E(Pc) protein levels, resulting in a decrease in activity of the Tip60 histone acetyltransferase at the DSB and a shift toward homologous recombination. Likewise, a heterozygous esc mutation would result in less of the esc/E(z) complex. Since the Rpd3 protein is found in several different complexes aside from the Esc/E(z) complex, a decrease in the amount of Esc/E(z) complex would result in more free Rpd3 protein in the cell. The excess free Rpd3 protein could deacetylate more of the histone H4 at the DSB and thus shift DSB repair toward homologous recombination. In either instance, a decrease in the levels of Rpd3 protein in the presence of mutations in either esc or E(Pc) might be expected to result in more complete histone H4 acetylation and to restore the normal balance between homologous recombination and nonhomologous end-joining (Holmes, 2006).

It is becoming increasingly clear that PcG proteins have functions beyond the regulation of homeotic genes. Many recent studies have identified deregulation of different PcG proteins (Ezh2, Pcl, Bmi-1) in tumorigenesis, suggesting increased proliferation as a possible mechanism. If Esc or E(Pc) proteins were highly expressed in mammalian cancer, one might expect more frequent nonhomologous end-joining and, consequently, genome instability. Conversely, loss of one copy of either gene may provide a survival advantage under challenge with ionizing radiation or radiomimetic drugs. It is thus possible that the increased proliferation of tumor cells with mutations in these genes is, in part, not a direct result of increased expression of these genes, but rather a secondary effect of genome instability caused by decreases in gene conversion and increases in error-prone nonhomologous end-joining (Holmes, 2006).

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date revised: 15 April 2011

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