Transcriptional Regulation

During embryogenesis Polycomb is found in all tissues, though in later stages it preferentially accumulates in the CNS. Polycomb and Ubx are involved in a feedback-type regulation: Ultrabithorax, a homeotic target gene of Pc in its own domain of expression down-regulates Polycomb (Paro, 1993).

The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin

Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. The histone H2A variant of Drosophila melanogaster, Histone H2A variant (H2Av), localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. Neither H2Av accumulation nor histone H4 Lys 12 acetylation is affected by mutations in either Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin, requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place (Swaminathan, 2005).

The establishment of heterochromatin has so far been defined as a four-step process initiated by the RNAi machinery through the production of small RNAs homologous to centromeric DNA repeats that are recruited to prospective heterochromatic regions as part of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The next step described in this process thus far is the deacetylation and subsequent methylation of histone H3 Lys 9, which serves to recruit HP1. HP1 then recruits the Suv4-20 methyltransferase to trimethylate histone H4 at Lys 20. The work described here suggests that heterochromatin formation is more complex than previously thought, and it involves at least two additional steps. One step requires recruitment of H2Av or replacement of the canonical histone H2A for the H2Av variant. This requirement is highlighted by the observation that mutations in the His2Av gene act as suppressors of position effect variegation by modulating the silencing effect of heterochromatin on the adjacent white gene (Swaminathan, 2005).

The replacement of H2A for H2Av is not specific to heterochromatin, and it may also take place in silenced regions of the euchromatin, since it appears that His2Av behaves genetically as a PcG gene. PcG proteins are responsible for the maintenance of epigenetic silencing of the homeotic genes during Drosophila development. The His2Av gene can be classified as a PcG gene, since mutations in His2Av enhance the phenotype of Pc mutants, suppress the phenotype of mutations in trxG genes, and cause ectopic expression of the Ant gene. The involvement of H2Av in Pc-mediated silencing is not completely unexpected, since H2Av is critical for the establishment of pericentric heterochromatin and both processes share similar strategies. Heterochromatin-induced silencing requires methylation of H3 at Lys 9 by the Su(var)3-9 histone methyltransferase, whereas Pc-induced silencing involves the recruitment of the ESC-E(z) complex to methylate H3 at Lys 27. Although the modified residues are different, in both cases the modification serves as a tag to bind chromo domain-containing proteins, HP1 in the case of pericentric heterochromatin and Pc in euchromatic silencing. Given the parallels between the two processes, it was surprising to find that replacement of H2Av was required for subsequent H3 Lys 9 methylation in heterochromatin but not for H3 Lys 27 methylation in silenced regions of euchromatin. This later conclusion is supported by the observation that neither H3 Lys 27 methylation nor E(z) recruitment is affected by mutations in His2Av (Swaminathan, 2005).

It has been shown that the Drosophila H2Av variant is distributed in a nonrandom manner in third instar polytene chromosomes. H2Av is present in the heterochromatic chromocenter and is associated with both transcribed and nontranscribed genes in polytene chromosome bands and interbands (Leach, 2000). To gain further insights into the function of H2Av, it was decided to test whether His2Av behaves genetically as a trithorax-Group (trxG) or Polycomb-Group (PcG) gene. In Drosophila, expression patterns of homeotic genes are maintained by the PcG and trxG proteins. Since H2Av is present in nontranscribed euchromatic regions (Leach, 2000), whether this histone variant is involved in Pc-mediated silencing was determined by examining whether mutations in the His2Av gene enhance the phenotype of Pc mutants. Adult flies from a strain heterozygous for Pc, Df(3R)Pc/+, show a partial transformation of the second leg into the first leg, visualized by the appearance of sex combs in the second leg of male flies. When flies are also heterozygous for a mutation in the His2Av gene, the frequency and severity of these transformations increase dramatically. Out of 100 flies of the genotype Df(3R)Pc+/+ His2Av05146 examined, 33% had extra sex combs in all four second and third legs and 40% had extra sex combs in the second legs and one of the third legs. Out of 220 flies of the genotype Df(3R)Pc+/+ His2Av810 tested, 18% showed transformations of second into first leg and 72% showed transformation of both second and third legs into first. These results suggest that mutations in His2Av enhance the Pc phenotype and therefore His2Av might be classified as a PcG gene. To confirm this possibility, genetic interactions between His2Av and trxG mutants were examined. If His2Av is a PcG gene, mutations in His2Av should suppress the phenotype of trxG genes. The effect of His2Av05146 and His2Av810 was examained on two different combinations of trG genes, ash1VF101 trxb11/++ and brm2 trxE2/++. Flies of the genotype ash1VF101 trxb11/++ show transformations of third leg into second leg by the appearance of an apical bristle on the third leg in 66% of 1000 flies examined. This frequency decreases to 37% in ash1VF101 trxb11+/++ His2Av05146 flies and to 29% in ash1VF101 trxb11+/++ His2Av810 flies. Similarly, flies of the genotype brm2, trxE2/++ show a 43% frequency of haltere to wing or third leg to second leg transformations, and this frequency is reduced to 22% in brm2 trxE2+/++ His2Av05146 flies and to 21% in brm2 trxE2+/++ His2Av810 flies. These data suggest that mutations in His2Av suppress the phenotype of trxG mutations and, together with the previously observed enhancement of the Pc phenotype, support the hypothesis that His2Av is a PcG gene (Swaminathan, 2005).

PcG gene products repress transcription of homeotic genes outside of their normal expression boundaries. If H2Av is a PcG protein, ectopic expression of homeotic genes in His2Av mutants can be expected. To test this possibility, the distribution of Antennapedia (Antp) protein was examined in flies homozygous for the His2Av810 mutation. Antp localizes in the ventral ganglion of wild-type larvae in three bands of cells corresponding to the three thoracic segments. In the case of the His2Av810 mutant, this pattern is altered and the Antp protein is present further posteriorly through the ventral ganglion. A second homeotic protein, Ultrabithorax (Ubx), is involved in the development of the third thoracic and first abdominal segments, and it is expressed posterior to the Antp expression in the ventral ganglion of wild-type larvae. This pattern is not disrupted in the His2Av810 mutant; the band of Ubx expression appears to be similar in intensity and spatial distribution to that of wild-type larvae. These results suggest that H2Av might be required to maintain proper expression of homeotic genes in the anterior part of the animal, where Antp is expressed, but not in more posterior segments where Ubx expression occurs. The results also confirm the hypothesis suggesting that His2Av is a PcG gene (Swaminathan, 2005).

Recruitment of PcG complexes to silenced regions of the genome requires methylation of Lys 27 of histone H3. To test whether H2Av replacement is required for Pc recruitment, the distribution of this protein in wild type versus His2Av mutants was compared. Pc localizes to ~100 sites on polytene chromosomes of wild-type-OR third instar larvae. In contrast, chromosomes from larvae homozygous for the His2Av810 allele show a reduction in the number of Pc sites as well as in the amount of protein present at these sites. As a control, the Su(Hw) protein is present at similar levels in polytene chromosomes of wild-type and His2Av810 flies. To test whether this decreased accumulation of Pc in polytene chromosomes is due to reduced synthesis of Pc protein or reduced recruitment of the protein to the chromosome, Western analyses of protein extracts obtained from wild-type and His2Av810 mutant larvae were carried out. There is no significant difference in the levels of Pc protein between these two strains, suggesting that the observed effect is due to the inability of Pc to be recruited to the chromosomes in the absence of H2Av (Swaminathan, 2005).

Recent results suggest that H3 trimethylated at Lys 27 facilitates Pc binding to silenced regions and this modification is carried out by the Enhacer of zeste [E(z)] protein present in the ESC-E(z) complex. Since a reduction in Pc on polytene chromosomes was observed in His2Av mutants, whether recruitment of the ESC-E(z) complex is also impaired in these mutants was examined. In wild type, E(z) can be observed at multiple sites throughout the genome. The levels and localization of E(z) do not appear to be altered in the His2Av810 mutant compared to wild type. Whether H3 Lys 27 methylation is affected by mutations in His2Av was examined. The levels and distribution of this modification appear to be the same in polytene chromosomes from wild-type and His2Av810 mutant larvae. This result was confirmed by Western analysis, which shows equal levels of H3 trimethylated at Lys 27 in wild-type and His2Av810 mutant larvae. These results suggest that H2Av is required upstream of Pc recruitment in the process of Pc-mediated silencing. Since neither recruitment of the E(z) complex nor H3 Lys 27 methylation seem to be affected in His2Av mutants, H2Av replacement might take place after H3 Lys 27 methylation and before Pc recruitment. Alternatively, Pc repression might require at least two parallel and independent pathways, one involving H2Av recruitment and a second one leading to H3 Lys 27 methylation, both of which might be required for proper Pc recruitment (Swaminathan, 2005).

Given the observed accumulation of H2Av in the centromeric heterochromatin, to test the possible involvement of H2Av in heterochromatic silencing it was determined whether mutations in the His2Av gene can act as modifiers of variegated phenotypes caused by the presence of a gene next to heterochromatin. The In(1)wm4 allele is caused by an inversion that positions the white gene next to the centromeric heterochromatin of the X chromosome. This rearrangement results in the characteristic variegated phenotype. Mutations in the His2Av gene act as dominant suppressors of this phenotype, with flies of the genotype In(1)wm4/In(1)wm4; His2Av810/+ showing a dramatic increase in eye pigmentation when compared to In(1)wm4 alone. The presence of the H2Av histone variant in the centromeric heterochromatin and its requirement for the variegated phenotype of the In(1)wm4 mutation suggest that H2Av plays an important role in the establishment and/or maintenance of heterochromatin (Swaminathan, 2005).

Formation of heterochromatin requires deacetylation of H3 Lys 9 followed by methylation of the same residue and recruitment of HP1. The heterochromatin of Drosophila chromosomes is enriched in dimethylated and trimethylated histone H3 in the Lys 9 residue. To analyze the possible role of H2Av in heterochromatin assembly, the localization was examined of H3 dimethylated at Lys 9 in polytene chromosomes from larvae carrying a mutation in the His2Av gene. Antibodies against histone H3 dimethylated in Lys 9 stain the pericentric heterochromatin in wild-type larvae. Interestingly, polytene chromosomes from His2Av810 mutants show a decrease in the amount of methylated H3 Lys 9, whereas the presence of Su(Hw), used as a control, is the same in chromosomes from wild-type and His2Av810 mutant larvae. Since modification of this residue is important for HP1 recruitment, whether localization of HP1 in heterochromatin is also affected by mutations in His2Av was examined. In wild-type larvae, HP1 localizes preferentially to the pericentric heterochromatin of the chromocenter, but accumulation of HP1 is dramatically reduced in the His2Av810 mutant (Swaminathan, 2005).

To confirm these results, Western analyses of protein extracts obtained from wild-type and His2Av mutant larvae was carried out using antibodies against HP1 and histone H3 dimethylated in Lys 9. The results show little or no accumulation of histone H3 methylated in Lys 9, and lower levels of HP1 in the His2Av810 mutant. Methylation of histone H3 at the Lys 9 residue is carried out by the Su(var)3-9 histone methyltransferase, and HP1 is encoded by the Su(var)2-5 gene. In order to ensure that the observed effects on the levels of HP1 or the methylation of H3 Lys 9 were not caused by alterations in transcription of Su(var)3-9 or Su(var)2-5 due to the His2Av mutation, quantitative RT-PCR analyses of RNA obtained from wild-type and His2Av810 mutant third instar larvae were carried out . The results show that there are no significant changes in the levels of Su(var)3-9 or HP1 RNAs in His2Av810 mutant larvae when compared to wild type. These results and those from immunocytochemistry analyses confirm a role for H2Av in the methylation of H3 Lys 9 and subsequent recruitment of HP1 (Swaminathan, 2005).

Based on the observed effects of His2Av mutations on H3 Lys 9 methylation and HP1 recruitment, it appears that the presence of H2Av in heterochromatin might be required prior to these two events. To confirm this hypothesis, the pattern of H2Av distribution on polytene chromosomes from larvae carrying mutations was examined in the Su(var)2-5 and Su(var)3-9 genes. In both cases, H2Av localization appears normal, suggesting that the presence of H2Av is required prior to H3 Lys 9 methylation and HP1 recruitment during the establishment of heterochromatin (Swaminathan, 2005).

An ectopic heterochromatin domain can be created by insertion into euchromatin of closely linked multiple copies of a P-element transposon containing the white gene. HP1 is recruited to this site, suggesting that ectopic heterochromatin formation by the transgene array follows the same pathway as normal constitutive heterochromatin. To test whether H2Av is also involved in ectopic heterochromatin formation or if its role is specific to centromeric heterochromatin, the presence of H2Av at the site of integration of transgene repeats was examined. In a strain carrying only one transgene insertion, the white gene present in the P transposon is expressed at normal levels, but in strains carrying an array of six closely linked transgenes, expression of the white gene shows a characteristic variegated phenotype. Mutations in His2Av suppress this variegated phenotype, showing a red pigmentation of the eye closer to that of wild-type flies. This result suggests a requirement for H2Av in the establishment of ectopic heterochromatin caused by transgene arrays (Swaminathan, 2005).

To further test this conclusion, it was determined whether H2Av is indeed present at the site of transgene insertion. For this, simultaneous fluorescence in situ hybridization (FISH) was performed using the white gene as a probe and immunolocalization was performed using antibodies against H2Av. The FISH signal marks the site of insertion of the transgene, which can then be compared to that of H2Av immunostaining. Analysis of polytene chromosomes from a fly strain carrying a single-copy transgene (strain 6-2) shows that the site of insertion is located in an interband, where the chromatin is decondensed. In this strain, H2Av is not present at the site of insertion, in agreement with the normal expression of the white gene observed in these flies. When the same experiment was performed with polytene chromosomes from a strain carrying an array of six transposons at the same chromosomal location (strain DX1), the site of insertion was found to be associated with a DAPI-staining band as well as H2Av. This finding confirms a role for H2Av in ectopic heterochromatin formation, and suggests that compaction of chromatin at an ectopic site as a consequence of the presence of a transgene array follows the same pathway as that used for the formation of centromeric heterochromatin (Swaminathan, 2005).

Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs

During the regeneration of Drosophila imaginal discs, cellular identities can switch fate in a process known as transdetermination. For leg-to-wing transdetermination, the underlying mechanism involves morphogens such as Wingless that, when activated outside their normal context, induce ectopic expression of the wing-specific selector gene vestigial. Polycomb group (PcG) proteins maintain cellular fates by controlling the expression patterns of homeotic genes and other developmental regulators. Transdetermination events are coupled to PcG regulation. The frequency of transdetermination is enhanced in PcG mutant flies. Downregulation of PcG function, as monitored by the reactivation of a silent PcG-regulated reporter gene, is observed in transdetermined cells. This downregulation is directly controlled by the Jun amino-terminal kinase (JNK) signalling pathway, which is activated in cells undergoing regeneration. Accordingly, transdetermination frequency is reduced in a JNK mutant background. This regulatory interaction also occurs in mammalian cells, indicating that the role of this signalling cascade in remodelling cellular fates may be conserved (Lee, 2005).

Imaginal discs are clusters of cells that form the precursors of adult cuticular structures. On fragmentation and cultivation, a disc structure regenerates and forms the appendage, such as a leg or a wing, for which it was initially determined. Transdetermination events occur at sites of regeneration and on the ectopic expression of morphogens. Misregulation of PcG function causes homeotic transformations that are often phenocopied in transdetermination events. To determine whether PcG proteins are involved in transdetermination events, leg-to-wing fate changes were induced by ectopically overexpressing wingless (wg) and visualizing the transdetermined tissue by staining for ectopic expression of vestigial (vg) coupled to a lacZ reporter gene. In this assay, wild-type discs showed a transdetermination rate of 2.3% (2/87), whereas Polycomb (Pc) and Sex combs on midleg (Scm) heterozygous mutant discs showed an increased transdetermination rate of 17.7% (11/62) and 11.0% (10/91), respectively (Lee, 2005).

The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).

To show that transdetermination takes place only in cells with downregulated PcG function, fragmented leg discs of the FLW-1 line were stained for lacZ expression and for Vg in order to visualize the transdetermination to wing fate. It was consistently observed that the Vg staining lay within the lacZ expression domain, suggesting that PcG genes are downregulated in the blastema, enabling PRE-silenced genes to be reactivated according to new morphogenetic cues (Lee, 2005).

To investigate direct targets of PcG regulation that, when reactivated, might contribute to transdetermination, the PREs predicted at the wg and vg genes were tested and both were found to be controlled by PcG proteins. The fact that both the transgenic vg-lacZ reporter construct (which lacks the PRE) and the endogenous vg gene were upregulated in the blastema suggests that PcG proteins may affect vg expression both indirectly (for example, through wg) and directly by means of the vg PRE (Lee, 2005).

JNK signalling in Drosophila is crucial for wound healing and is implicated in many different developmental processes, such as dorsal and thorax closure. hemipterous encodes the JNK kinase (JNKK) that activates the Drosophila JNK Basket. Products of DJun and kayak (the Drosophila homologue of Fos) form the AP-1 transcription factor. A downstream target of JNK signalling is puckered (puc), which encodes a phosphatase that selectively inactivates Basket and thus functions in a negative feedback loop. The expression of puc thus mirrors JNK activity. Because wound healing takes place after fragmentation, it was reasoned that activation of the JNK pathway might be causing the downregulation of PcG proteins in the blastema. The pucE69 line, which carries a P(lacZ) insertion at the puc locus, was used to monitor JNK activity. During the third-instar larval stage puc is not expressed and thus JNK signalling was not activated in leg discs. As expected, however, puc was expressed on fragmentation in all cells at the annealing cut edge (Lee, 2005).

To check whether cells that have activated the JNK pathway also show transdetermination, fragmented leg discs of flies carrying the puc-lacZ reporter and vgBE-Gal4; UAS-GFP constructs were transplanted. In these flies, cells that adopted a wing fate were identified by their expression of green fluorescent protein (GFP). Two days after fragmentation, weak residual puc-lacZ staining was still visible in the central region of the disc. puc-lacZ staining is known to decline rapidly after wound healing is completed. It was found that stronger staining was visible along the cut site, probably owing to ongoing wound healing. On comparison of puc-lacZ staining and GFP fluorescence, JNK-active cells showed a substantial overlap with transdetermined cells; thus, it is concluded that JNK signalling is activated in cells that undergo transdetermination (Lee, 2005).

JNK signalling affects the transcription of numerous genes, including those encoding chromatin regulating factors. Therefore whether JNK signalling can downregulate the PcG proteins required for transdetermination was examined. A constitutively active form of hep was overexpressed in UAS-hepact; hsGal4 flies by a heat-shock pulse. Activating the JNK pathway caused a downregulation of some PcG genes, such as Pc, ph-p and E(Pc). No downregulation of these genes was observed in wild-type larvae before and after heat shock, indicating that this was not an unspecific heat-shock response. Expression was examined of two genes of the Trithorax group (ash1 and brm) that function antagonistically to PcG proteins, but found no upregulation on JNK induction (Lee, 2005).

To show further that JNK has a specific effect on PcG proteins, the analogous experiment was carried out in mammalian cells. The JNK pathway can be activated in mouse embryonic fibroblasts by exposing the cells to ultraviolet light. The expression of MPh2 (mouse polyhomeotic2) was examined because this mammalian PcG gene is expressed in these cells. The expression of MPh2 was decreased on JNK induction, but after treatment with a specific JNK inhibitor it was partially restored. In addition, to show that the downregulation of PcG genes is directly controlled by AP-1, chromatin immunoprecipitation was carried out using antibodies against Fos on chromatin from UAS-hepact; hsG4 and kay1 mutant flies. Enrichment of Fos on the promoter region of ph-p was observed, but no enrichment in chromatin from flies lacking Fos. This finding suggests that AP-1 binds directly to this region to regulate negatively the transcription of ph-p (Lee, 2005).

If activation of JNK signalling in the blastema indeed leads to a downregulation of PcG genes, then impairment of the JNK pathway should result in reduced efficiency of transdetermination. The transdetermination behaviour of wild-type discs was compared with that of discs bearing mutations in the JNKK hep. The transdetermination events were classified into three categories: large regions, small regions, and no regions of transdetermination. In wild-type discs only large regions were detected. In males hemizygous for hep1 (a weak hypomorphic allele), most transplanted leg discs had large transdetermined regions; however, a substantial proportion showed only small regions of transdetermination and a few showed no transdetermination event. In flies heterozygous for hepr75 (a null allele which is hemizygous lethal), most discs showed no or only small regions of transdetermination, and large regions were rarely seen. The morphology of the regenerated discs seemed unaffected in these mutants, indicating that the decline of transdetermination efficiency was not due to inefficient wound healing (Lee, 2005).

This study has shown that PcG genes are downregulated by JNK signalling. Because many developmental regulators need to be switched, the role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting the chromatin to a reprogrammable state. Transdetermination has been ascribed to the action of ectopic morphogens, which induce cells to activate incorrect gene cascades. Without doubt, wg and decapentaplegic signalling must be crucially involved in this process, because transdetermination does not result from any random cut but occurs preferentially when cuts are made through particular regions of the disc called 'weak points', which are regions of high morphogen. Inappropriate or overextreme downregulation of the PcG system by JNK in sensitive cells of the weak points thus may create such aberrant local patterns. Indeed, the data indicate that at least the two patterning genes, wg and vg, may be direct targets of the PcG. Notably, hyperactive Wnt signalling can also induce a switch in lineage commitment in mammals, implying that signalling pathways are a potent inducer of cell fate changes in many organisms (Lee, 2005).

Another study has shown that regenerating and transdetermining cells in the blastema have a distinct cell-cycle profile in contrast to the surrounding normal disc cells. It has been proposed that this change in cell-cycle regulation is a prerequisite for the change in cell fate. Indeed, PcG targets include genes involved in cell-cycle regulation, suggesting that this initial step is part of the complete reprogramming cascade required for the regenerating cells to achieve multipotency. Downregulation of PcG silencing by JNK seems to be a fundamental, evolutionarily conserved mechanism of cell fate change and thus may also have implications for studies of stem cell plasticity and tissue remodelling (Lee, 2005).

Targets of Activity (part 1/2)

The Polycomb protein maintains the segmental expression limits of the homeotic genes in the bithorax complex, consisting of abdominal-A, Abdominal-B and Ultrabithorax. Polycomb-binding sites within the bithorax complex were mapped by immunostaining of salivary gland polytene chromosomes. Polycomb binds to four DNA fragments, one in each of four successive parasegmental regulatory regions. Thus, Polycomb acts directly on discrete multiple sites in bithorax regulatory DNA (Chiang, 1995).

Parasegmental (PS)-specific expression of the homeotic genes of the bithorax-complex (BX-C) appears to depend upon the subdivision of the complex into a series of functionally independent cis-regulatory domains. Fab-7 is a regulatory element that lies between iab-6 and iab-7 (the PS11- and PS12-specific cis-regulatory domains, respectively). Deletion of Fab-7 causes ectopic expression of iab-7 regulated abdominal-A in PS11 (where normally only iab-6 is active). Two models have been proposed to account for the dominant Fab-7 phenotype. The first considers that Fab-7 functions as a boundary element that insulates iab-6 and iab-7. The second model posits that Fab-7 contains a silencer element that keeps iab-7 repressed in parasegments anterior to PS12 (Mihaly, 1997).

Using a P-element inserted in the middle of the Fab-7 region (the bluetail transposon), an extensive collection of new Fab-7 mutations have been generated that allow the subdivision of Fab-7 into a boundary element and a Polycomb-response element (PRE). The boundary lies within 1 kb of DNA on the proximal side of the blt transposon (towards iab-6). Deletions removing this element alone cause a complex gain- and loss-of-function phenotype in PS11; in some groups of cells, both iab-6 and iab-7 are active, while in others both iab-6 and iab-7 are inactive. Thus, deletion of the boundary allows activating as well as repressing activities to travel between iab-6 and iab-7. Evidence is provided that the boundary region contains an enhancer blocker element. The Polycomb-response element lies within 0.5 kb of DNA immediately distal to the boundary (towards iab-7). Deletions removing the PRE alone do not typically cause any visible phenotype as homozygotes. Interestingly, weak ectopic activation of iab-7 is observed in hemizygous PRE deletions, suggesting that the mechanisms that keep iab-7 repressed in the absence of this element may depend upon chromosome pairing (Mihaly, 1997).

These results help to reconcile the previously contradictory models on Fab-7 function and to shed light on how a chromatin domain boundary and a nearby PRE concur in the setting up of the appropriate PS-specific expression of the abd-A gene of the BX-C. It is suggested that there are two phases to BX-C regulation. During the first phase, gap and pair-rule genes select the activity state of the iab-6 and iab-7 cis-regulatory domains in PS11 and PS12. Once the activity states have been selected, BX-C regulation switches to the maintenance system. In PS11, where iab-6 is actived while iab-7 is not, the maintenance system must keep iab-7 turned off. This is presumably accomplished by the assembly of a Polycomb group protein silencing complex on iab-7. The Fab-7 boundary must prevent this iab-7 silencing complex from nucleating the assembly of a Polycomb-group protein in iab-6. This is presumably accomplished by blocking interactions between the two domains. In PS-12, where iab-7 is activated, the on state must be maintained (perhaps throught the action of proteins encoded by members of the trithorax group) (Mihaly, 1997).

Fab-7 is a genetically identified element of the BX-C necessary to regulate spatial transcription of the Abdominal-B (Abd-B) gene. Polycomb group (PcG) and trithorax group (trxG) gene products are responsible for the maintenance of repressed and active expression patterns of many developmentally important regulatory genes, including Abd-B. In Drosophila embryos, Polycomb (Pc) protein and the trxG protein GAGA factor colocalize at the Fab-7 DNA element of the bithorax complex. There is a strong enrichment in GAGA factor and Pc at the Fab-7 site in 11-16 hr old embryos, as compared to mock immunoprecipitations. A major GAGA factor binding site was localized in a 422 bp fragment, which contains six putative GAGA binding sites. This fragment is located within the putative boundary element. A lower level of binding was detected in the flanking 1230 bp fragment, which contains three GAGA target consensus sequences and also the putative PRE. Little or no association with all other flanking sequences is observed. In contrast, Pc is found to be associated with the entire 3.6 kb region, with a rather broad peak centered at the boundary and the PRE regions. Peak binding for Pc and GAGA factor colocalize. Thus, the physical distribution of these two proteins at Fab-7 does not allow discrimination of the apparent insulator and PRE function identified by transgenic constructs. This might indicate that the chromosomal elements through which PcG and trxG proteins act (the PRE) are located in sequences partially overlapping the putative boundary region (Cavalli, 1998).

In transgenic lines, the Fab-7 element induces extensive silencing on a flanking GAL4-driven lacZ reporter and mini-white genes. The Fab-7 fragment acts as a silencer preventing trans-activators like GAL4 from binding to the UAS-site when Pc protein is bound at the PRE. However, a short single pulse of GAL4 during embryogenesis is sufficient to release PcG-dependent silencing from the transgene. Such an activated state of Fab-7 is mitotically inheritable through development and can be transmitted in a GAL4-independent manner to the subsequent generations through female meiosis. In those flies where repression is reestablished upon meiosis, the repressed state can be reactivated again. This complete reversibility strongly suggests that the observed partial efficiency of transmission of the active state does not depend on heterogeneity in the genetic background of the fly line, but on a stochastic process whereby some of the chromatin templates may lose the epigenetic information upon meiotic transmission. Crosses using GAL4-less females strongly suggest that meiotic transmission of the activated Fab-7 state is not dependent on a preondurance of GAL4 protein. It is concluded that Fab-7 is a switchable chromosomal element, which can convey memory of epigenetically determined active and repressed chromatin states (Cavalli, 1998).

Although in the test system described here only maternal inheritance of Fab-7-dependent epigenetic regulatory states was observed, paternal inheritance of heterochromatin states has also been documented in Drosophila. Therefore, paternal inheritance of chromatin states is possible, and whether PcG/trxG-mediated meiotic inheritance is truly restricted to the female germline for all of their regulated sequences remains a fascinating question for future investigation. It is proposed that chromosomal elements such as Fab-7, where PcG and trxG proteins perform a coordinate maintenance function, be termed "cellular memory modules" (CMMs). CMMs may be thought of as switchable elements able to induce and heritably propagate both silenced and open chromatin conformations. The respective chromatin status determined by a regulatory cascade of transcription factors during early embryogenesis might be the primary switch. Activated transcription would drive a CMM into the trxG-dependent open chromatin mode, while inactive states would be maintained as silent chromatin (Cavalli, 1998).

The finding of meiotic inheritance in PcG/trxG-dependent regulation is surprising since these proteins control genes involved in developmental decisions. In the embryo, the zygotic genome has to develop different spatial patterns of homeotic gene expression. Therefore, the developing embryo must be able to erase the epigenetic information of its parental gametes in order to allow differentiation of a variety of cell lineages. It could be argued that PcG silencing at CMMs is the default state; that is, in the germ line, all CMMs remain "marked" by certain elements of the PcG silencing complex. As such, in the early zygote the occupation of all CMMs by PcG proteins would be retained, perpetuating silencing as the default state. Recent evidence speaks in favor of such a mechanism. In somatic cells, differential transcription induced by patterning factors would switch CMMs into the active mode, which would then be heritably maintained in subsequent cell generations. In the particular transgene combination used in this study, the strong GAL4 induction might have completely removed the PcG-silencing tag from this PRE, which subsequently remains in the active state through several rounds of mitotic and meiotic divisions until it becomes reinactivated by stochastic processes. The finding that a defined Drosophila chromosomal element can transmit an epigenetic state to the next generations in the absence of any apparent covalent modifications of the DNA suggests that chromatin proteins can faithfully maintain an epigenetic state, and will allow a detailed molecular analysis of this type of inheritance (Cavalli, 1998).

The Drosophila Polycomb and trithorax group proteins act through chromosomal elements such as Fab-7 to maintain repressed or active gene expression, respectively. A Fab-7 element is switched from a silenced to a mitotically heritable active state by an embryonic pulse of transcription. Here, histone H4 hyperacetylation has been found to be associated with Fab-7 after activation, suggesting that H4 hyperacetylation may be a heritable epigenetic tag of the activated element. Activated Fab-7 enables transcription of a gene even after withdrawal of the primary transcription factor. This feature may allow epigenetic maintenance of active states of developmental genes after decay of their early embryonic regulators (Cavalli, 1999).

Fab-7-dependent chromosomal memory of silent or open chromatin states occurs in transgenic Drosophila lines such as FLW-1 and FLFW-1. These lines carry a heat shock-inducible GAL4 driver (hsp70-GAL4) regulating a GAL4-dependent lacZ reporter (UAS-lacZ) flanked by Fab-7 and the mini-white gene. Silencing imposed by Fab-7 on the flanking reporter genes is dependent on the components of the PcG, since heterozygous mutant PcG genes show a relief of white gene repression. Conversely, white gene activity requires the trxG because heterozygous mutations in the different members tested result in a down-regulation of expression. A GAL4 pulse during embryogenesis can impose a mitotically stable reprogramming of the Fab-7 cellular memory module (CMM) from a silenced to an open chromatin state. The maintenance of the activated Fab-7 state is dependent on trithorax (trx) but not on Polycomb (Pc). In a heterozygous Pc- background, Fab-7 can be switched by a GAL4 pulse and be stably maintained, resulting in strong white expression. In contrast, a trx- mutation completely abolishes the mitotic transmission (Cavalli, 1999).

To assess whether the epigenetically activated Fab-7 state correlates with a permanent loss of PcG proteins from the chromatin template, a strong GAL4 induction pulse was administered during embryogenesis in the FLFW-1 line. Polytene chromosomes of third instar larvae were immunostained with antibodies directed against PcG proteins. Surprisingly, all of the PcG proteins tested, Polycomb (Pc) and Posterior sex combs (Psc), Polyhomeotic (Ph), and Polycomb-like (Pcl), are still strongly bound to the Fab-7 transgene irrespective of the epigenetic state. Thus, an epigenetically activated state can be stably propagated in the presence of the protein components of the PcG. These data support previous observations that have demonstrated binding of Pc at cytological sites containing potentially active genes in polytene chromosomes and binding of Ph and Psc proteins at an actively transcribed gene in Drosophila Schneider cells. It has been reported that certain PcG genes may function as activators in specific tissues and at specific developmental times by genetic analyses. Although a role for Pc protein in the maintenance of the activated state of Fab-7 is not observed, it may be possible that other PcG proteins are involved in this process (Cavalli, 1999).

The distribution of Polycomb protein has been mapped at high resolution on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Sheared chromatin was immunoprecipitated and amplified by linker-modified PCR, before using as a probe on a Southern of the entire PX-C walk. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Among the identified sites are peak F (the bxd PRE) and peak G (the bx PRE), both of which contain GAGA consensus sequences. Three other sites, E, D and C correspond to iab2, iab3 and iab4. No PC binding is seen in the regulatory domains iab6, iab7 or iab8, indicating that these domains positively regulate Abd-B expression. These results suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. The Abd-B sites include the iab7/iab8 regulatory region, and the Fab-7 PRE. The Fab-7 PRE does not bind Polycomb. It is proposed that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs (silencing elements) and as trithorax group response elements. It is suggested that a GAGA site in the Antennapedia promoter is both a PRE (binding PC protein) and a TRE (binding GAGA) factor (Strutt, 1997a).

The suppressor of Hairy-wing [su(Hw)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the su(Hw) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. These studies investigate whether repression resulting from the Polycomb group genes (derived from a gypsy element) can be blocked by the su(Hw) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group response element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. The su(Hw) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome (Mallina, 1998).

Transcriptional silencing by the Polycomb Group of genes maintains the position-specific repression of homeotic genes throughout Drosophila development. The Polycomb Group of genes characterized to date encode chromatin-associated proteins that have been suggested to form heterochromatin-like structures. By studying the expression of reporter genes, a 725 bp fragment (called MCP725) in the homeotic gene Abdominal-B has been identified. It accurately maintains position-specific silencing during proliferation of imaginal cells. Complete repression of MCP725 directed gene expression is found in the wing disc (parasegments 4 and 5); repression in the anterior compartment (ps5) of the haltere disc and strong expression in the posterior compartment of the haltere disc (ps6). Therefore, the MCP725 element is a silencer that functions throughout proliferation of the imaginal discs. MCP725 contains sequences that have been proposed to act as a chromatin boundary influenceing Abd-B gene expression. Data presented here suggests that MCP725 does not act as a boundary element. Silencing by MCP725 requires the Polycomb and the Polycomblike genes, indicating that it contains a Polycomb response element. To investigate the mechanisms of transcriptional silencing by MCP725, its temporal requirements were studied by removing MCP725 from the transgene at various times during development. Excision of MCP725 during larval stages leads to loss of silencing. These findings indicate that the silencer is required for the maintenance of the repressed state throughout cell proliferation. They also suggest that propagation of the silenced state does not occur merely by templating of a heterochromatin structure by virtue of protein-protein interactions. Rather, they suggest that silencers play an active role in the maintenance of the position-specific repression throughout development. It is thought that silencers like MCP725 might serve as DNA binding sequences for proteins that functionally replace the position-specific repressor role of the gap genes at later stages in development. These proteins, if they exist, would likely have DNA binding specificity and be expressed in a spatially restricted pattern throughout development. Candidates for these include either the remaining uncharacterized members of the PcG genes or the homeotic proteins, which themselves might replace the role of the gap proteins as repressors. This latter suggestion is attractive because (1) homeotic proteins are transcriptional repressors as well as activators; (2) are known to bind the promoters of other homeotic gene family members; (3) are expressed and functionally reqired throughout development, and (4) they show highly complementary expression patterns with one another (Busturia, 1997).

During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence-specific DNA-binding proteins that are Trithorax group activators of the homeotic gene Ultrabithorax. Zeste and GAGA DNA-binding sites at the proximal promoter are also required to maintain, but not to initiate, repression of Ubx. Furthermore, the repression mediated by Zeste DNA-binding site is abolished in zeste null embryos. These data imply that Zeste and probably GAGA mediate Polycomb repression. A model is presented in which the dual transcriptional activities of Zeste and GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter (Hur, 2002).

Zeste, GAGA and a third transcription factor, NTF-1 (Grainy head), activate promoter constructs of the Ubx gene in embryos via an intermingled cluster of sites between nucleotides -200 to -31. However, the constructs that were used in these experiments contain only a small subset of the Ubx cis regulatory region, and while they reproduce many features of Ubx expression, they do not respond to Polycomb repression when inserted at many chromosomal locations. Consequently, they have not permitted a rigorous analysis of the role of the proximal promoter factors in maintaining repression. To address this question, larger constructs have been used that contain the 22 kb of DNA upstream of the Ubx mRNA start site. These constructs do not suffer from significant position effect variation; they more closely approximate the expression pattern of the endogenous Ubx gene than the shorter constructs; they maintain efficient repression in late embryos as shown by the lack of ß-galactosidase reporter gene expression in more anterior and posterior regions, and they are genetically under the control of PcG genes (Hur, 2002).

Deletion of nucleotides -200 to -31 essentially abolishes transcription from the large Ubx promoter constructs, indicating a crucial role for factors binding to the proximal promoter. To determine the role of each factor separately, three constructs were prepared, each containing binding sites for either Zeste, GAGA or NTF-1 inserted between the deletion end points of the above construct. Importantly, biochemical, in vivo u.v. crosslinking, and genetic experiments strongly suggest that the DNA-binding sites used in these constructs are recognized only by their cognate factor, and not by any other sequence-specific DNA-binding activities. Binding sites for each factor separately activate transcription of the large constructs during late embryogenesis. Strikingly, constructs containing only GAGA- or Zeste-binding sites at the proximal promoter are not expressed in the anterior or posterior of the embryo, whereas constructs bearing only NTF-1 sites are strongly transcribed in these terminal regions (Hur, 2002).

Ectopic expression of Ubx in anterior and posterior regions is generally caused by a failure of the initiating repressors or the Polycomb maintenance system. One interpretation of this result is that Zeste and GAGA are required for at least one form of repression, while NTF-1 is not. It is also possible, however, that Zeste and GAGA are not repressors. Instead, it may be that they are unable to activate expression in anterior or posterior regions, even though they are expressed at similar levels throughout the embryo. To distinguish between these two possibilities, constructs were examined that contained either Zeste and NTF-1 sites or GAGA and NTF-1 sites. These constructs are expressed in the central region of the embryo; but, importantly, they are not significantly expressed in anterior or posterior regions. Since NTF-1 can activate Ubx transcription in these terminal regions, the absence of terminal expression is consistent with GAGA and Zeste directly repressing transcription in addition to their activation function (Hur, 2002).

The PcG genes are an essential part of system that maintains repression of the endogenous Ubx gene. To confirm that these genes also act on these transgenes, the 22UZ Zeste and 22UZ GAGA constructs were crossed into PcG mutant embryos. Both transgenes are derepressed in late stage embryos lacking the Polycomb gene. Similar results were obtained in embryos lacking another PcG gene, extra sex combs. Thus, Zeste -- and probably also GAGA -- act together with the Polycomb system to maintain repression of Ubx (Hur, 2002).

The data presented in this paper are consistent with the earlier genetic data that suggested that some trxG and PcG proteins may have dual activities. Further support for this idea comes from recent biochemical experiments that have shown that GAGA is complexed with two PcG proteins in Drosophila nuclear extracts and Zeste is part of a multisubunit complex that contains Polycomb. In addition, PcG proteins are frequently associated in vivo with promoter regions that include Zeste or GAGA DNA recognition sites, including the Ubx proximal promoter examined in this paper. Most PcG proteins do not recognize specific DNA sequences; thus, the interaction with Zeste and GAGA may serve to recruit PcG proteins to promoters (Hur, 2002).

But is it essential that some proteins, such as Zeste and GAGA, participate in both repression and activation, or is it mere coincidence? This joint participation may be essential. At the transition between the initiating repressors and the Polycomb system, one possibility is it that Polycomb proteins are recruited to or activated on only those genes that are bound by initiating repressors; the initiating repressors may physically bind to PcG proteins to recruit them. However, Polycomb repression can be established on Ubx promoter constructs that lack initiating repressors elements, provided that initiating enhancer elements are also absent. In other words, at the transition between the establishment and maintenance of the Ubx expression pattern, the Polycomb systems reads the absence of activation, rather than the presence of repression or repressors (Hur, 2002).

Hierarchical recruitment of polycomb group silencing complexes

Polycomb group (PcG) proteins maintain the transcriptional silence of target genes through many cycles of cell division. This study provides evidence for the sequential binding of PcG proteins at a Polycomb response element (PRE) in proliferating cells in which the sequence-specific DNA binding Pho and Phol proteins directly recruit E(z)-containing complexes, which in turn methylate histone H3 at lysine 27 (H3mK27). This provides a tag that facilitates binding by a Pc-containing complex. In wing imaginal discs, these PcG proteins also are present at discrete locations at or downstream of the promoter of a silenced target gene, Ubx. E(z)-dependent H3mK27 is also present near the Ubx promoter and is needed for Pc binding. The location of E(z)- and Pc-containing complexes downstream of the Ubx transcription start site suggests that they may inhibit transcription by interfering with assembly of the preinitiation complex or by blocking transcription initiation or elongation (L. Wang, 2004; full text of article).

maintenance of teashirt repression requires Polycomb group mediated gene silencing

Secreted signaling molecules such as Wingless (Wg) and Decapentaplegic (Dpp) organize positional information along the proximodistal (PD) axis of the Drosophila wing imaginal disc. Responding cells activate different downstream targets depending on the combination and level of these signals and other factors present at the time of signal transduction. Two such factors, teashirt (tsh) and homothorax (hth), are initially co-expressed throughout the entire wing disc, but are later repressed in distal cells, permitting the subsequent elaboration of distal fates. Control of tsh and hth repression is, therefore, crucial for wing development, and plays a role in shaping and sizing the adult appendage. Although both Wg and Dpp participate in this control, their specific contributions remain unclear. In this report, tsh and hth regulation were analyzed in the wing disc; Wg and Dpp act independently as the primary signals for the repression of tsh and hth, respectively. In cells that receive low levels of Dpp, hth repression also requires Vestigial (Vg). Furthermore, although Dpp is required continuously for hth repression throughout development, Wg is only required for the initiation of tsh repression. Instead, the maintenance of tsh repression requires Polycomb group (PcG) mediated gene silencing, which is dispensable for hth repression. Thus, despite their overall similar expression patterns, tsh and hth repression in the wing disc is controlled by two very different mechanisms (Zirin, 2004).

Analysis of Su(z)12daed and Pc mutant clones indicates that the maintenance of tsh repression is mediated by a heritable silencing mechanism. By inducing Pc mutant clones in third instar discs, it was demonstrated that this ectopic tsh expression represents a failure to maintain rather than a failure to establish repression. The weak hth levels observed in some PcG mutant clones may be due to the ability of tsh to upregulate hth. This interpretation is supported by the fact that hth expression is seen only in large Pc mutant clones, and only in cells expressing the highest levels of tsh. The general absence of hth expression in PcG mutant clones, together with the ectopic hth expression resulting from late Dpp pathway disruption, points to the need for continuous signaling input to maintain hth repression. By contrast, tsh requires PcG gene activity, but not continuous Wg or Dpp input, to maintain its repression during the third instar (Zirin, 2004).

At this stage the possibility that the affects of PcG mutant clones on tsh repression described here are indirectly due to the de-repression of another factor cannot be ruled out. It is suggested that this is unlikely, however, in part because the spatial distribution of tsh de-repression in PcG mutant clones differs significantly from reports of Hox gene de-repression. Additionally, the ectopic tsh expression in Pc mutant clones is repressible by Nrt-Wg, indicating that tsh is still subject to regulation by Wg signaling (Zirin, 2004).

Inheritance of Polycomb-dependent chromosomal interactions in Drosophila: Evidence using the Fab-7 cellular memory module

Maintenance of cell identity is a complex task that involves multiple layers of regulation, acting at all levels of chromatin packaging, from nucleosomes to folding of chromosomal domains in the cell nucleus. Polycomb-group (PcG) and trithorax-group (trxG) proteins maintain memory of chromatin states through binding at cis-regulatory elements named PcG response elements or cellular memory modules. Fab-7 is a well-defined cellular memory module involved in regulation of the homeotic gene Abdominal-B (Abd-B). In addition to its action in cis, it has been shown, by three-dimensional FISH, that the Fab-7 element leads to association of transgenes with each other or with the endogenous Fab-7, even when inserted in different chromosomes. These long-distance interactions enhance PcG-mediated silencing. They depend on PcG proteins, on DNA sequence homology, and on developmental progression. Once long-distance pairing is abolished by removal of the endogenous Fab-7, the derepressed chromatin state induced at the transgene locus can be transmitted through meiosis into a large fraction of the progeny, even after reintroduction of the endogenous Fab-7. Strikingly, meiotic inheritance of the derepressed state involves loss of pairing between endogenous and transgenic Fab-7. This suggests that transmission of nuclear architecture through cell division might contribute to inheritance of chromatin states in eukaryotes (Batignies, 2003).

In most experiments throughout this work, two constructs were used carrying a 3.6-kb Fab-7 fragment cloned in two different orientations (p5F24 and p5F3 transgenes) upstream to lacZ and mini-white reporter genes. These transgenes were inserted at different genomic locations and combined with deletions or with mutations in PcG or trxG genes. For simplicity, transgenic lines will be named after the CMM element present in the transgene and the chromosomal arm of insertion of the transgenes. For instance, one previously published line, 5F24 will be renamed here as Fab-X, to indicate insertion of Fab-7 in the X chromosome. As expected for CMM-mediated silencing, pairing-sensitive repression is observed and the eye color of homozygous females is strongly variegated, whereas in heterozygous females or in males (hemizygous) repression is weaker. Precise mapping of the transgene insertion indicates that the Fab-X line harbors two copies of the p5F24 transgene inserted in tandem 9.6 kb upstream of the scalloped (sd) gene. The sd gene product is required for wingblade development in Drosophila. Reduced expression of this gene leads to a characteristic wing phenotype (sd phenotype) that can have different degrees of severity ranging from small lesions in the wing margin to complete destruction of wing morphology. The insertion of the Fab-7 CMM at a distance of 18.4 kb from sd (8.8 kb DNA spanning the lacZ and mini-white regions plus 9.6 kb from the transgene insertion site to the sd promoter) induces a mutant phenotype, resulting in destruction of the wingblade. This phenotype is temperature sensitive and pairing dependent, since it is observed with a strong penetrance of up to 95% in homozygous females raised at 29°C, whereas it is almost absent in heterozygous females or hemizygous males. Both features are typical of PcG-mediated silencing and parallel effects on mini-white. The incomplete penetrance of the sd phenotype does not depend on genetic heterogeneity of the flies, preventing silencing in a fraction of the population. When flies were raised at 29°C and Fab-X females with wild-type wings were selected and remated with Fab-X males, the next generation females showed a sd phenotype with similar penetrance as nonselected Fab-X females. Finally, as observed for the white eye phenotype in the Fab-X line, the sd phenotype is strongly attenuated by mutations in PcG genes, whereas it is enhanced by a mutation in the trx gene (Batignies, 2003).

Surprisingly, repression of sd depends also on the presence of an intact copy of the endogenous Fab-7 element in the Abd-B locus, located in the right arm of the third chromosome (chromosome 3R). A genomic deletion of 4 kb encompassing the 3.6-kb Fab-7 transgenic element was introduced in the homozygous state into the Fab-X line to give the Fab-X; Fab-71 line. When raised at 29°C, Fab-X; Fab-71 females showed derepressed eye color and showed only 6%-12% of sd wing phenotype compared with 90%-95% of Fab-X females, suggesting that silencing of mini-white and sd is reduced. As a control, the presence of the Fab-7 deletion had no effect on the wing phenotype of a mutant line for the sd gene, indicating that endogenous Fab-7 does not play any role in sd regulation in the absence of the X-linked transgene. Moreover, a derepression both of mini-white and of sd was observed by introducing a homozygous Fab-712 deletion into Fab-X. This mutation deletes 1.5 Kb of DNA from the same region and has a similar effect on regulation of its endogenous target gene Abd-B, but it has an independent origin and genetic background from Fab-71. Therefore, derepression depends specifically on removal of Fab-7 (Batignies, 2003).

The results presented here show that a CMM element of 3.6 kb can mediate long-distance associations between distant chromosomal regions in embryonic nuclei. These interactions depend strongly on chromatin components of the PcG and on DNA sequence homology. Importantly, when disrupted in one generation, these pairing interactions are inefficiently re-established even upon reintroduction of sequence homology, and a large portion of the progeny maintains the loci unpaired in subsequent generations. This phenomenon is reversible, suggesting that PcG-mediated chromatin regulation is an equilibrium process that depends on the concentration of regulatory components and on the previous history of the cell. Perturbation of the balance between these regulatory cues might favor establishment as well as inheritance of active or repressed states at target genes. Several cases of inheritance of chromatin composition features have been reported in eukaryotes. The data described here suggest that inheritance of Fab-7 regulatory states depends not only on chromatin components, but also on nuclear compartmentalization of chromosomal domains (Batignies, 2003).

PcG proteins have been implicated previously in phenomena involving long-distance interactions among independent loci. Phenotypic interactions were documented both at transgenes containing CMM elements as well as in the phenomenon of cosuppression. In this last case, PcG proteins as well as mechanisms of RNA-dependent posttranscriptional gene silencing were shown to be involved. However, whether direct long-distance associations occur in these cases is presently unknown. In contrast, long-distance pairing has been observed in up to 30% of embryonic nuclei containing a euchromatic translocation of a region of ~900 kb, spanning the BX-C and flanking genes from chromosome 3R to chromosome X. Although the DNA sequence determinants and the proteins responsible for pairing in this large chromosomal region were not identified, several CMM, including Fab-7, are present in the BX-C and could contribute to this interaction. On the basis of these results, it is suggested that PcG proteins may mediate long-range pairing interactions in the case of Fab-7 as well as in transgenes containing other CMM and in some cases of cosuppression and silencing of repetitive DNA elements (Batignies, 2003).

Long-distance associations may not only involve transgenes, but also natural genes regulated by the Polycomb pathway. PcG proteins are distributed in specific nuclear compartments that have been termed PcG bodies. Although the significance of these bodies is presently unclear, it is speculated that endogenous PcG target genes may undergo physical associations at nuclear PcG bodies dedicated to their regulation. Compartmentalization of PcG target genes may not be required for primary recruitment of PcG complexes, but it may rather stabilize PcG and trxG-mediated gene regulation. This phenomenon may not be unique in eukaryotic nuclei, since evidence for gene clustering at specific intranuclear organelles has been found in vertebrates. These clustering phenomena were suggested to involve positioning of genes coregulated by the same set of proteins in the same nuclear compartments. PcG proteins may represent one class of factors acting in this manner (Batignies, 2003).

A hint for involvement of the Fab-7 element in heterologous associations at PcG bodies comes from experiments showing that in Fab-X; Fab-71 larvae, the Fab-7 transgene pairs with the BX-C locus to some extent, even in the absence of the endogenous Fab-7. This suggests that in the absence of sequence homology, Fab-7 may interact with other CMM present in the BX-C, albeit more weakly. An increase in Fab-7-dependent sd silencing is detected in the presence of a transgene containing the Mcp sequence from the BX-C, suggesting that these two elements may be able to interact (Batignies, 2003).

How is pairing achieved? First, homologous CMM must come in physical proximity. This may depend on constrained brownian motion of chromosomal territories. However, other processes may help this long-distance search. In particular, it may be postulated that genes containing CMM localize to PcG bodies. It is speculated that these bodies might not be immobile, but, as in the case of splicing speckles, they may rather undergo occasional movements, splitting, and mergers, although perhaps with different kinetics. Genes localized within these bodies may reside there for a certain time and then leave one PcG body to incorporate another one. Such a dynamic behavior may allow PcG target genes to explore part of the nucleus, but would, at the same time, allow them to stay in the vicinity of other PcG target genes and prevent them from diffusing away randomly in the nucleoplasm. This may increase the probability for a CMM to explore contacts with other CMM (Batignies, 2003).

Once proximity is established, strong association might be established by regulatory components of CMM chromatin. The identification of a 3.6-kb DNA sequence as a sufficient region of homology to induce long-range physical associations will allow, for the first time, to dissect DNA sequences and chromatin factors responsible for pairing at high resolution. The 3.6-kb Fab-7 element contains a chromatin boundary that can attenuate enhancer-promoter communications, and a PRE. GAGA factor binds to both the PRE and the boundary region of Fab-7. Moreover, this protein is able to bind cooperatively to DNA to form oligomers, bringing distant DNA sequences close together. Thus, GAGA factor-binding sites may be partly responsible for long-range interactions. Similarly, putative binding sites for Zeste, a protein that mediates trans-sensing phenomena, are also present in Fab-7, and they may contribute to pairing. Other chromatin components described previously to act at this element, such as proteins of the trxG, chromatin condensation proteins, and DNA topoisomerase II may also be involved in pairing of Fab-7 (Batignies, 2003).

However, all of these proteins associate also with other CMM in the genome. How do they distinguish between DNA sequence homologous and nonhomologous CMM? One possibility is that chromatin regulation and the DNA sequence determine a specific array of proteins and of histone modifications associated with it. For a given locus, this may result in the formation of a unique order of chromatin tags that can only be found at loci sharing strong sequence homology. Some of these components may undergo dimerization or oligomerization, leading to specific contacts that may maintain homologous chromatin stably associated. Similar contacts may also be involved in chromosome pairing in somatic cells or during meiosis (Batignies, 2003).

A remarkable finding involving long-distance pairing of the Fab-7 CMM is transmission through meiosis. What could be the role of this meiotic inheritance of chromatin states? This is particularly intriguing in the case of a CMM regulating a homeotic gene, as expression of homeotic genes must be reset at every generation in order to establish appropriate gene expression patterns along the anteroposterior embryonic axis. However, meiotic inheritance was reported previously to involve a phenotype associated with a chromosomal rearrangement at the BX-C locus, although no molecular determinant for this phenomenon could be found. In Caenorhabditis elegans, PcG proteins establish a germ-line-specific gene silencing that is heritable through meiosis. One possible way in which meiotic inheritance could be important in Drosophila homeotic gene regulation is to maintain a default silenced state during early embryogenesis. At the onset of homeotic gene transcription, spatial-specific transcriptional repressors maintain homeotic genes repressed outside of the appropriate expression domains. Maintenance of repression is crucial, as failure could cause homeotic transformations. Inheritance of chromatin silencing may stabilize this repression and contribute to developmental homeostasis (Batignies, 2003).

The fact that pairing interactions involving a chromosomal element regulated by PcG/trxG proteins are heritable raises the question of how transmission of chromatin architectural features is possible through cell division. Two different, but not mutually exclusive, mechanisms may contribute to explain this novel form of inheritance. Chromosomal contacts may depend on specific, heritable chromatin marks deposited in cis on the templates undergoing pairing. These marks may allow contacts to re-establish after they are broken during chromosome metabolism at mitosis and meiosis. Perturbation of these marks may change chromatin at Fab-7 and make it incapable of establishing pairing interactions with its homolog sequence in another chromosome. Chromatin marks, such as histone acetylation, histone methylation, and association of Swi6 protein to the mating type locus in Schizosaccharomyces pombe were shown previously to be heritable through meiosis. Initial chromatin characterization in the presence or absence of Fab-7-pairing interactions showed recruitment of PcG proteins to the transgene and to the region surrounding its site of insertion in both cases, and did not reveal significant changes in PcG protein binding or in histone modifications (Batignies, 2003).

A second mechanism for inheritance of long-range chromosomal interactions may depend on stable transmission of the relative chromosome positions and specific gene contacts through cell division. It was shown recently that global chromosome positioning can be transmitted in mammalian cells through the whole-cell cycle and mitosis, although the fidelity of mitotic transmission may depend on cell type. The data show that long-distance pairing is dynamic during development; it has a relatively weak frequency during embryonic stages, and it increases at larval stages. This dynamics may depend on the increased length of the cell cycle or on more robust PcG silencing in larvae, and it suggests that the actual physical contact between chromosomes may be lost, but regulation of nuclear compartmentalization may favor re-establishment of long-distance pairing at each cell generation (Batignies, 2003).

In summary, the present study suggests that features of the nuclear architecture of PcG target genes can be transmitted through cell division. It is proposed that this may represent a novel form of epigenetic inheritance that may be used to convey cellular memory of chromatin states in eukaryotic organisms (Batignies, 2003).

The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes

Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. This study investigated the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

Eukaryotic DNA is packaged into a periodic nucleoprotein complex termed chromatin. The nucleosome is the basic repeating unit of chromatin, and the nucleosomal core consists of 146 bp of DNA wrapped around an octamer of histones H2A, H2B, H3, and H4. In addition to the core histones, chromatin contains other components such as linker histones and high mobility group proteins. Chromatin is involved in the regulation of transcription and other DNA-directed processes via posttranslational modifications of core histones, the reorganization of nucleosomes by chromatin remodeling factors, and the alteration of higher-order structures (Fyodorov, 2004 and references therein).

The assembly of chromatin is a fundamental biological process that occurs in proliferating cells during DNA replication and in quiescent cells during maintenance and repair of chromosomes. During DNA replication, chromatin structure is transiently disrupted at the replication fork, and the preexisting nucleosomes are segregated randomly between the daughter DNA strands. Then, additional nucleosomes are formed with newly synthesized histones. In this process, it appears that histones H3 and H4 are deposited prior to the incorporation of histones H2A and H2B. Chromatin assembly also occurs in nonreplicating DNA, and several examples of replication-independent assembly of chromatin have been described. These latter processes may occur during histone replacement, DNA repair, and transcription (Fyodorov, 2004 and references therein).

The basic chromatin assembly process is mediated by core histone chaperones and an ATP-utilizing motor protein. The histone chaperones include CAF-1 (chromatin assembly factor-1), NAP-1 (nucleosome assembly protein-1), Asf1 (anti-silencing function-1; see Drosophila Asf1), nucleoplasmin, N1/N2, and Hir (histone regulatory) proteins. These proteins appear to deliver the histones from the cytoplasm to the sites of chromatin assembly in the nucleus. The ATP-utilizing assembly factor ACF (ATP-utilizing chromatin assembly and remodeling factor) can catalyze the transfer of histones from the chaperones to the DNA to yield periodic nucleosome arrays. The assembly reaction can also be catalyzed by purified RSF (remodeling and spacing factor), which appears to possess both chaperone and motor activities (Fyodorov, 2004).

This work investigates the biological function of ACF. ACF was purified from Drosophila embryos as an activity that mediates the ATP-dependent assembly of regularly spaced nucleosome arrays in vitro. During the assembly process, ACF commits to and translocates along the DNA template. ACF consists of two subunits, Acf1 and ISWI, which cooperatively catalyze nucleosome assembly in conjunction with histone chaperone proteins NAP-1 or CAF-1. Acf1 is the larger subunit of ACF, and it possesses WAC, DDT, WAKZ, PHD finger, and bromo-domain motifs. ISWI belongs to the SNF2-like family of DNA-dependent ATPases, and is a subunit of the ACF, CHRAC (chromatin accessibility complex), NURF, and TRF2 complexes. NURF and TRF2 complexes share only the ISWI subunit with ACF, whereas CHRAC is closely related to ACF. CHRAC was purified on the basis of its ability to increase the access of restriction enzymes to DNA in chromatin, and it consists of Acf1, ISWI, and two small subunits, CHRAC-14 and CHRAC-16, which are detected only during early embryonic development. The biochemical activities of ACF and CHRAC are indistinguishable. These Acf1-containing species will be referred to as 'ACF/CHRAC'. To study the function of ACF/CHRAC in vivo, a genetic analysis of the Drosophila acf1 gene was performed. The results indicate that Acf1 programs ACF/CHRAC to perform functions that are distinct from those of the NURF complex, which shares a common ISWI ATPase subunit with ACF/CHRAC. In addition, the phenotypes of flies lacking Acf1 suggest that ACF/CHRAC does not disrupt chromatin, as might be expected for a nucleosome remodeling factor, but rather promotes the formation of chromatin, as would be expected for a chromatin assembly factor (Fyodorov, 2004).

Polycomb regulation is caused by chromatin-dependent transcriptional silencing. The identity of body segments in Drosophila is specified by homeotic genes of the Antennapedia and bithorax complexes, which are in turn subject to regulation by Polycomb and trithorax group (PcG and trxG) genes. PcG genes encode protein complexes that can maintain chromatin-dependent transcriptional silencing via cis-acting DNA elements termed Polycomb response elements, or PREs (Fyodorov, 2004).

To determine the influence of Acf1 on Polycomb regulation, whether the loss of Acf1 affects transcriptional repression by the Ubx PRE in a PRE-miniwhite reporter gene was examined. In the wild-type control background(acf13/acf13), the expression of the PRE-miniwhite reporter gene was strongly repressed, with pigments limited to a small part of the adult fly eye. In the absence of Acf1 (acf11/acf11), partial activation was observed of the PRE-miniwhite reporter gene with pigments distributed over a larger area of the eye. This observed derepression in the homozygous acf11 background is comparable to derepression in a heterozygous Pc background (Fyodorov, 2004).

Whether acf1 interacts genetically with the segmentation function of Pc was investigatede. The appearance of extra sex combs on distal portions of the second and third legs in F1 males was scored in the progeny from a cross between males with a heterozygous deficiency for Pc (Df(3L)Asc) and females homozygous for acf1 alleles. The mutation of acf1 significantly enhanced this Pc phenotype in a manner similar to that seen with other enhancers of the Pc gene. Whereas only about 18% or 17% of the Df(3L)Asc/+; acf13/+ or Df(3L)Asc/+; acf14/+ males had extra sex combs on second and/or third pairs of legs (from the total number of male progeny scored, 61% or 58% of the Df(3L)Asc/+; acf11/+ or Df(3L)Asc/+; acf12/+ male flies had the extra sex comb phenotype). In addition, >50% of males in the latter two crosses exhibited ectopic pigmentation of their A3 and A4 abdominal tergites, which was never observed in crosses with acf13 or acf14 mothers. These results, combined with the derepression of PRE-mediated miniwhite silencing, demonstrate that acf1 is a Polycomb enhancer and suggest that ACF/CHRAC is involved in the assembly and/or maintenance of repressive chromatin in Polycomb-responsive loci (Fyodorov, 2004).

The identity of Drosophila abdominal segments A5-A8 is determined by homeotic selector genes of the bithorax complex. For instance, in Pc/acf1 males, the posteriorly directed homeotic transformation may be caused by an increase in the expression of the bithorax complex gene Abd-B on loss of Acf1. In contrast, the anterior transformation phenotype of ISWI/+; acf1/acf1 and nap1/nap1; acf1/acf1 animals is reminiscent of mutations in various trithorax group genes, which include the brm and kis genes that encode ATPase subunits of chromatin remodeling complexes. This anterior transformation is likely to result from a decrease in expression of Abd-B on loss of Acf1. These data suggest that Acf1 may be involved in repression or activation of Abd-B in different contexts. Transcriptional repression of Abd-B by Acf1 is consistent with its function in the assembly of repressive chromatin. In fact, genetic evidence in yeast as well as polytene chromosome localization studies in Drosophila primarily implicate ISWI-containing complexes in transcriptional repression in vivo. Transcriptional activation of Abd-B by Acf1 could be due to its chromatin remodeling function, which could potentially facilitate transcription, or to an indirect effect, such as the repression of a transcriptional repressor of Abd-B (Fyodorov, 2004).

Surprisingly, Acf1 is not absolutely required for viability. Chromatin from homozygous acf1 mutant embryos exhibits less nucleosomal periodicity as well as a shorter repeat length than chromatin from wild-type embryos. Extracts from Acf1-deficient embryos assemble nucleosomes in vitro much less efficiently than wild-type extracts, and also that the deficiency in chromatin assembly can be rescued on addition of purified recombinant ACF or Acf1. These findings indicate that ACF/CHRAC is a major chromatin assembly activity in Drosophila, but also that Acf1-deficient flies contain other ATP-utilizing chromatin assembly factor(s) that are able to sustain partial viability (Fyodorov, 2004).

The analysis of the Acf1 null flies revealed that ACF/CHRAC performs different biological functions than NURF, even though ACF/CHRAC and NURF both share a common ISWI ATPase. Hence, the unique subunits of ACF/CHRAC and NURF can program the basic motor function of ISWI to perform specific biological tasks in vivo (Fyodorov, 2004).

ATP-utilizing motor proteins could potentially assemble or disrupt chromatin structure. Through multiple lines of investigation, the function of ACF/CHRAC was studied in vivo. (1) Whether there are genetic interactions between acf1 and nap1 was investigated, because the ACF/CHRAC motor protein and the NAP-1 histone chaperone function together in chromatin assembly in vitro. Double mutant nap1/nap1; acf1/acf1 flies exhibit a homeotic transformation that is not seen in the corresponding single mutant flies. These results are consistent with the biochemical activities of ACF/CHRAC and NAP-1 in the chromatin assembly process (Fyodorov, 2004).

(2) The effect of Acf1 on heterochromatic transcriptional silencing was tested. In these experiments, suppression of pericentric position-effect variegation was detected on loss of Acf1. It was additionally found that Acf1-deficient flies exhibit reduced levels of Polycomb-mediated transcriptional silencing. These findings indicate that ACF/CHRAC is important for the establishment and/or maintenance of repressive chromatin states (Fyodorov, 2004).

(3) Whether Acf1 enhances or disrupts chromatin-mediated repression of DNA replication was investigated. Shortening of S phase was observed in Acf1-deficient embryos and larval neuroblasts, consistent with a role of ACF/CHRAC in the assembly rather than disruption of chromatin in vivo. The effect of chromatin structure on the duration of S phase in larvae was investigated with a deficiency that uncovers the histone gene cluster. These animals contain reduced levels of histones and exhibit an acceleration of late S phase progression in larval neuroblasts relative to that in wild-type flies. Thus, the mutation of acf1 as well as the reduction in the level of histones each correlate with an increase in the rate of S phase progression. These data collectively support a role of Acf1 in the assembly of histones into chromatin (Fyodorov, 2004).

In summary, several independent lines of experimentation implicate Acf1 in the formation of chromatin in vivo. These experiments provide evidence for the function of ACF/CHRAC (and other ATP-utilizing factors) in the assembly of chromatin in conjunction with the NAP-1 histone chaperone. They also include the unexpected finding of a role of ACF/CHRAC in Polycomb-mediated silencing as well as the discovery of mutations (acf1 and Df(2L)DS6) that result in an unusual increase in the rate of S phase. Lastly, the loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin, which support a role of Acf1 in the assembly of repressive chromatin. Hence, the collective biochemical and genetic data indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus (Fyodorov, 2004).

Polycomb mediates Myc autorepression and its transcriptional control of many loci in Drosophila

Aberrant accumulation of the Myc oncoprotein propels proliferation and induces carcinogenesis. In normal cells, however, an abundance of Myc protein represses transcription at the c-myc locus. Cancer cells often lose this autorepression. This study examined the control of myc in Drosophila and show here that the Drosophila ortholog, dmyc, also undergoes autorepression. The developmental repressor Polycomb (Pc) is required for dmyc autorepression, and this Pc-dMyc-mediated repression spreads across an 875-kb region encompassing the dmyc gene. To further investigate the relationship between Myc and Polycomb, microarrays were used to identify genes regulated by each, and a striking relationship was identified between the two: A large set of dMyc activation targets is normally repressed by Pc, and 73% of dMyc repression targets require Pc for this repression. Chromatin immunoprecipitation confirmed that many dMyc-Pc-repressed loci have an epigenetic mark recognized by Pc. These results suggest a novel relationship between Myc and Polycomb, wherein Myc enhances Polycomb repression in order to repress targets, and Myc suppresses Polycomb repression in order to activate targets (Goodliffe, 2006).

The first Myc-regulated gene ever identified was c-myc itself. The mechanism of autorepression has remained elusive, and the present study offers new insight into this feedback regulatory loop. myc autorepression is conserved from mammals to flies and that it requires the Pc complex. The myc autoregulation loop is frequently disrupted in cancer cells, and furthermore, it has been suggested that gene repression correlates better with Myc biological activity than does gene activation. The data suggest that autorepression and general repression by Myc are mediated by the same mechanism and that both are dependent on the PcG. Indeed, dMyc repressed genes have the hallmark chromatin modification of Pc-repressed genes. Members of the PcG have previously been implicated in cancer, including Bmi-1 (homologous to Psc), which cooperates with Myc in lymphomagenesis and represses expression of the p16 CDK inhibitor. However, no previous connection has been made between general Myc-mediated repression and the PcG. The large chromosomal domain surrounding the dmyc locus that is repressed in concert with dmyc itself is consistent with a PcG-mediated mechanism, since repression by Pc is known to act over long distances. Interestingly, repression within this domain is not absolute, since some interspersed genes can resist repression or even be activated. The possibility cannot be excluded that each of the genes in the domain is independently repressed by elevated dMyc expression, but their proximity to dmyc itself seems more consistent with a regional effect (Goodliffe, 2006).

An unexpected outcome of these studies was the striking observation that one-third of the genes that score as dMyc-activated in early stage embryos were also scored as repressed by Pc, since ablation of Pc by RNAi activated the genes to a similar extent as transgenic dmyc overexpression. Similarly, approximately one-half of the Pc repressed genes were also activated by transgenic dmyc overexpression. The overlap in these two gene sets is statistically highly significant and suggests a mechanistic overlap in the gene response. Since dmyc overexpression was provided via transgene, whereas ablation of Pc was achieved by RNAi, the overlap in gene response is unlikely to be a consequence of experimental manipulation. It has not yet been determined if this response is a direct effect of either dMyc or Pc binding to the corresponding genes. Nevertheless, the microarray data suggest that, at the minimum, the two pathways converge on a common cellular network (Goodliffe, 2006).

For both dMyc-activated and -repressed genes, the Polycomb complex provides an essential context for Myc regulation, but the direction of that regulation depends on Myc itself and the nature of its interaction with a particular target. In the simplest view, Myc repression might work by enhancing Pc's generally negative effects on transcription, whereas it appears to activate other genes by opposing those same effects (Goodliffe, 2006).

PRE-mediated bypass of Two Su(Hw) insulators targets PcG proteins to a downstream promoter

Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. In contrast, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter (Comet, 2006).

The present work suggests two complementary mechanisms for promoter silencing by PcG proteins. (1) The data show directly that PcG proteins recruited at a PRE can spread over several kilobases along the flanking chromatin. Therefore, promoters located within short distances from PREs might be silenced by PcG spreading and interference with the transcription machinery. However, PcG spreading induced by the Ubx PRE did not extend beyond few kilobases in these experiments, and ChIP on chip also showed limited extension of PcG protein binding from known PREs. This limited spreading might depend on genomic sequences or proteins bound to them that might attenuate chromatin association of PcG proteins. Thus, spreading alone might not be sufficient for silencing promoters located several tens of kilobases away, as in the case of the Ubx gene, suggesting that additional mechanisms allow PcG proteins to gain access at distant promoters. It was found that pairing of two Su(Hw) insulators can induce promoter association of PcG complexes without PcG-mediated coating of the insulated domain. (2) This suggests an additional mechanism of PRE-dependent promoter silencing, whereby PREs located at large distances from their promoters might contact them via looping of intervening domains. This looping might be favored by natural regulatory elements present at these loci, which might play a role similar to the pair of Su(Hw) insulators used in this study (Comet, 2006).

The endogenous distribution of PcG proteins might reflect spreading from a PRE into the flanking genomic region as well as their ability to bypass insulators. At the two endogenous target loci en and ph, where PREs are located in the promoter region, the distribution of PC and PH suggests spreading from the PREs. The distribution of PC and PH was characterized at Ubx, a locus where the PRE is over 20 kb upstream from the Ubx promoter. In addition to Ubx, this region contains the bxd locus, driving the production of noncoding transcripts. PC and PH binding shows a peak at the bxd transcription start site downstream to the PRE, in addition to the previously described peaks corresponding to the PRE and the Ubx promoter. Furthermore, binding of PH and PC drops between the bxd peak and the Ubx promoter and rises again at the promoter. This distribution is consistent with spreading from the PRE for short-distance chromatin silencing, and direct targeting of PRE bound PcG complexes to the downstream promoter to drive silencing over larger distances (Comet, 2006).

The sharp transitions in PcG protein binding detected at insulators are surprising, especially considering that the PRC1 complex is larger than 1 MDa, a size equivalent to several nucleosomes. The block in PcG spreading might depend on a physical barrier imposed by protein complexes tightly bound to the insulator. The bypass of the insulated domain might be explained by topological features imposed by insulators on three-dimensional chromatin folding. The Su(Hw) and Mod(mdg4) proteins that regulate the Su(Hw) insulator are organized into discrete “insulator bodies” in the cell nucleus. PcG proteins are also organized into “PcG bodies” that might be the sites of PRE-mediated silencing. A single Su(Hw) insulator located near a PRE might thus exclude the downstream domain from the PcG body associated to the PRE. A second insulator paired with the first one in the insulator body might bring the downstream promoter at the PRE-associated PcG body, while excluding from it the intervening chromatin domain. This type of regulation of three-dimensional chromatin folding by insulator elements might modulate gene expression at a number of loci in Drosophila and other species (Comet, 2006).

Systematic protein location mapping reveals five principal chromatin types in Drosophila cells

Chromatin is important for the regulation of transcription and other functions, yet the diversity of chromatin composition and the distribution along chromosomes are still poorly characterized. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, this study shows that the genome is segmented into five principal chromatin types (see Chromatin types are characterized by distinctive protein combinations and histone modifications) that are defined by unique yet overlapping combinations of proteins and form domains that can extend over > 100 kb. A repressive chromatin type was identified that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and H3K36 methylation and regulate distinct classes of genes. Finally, evidence is provided that the different chromatin types help to target DNA-binding factors to specific genomic regions. These results provide a global view of chromatin diversity and domain organization in a metazoan cell (Filion, 2010).

By systematic integration of 53 protein location maps this study found that the Drosophila genome is packaged into a mosaic of five principal chromatin types, each defined by a unique combination of proteins. Extensive evidence demonstrates that the five types differ in a wide range of characteristics besides protein composition, such as biochemical properties, transcriptional activity, histone modifications, replication timing, DNA binding factor (DBF) targeting, as well as sequence properties and functions of the embedded genes. This validates the classification by independent means and provides important insights into the functional properties of the five chromatin types (Filion, 2010).

Identifying five chromatin states out of the binding profiles of 53 proteins comes out as a surprisingly low number (one can form approximately 1016 subsets of 53 elements). It is emphasized that the five chromatin types should be regarded as the major types. Some may be further divided into sub-types, depending on how fine-grained one wishes the classification to be. For example, within each of the transcriptionally active chromatin types, promoters and 3' ends of genes exhibit (mostly quantitative) differences in their protein composition and thus could be regarded as distinct sub-types. However, these local differences are minor relative to the differences between the five principal types that are described in this study. It cannot be excluded that the accumulation of binding profiles of additional proteins would reveal other novel chromatin types. It is also anticipated that the pattern of chromatin types along the genome will vary between cell types. For example, many genes that are embedded in 'BLACK' chromatin (defined in Kc167 cells) are activated in some other cell types. Thus, the chromatin of these genes is likely to switch to an active type (Filion, 2010).

While the integration of data for 53 proteins provides substantial robustness to the classification of chromatin along the genome, a subset of only five marker proteins (histone H1, PC, HP1, MRG15 and BRM), which together occupy 97.6% of the genome, can recapitulate this classification with 85.5% agreement. Assuming that no unknown additional principal chromatin types exist in some cell types, DamID or ChIP of this small set of markers may thus provide an efficient means to examine the distribution of the five chromatin types in various cells and tissues, with acceptable accuracy (Filion, 2010).

Previous work on the expression of integrated reporter genes had suggested that most of the fly genome is transcriptionally repressed, contrasting with the low coverage of PcG and HP1-marked chromatin. BLACK chromatin, which consists of a previously unknown combination of proteins and covers about half of the genome, may account for these observations. Essentially all genes in BLACK chromatin exhibit extremely low expression levels, and transgenes inserted in BLACK chromatin are frequently silenced, indicating that BLACK chromatin constitutes a strongly repressive environment. Importantly, BLACK chromatin is depleted of PcG proteins, HP1, SU(VAR)3-9 and associated proteins, and is also the latest to replicate, underscoring that it is different from previously characterized types of heterochromatin (identified as BLUE and GREEN chromatin in this study) (Filion, 2010).

The proteins that mark BLACK domains provide important clues to the molecular biology of this type of chromatin. Loss of Lamin (LAM), Effete (EFF) or histone H1 causes lethality during Drosophila development. Extensive in vitro and in vivo evidence has suggested a role for H1 in gene repression, most likely through stabilization of nucleosome positions. The enrichment of LAM points to a role of the nuclear lamina in gene regulation in BLACK chromatin, consistent with the long-standing notion that peripheral chromatin is silent. Depletion of LAM causes derepression of several LAM-associated genes (Shevelyov, 2009), while artificial targeting of genes to the nuclear lamina can reduce their expression, suggesting a direct repressive contribution of the nuclear lamina in BLACK chromatin. D1 is a little-studied protein with 11 AT-hook domains. Overexpression of D1 causes ectopic pairing of intercalary heterochromatin (Smith, 2010), suggesting a role in the regulation of higher-order chromatin structure. SUUR specifically regulates late replication on polytene chromosomes (Zhimulev, 2003), which is of interest because BLACK chromatin is particularly late-replicating. EFF is highly similar to the yeast and mammalian ubiquitin ligase Ubc4 that mediates ubiquitination of histone H3, raising the possibility that nucleosomes in BLACK chromatin may carry specific ubiquitin marks. These insights suggest that BLACK chromatin is important for chromosome architecture as well as gene repression and provide important leads for further study of this previously unknown yet prevalent type of chromatin (Filion, 2010).

In RED and YELLOW chromatin most genes are active, and the overall expression levels are similar between these two chromatin types. However, RED and YELLOW chromatin differ in many respects. One of the conspicuous distinctions is the disparate levels of H3K36me3 at active transcription units. This histone mark is thought to be laid down in the course of transcription elongation and may block the activity of cryptic promoters inside the transcription unit. Why active genes in RED chromatin lack H3K36me3 remains to be elucidated (Filion, 2010).

The remarkably high protein occupancy in RED chromatin suggests that RED domains are 'hubs' of regulatory activity. This may be related to the predominantly tissue-specific expression of genes in RED chromatin, which presumably requires many regulatory proteins. It is noted that the DamID assay integrates protein binding events over nearly 24 hours, so it is likely that not all proteins bind simultaneously; some proteins may bind only during a specific stage of the cell cycle. It is highly unlikely that the high protein occupancy in RED chromatin originates from an artifact of DamID, e.g. caused by a high accessibility of RED chromatin. First, all DamID data are corrected for accessibility using parallel Dam-only measurements. Second, several proteins, such as EFF, SU(VAR)3-9 and histone H1 exhibit lower occupancies in RED than in any other chromatin type. Third, ORC also shows a specific enrichment in RED chromatin, even though it was mapped by ChIP, by another laboratory and on another detection platform. Fourth, DamID of Gal4-DBD does not show any enrichment in RED chromatin (Filion, 2010).

RED chromatin resembles DBF binding hotspots that were previously discovered in a smaller-scale study in Drosophila cells. Discrete genomic regions targeted by many DBFs have recently also been found in mouse ES cells , hence it is tempting to speculate that an equivalent of RED chromatin may also exist in mammalian cells. Housekeeping and dynamically regulated genes in budding yeast also exhibit a dichotomy in chromatin organization which may be related to the distinction between YELLOW and RED chromatin. The observations that RED chromatin is generally the earliest to replicate and strongly enriched in ORC binding, suggest that this chromatin type may be not only involved in transcriptional regulation but also in the control of DNA replication (Filion, 2010).

This analysis of DBF binding indicates that the five chromatin types together act as a guidance system to target DBFs to specific genomic regions. This system directs DBFs to certain genomic domains even though the DBF recognition motifs are more widely distributed. It is proposed that targeting specificity is at least in part achieved through interactions of DBFs with particular partner proteins that are present in some of the five chromatin types but not in others. The observation that yeast Gal4-DBD binds its motifs with nearly equal efficiency in all five chromatin types suggests that differences in compaction among the chromatin types represent overall a minor factor in the targeting of DBFs. Although additional studies will be needed to further investigate the molecular mechanisms of DBF guidance, the identification of five principal types of chromatin provides a firm basis for future dissection of the roles of chromatin organization in global gene regulation (Filion, 2010).

P-element homing is facilitated by engrailed polycomb-group response elements in Drosophila melanogaster

P-element vectors are commonly used to make transgenic Drosophila and generally insert in the genome in a nonselective manner. However, when specific fragments of regulatory DNA from a few Drosophila genes are incorporated into P-transposons, they cause the vectors to be inserted near the gene from which the DNA fragment was derived. This is called P-element homing. The minimal DNA fragment that could mediate homing was mapped to the engrailed/invected region of the genome. A 1.6 kb fragment of engrailed regulatory DNA that contains two Polycomb-group response elements (PREs) was sufficient for homing. Flies that contain a 1.5 kb deletion of engrailed DNA (enδ1.5) in situ, including the PREs and the majority of the fragment that mediates homing. Remarkably, homing still occurs onto the enδ1.5 chromosome. In addition to homing to en, P[en] inserts near Polycomb group target genes at an increased frequency compared to P[EPgy2], a vector used to generate 18,214 insertions for the Drosophila gene disruption project. It is suggested that homing is mediated by interactions between multiple proteins bound to the homing fragment and proteins bound to multiple areas of the engrailed/invected chromatin domain. Chromatin structure may also play a role in homing (Cheng, 2012).

Previous results indicated that a 2.6 kb fragment of en DNA, extending from -2.4 kb upstream through +188bp of the en transcription unit could mediate P-element homing to the en/inv domain. This study showa that a 1.6kb fragment that extends from -2.0 kb through -0.4 kb is sufficient for homing. It is suggested that homing is mediated by a complex array of proteins and/or chromatin structure (Cheng, 2012).

PcG proteins are thought to mediate long-range chromatin interactions at the Bithorax complex, between the Bithorax and Antennapedia complexes, and also between PcG targets on the same chromosome arm. It is noted that one study suggests that the interactions at the Bithorax complex are not mediated by PREs, but by closely associated insulator elements. Biochemical studies show that PcG protein complexes can interact in vitro. The current results suggest that PREs play a role in P[en] homing: 1) deletion of the 181-bp PRE in the transgene decreases the homing frequency and 2) P[enHSP1] insertions occur in PcG-regulated genes at a higher frequency than P{EYgy2} insertions. It is noted that both the eve and Bithorax homing fragments are thought to be insulator elements. The en homing fragment is located just upstream of the en promoter and it is considered unlikely to be an insulator. However, the insulator proteins GAGA Factor, CTCF, and Mdg4 are associated with this DNA in embryos. Therefore, it is possible that the homing fragment has some of the same properties as insulators (Cheng, 2012).

In a previous study it was found that embryonic lacZ expression from P[en3R] (called P[en1] in that study) occurred in stripes at a much higher frequency than with the enhancer trap P[lacW]. It was hypothesized that P[en3R] caused selective insertion of P[en3R], not just to en/inv, but also to many genes expressed in stripes. It is known now that both the en promoter and en PREs (or sequences closely associated with them) mediate interactions with distant enhancers. Thus, one reason for the enriched number of lacZ stripe patterns with P[en3R] could be its ability to work with distant enhancers. In support of this, when P[en3R] is inserted up to 140 kb and 5 transcription units away from the nearest en stripe enhancer (either upstream or downstream), P[en3R]-encoded lacZ is still expressed in en-like stripes. In contrast, when P[lacW] is inserted about 45kb upstream of the nearest en stripe enhancer, into tou, the gene adjacent to en, P[lacW]-encoded lacZ is not expressed in stripes. In fact, the PREs in P[en3] facilitate long-distance interactions with enhancers in many different regions of the genome. It is suggested that the high percentage of striped lacZ expression from P[en3R] insertions is due both to the ability of the en promoter and PREs to act with distant enhancers and also to increased insertion into PcG-regulated genes, many of which are developmental regulators and expressed in stripes (Cheng, 2012).

Surprisingly, enδ1.5 flies are homozygous viable and fertile. En expression appears normal in these flies. It is suggested that these PREs are redundant with inv PREs and that the en/inv H3K27me3 domain is not disrupted in enδ1.5 flies. Interestingly, P[en] homing still occurs in enδ1.5 flies. These data suggest that homing is not mediated solely by self-self interactions between the homing fragment in P[en] and the genomic homing fragment (Cheng, 2012).

It is suggested that P[en] homing is mediated by the interaction of multiple proteins bound to the en fragment within P[en] and proteins bound to the en genomic region, and that these interactions are facilitated by the H3K27me3 mark characteristic of PcG target genes. Note that since P[en] insertions into the en/inv target occur much more frequently than into other PcG-target genes, protein-protein interactions, specific for the en/inv region must be involved in homing. The smallest fragment that could mediate homing was 1.6kb, a size capable of binding many proteins. This suggests that P[en] homing is not caused by a binding of a single protein or protein complex. However, it is also possible that the 1.6 kb fragment is needed to form the chromatin structure that facilitates homing. Finally, it is suggested that P[en] interacts with multiple proteins bound to the en/inv domain, since homing still occurs in enδ1.5, where the majority of the genomic homing fragment has been deleted (Cheng, 2012).

P-element homing occurs in germ cells. En is not expressed in these cells. Recent results indicate that the H3K27me3 modification is present at many developmental loci in germ cells. P[en] homing suggests that, in addition to the H3K27me marks, there are specific proteins bound to en DNA in the germ cells. These proteins could be present to keep en silenced, or perhaps they are there to facilitate rapid initiation of en transcription in the embryo (Cheng, 2012).

Three-dimensional folding and functional organization principles of the Drosophila genome

Chromosomes are the physical realization of genetic information and thus form the basis for its readout and propagation. This study presents a high-resolution chromosomal contact map derived from a modified genome-wide chromosome conformation capture approach applied to Drosophila embryonic nuclei. The data show that the entire genome is linearly partitioned into well-demarcated physical domains that overlap extensively with active and repressive epigenetic marks. Chromosomal contacts are hierarchically organized between domains. Global modeling of contact density and clustering of domains show that inactive domains are condensed and confined to their chromosomal territories, whereas active domains reach out of the territory to form remote intra- and interchromosomal contacts. Moreover, specific long-range intrachromosomal contacts between Polycomb-repressed domains were systematically identified. Together, these observations allow for quantitative prediction of the Drosophila chromosomal contact map, laying the foundation for detailed studies of chromosome structure and function in a genetically tractable system (Sexton, 2012).

A simplified Hi-C procedure. a variation of the chromosome conformation capture (3C) technique, was developed for minimally biased profiling of chromosomal contacts on a genomic scale. Using this technique, chromosomal architecture was comprehensively and accurately characterized in Drosophila melanogaster embryonic nuclei. The derived chromosomal contact map relaxes the classical trade-off between coverage and resolution in the study of chromosome structure. The data provide sufficient resolution to observe local contact profiles derived from Chromosome conformation capture on chip (4C) and consistently deliver such resolution for essentially any genomic locus. The effective resolution limitations of the map depend on the features being studied. Demarcation of physical domains can be achieved within a precision of one or a few DpnII fragments (i.e., of ~1 kb), as many fragments with high expected contact probability contribute to their identification. In contrast, detection of long-range contacts with statistical confidence greatly depends on their absolute intensity compared to the background, which decays significantly with genomic separation. For example, based on the current sequencing depth, the decay in background contact probability with genomic distance, and the average DpnII restriction site density, it was estimated that a contact with 4-fold enrichment over the background could be confidently detected at a resolution of ~10 kb for genomic separations of 100 kb, a resolution of ~30 kb for genomic separations of 1 Mb, and ~125 kb for interchromosomal coassociations. Regardless of these considerations, and despite the fact that the experiment assayed a large and heterogeneous set of nuclei, the derived Hi-C map reveals a clear structure and allows for multiple chromosome folding principles to be explored systematically. The implications of the Drosophila map are therefore far reaching, and the analysis presented in this study can be viewed as a baseline on which further efforts directed to understand genomic and epigenomic patterns at particular cell states or genetic backgrounds can be developed (Sexton, 2012).

The Hi-C map is rich in local and global structure, describing contact frequencies that vary within five orders of magnitude. It was desirable to explain the distributions of contact frequencies in the map using quantitative models based on the simplest principles, and any progressive increases in model complexity were justified by proven discrepancies between the data and a simpler version of the model. One of the most remarkable patterns observed in the map was the partitioning of chromosomes into physical domains, which showed up in the matrix as diagonal submatrices with high contact intensities. A quantitative probabilistic model was used to show that contacts inside these domains are governed by a distinct regime that cannot be attributed to denser contacts or more compact chromosomal structure alone. Further analysis showed that physical domains form the backbones of a hierarchical chromosome structure, as the contact intensities between genomic elements are mostly determined by the identities of the domains containing them, rather than the element's location within the domain. Previous lower-resolution exploration of human chromosome architecture identified a global power law decay of contact frequency with genomic separation and used this to propose a fractal globule model of chromosome folding. Although this study observe a roughly similar global decay curve for Drosophila chromatin, higher-resolution analysis of contact decays within the context of physical domains challenges this model and suggests that, in scales of 10-100 kb, the predominant factor affecting chromosome folding is the modular organization. This promotes hierarchical chromosomal organization as an attractive paradigm to facilitate functional epigenetic organization but leaves open questions about the scales at which it may be observed in different genomes that vary significantly in size and gene density (Sexton, 2012).

Remarkably, the physical domains inferred from the Hi-C contact map were compatible with numerous linear epigenetic profiles describing enrichment for histone modification or DNA-binding factors. Thus the physical domains, which are key fundamental units of chromosome folding, are reflected and possibly caused by their underlying epigenetic marks. Large silent chromosomal regions that are either enriched with repressive histone marks (H3K27me3 or HP1/H3K9me2) or void of any detectable epigenetic enrichment were shown to form modular chromosomal entities, which are interspersed with small domains associated with active chromosomal marks. By analyzing the epigenomic marks at the borders of physical domains, it was observed that a transition in transcriptional activity (as indicated by peaks of H3K4me3) is sometimes sufficient to disturb the compaction of flanking repressive chromatin domains. This may result in the formation of 'punctuated' repressed domains, with active genes forming 'passive' physical boundaries. However, in most cases, it was found that insulator proteins, particularly CP190 and polytene chromosome structure. These findings appear to extend the structural function of Chromator to diploid embryonic nuclei. By providing an architectural context to epigenomic chromatin domains, the Hi-C map thus provides a reference epigenomic model, directing future efforts for analyses of the correlations between hundreds of measured linear epigenomic profiles (Sexton, 2012).

Chromosomes clearly fold in a complicated, heterogeneous regime. In order to make any reasoned claims about the significance of previously reported individual cases of long-range chromatin interactions, it is important to first understand the basic principles of what defines 'standard' folding of a chromosome fiber. This Hi-C dataset allows formulation and testing of hypotheses on chromatin folding with progressively more complex quantitative models. First, heterogeneity in contact density was accounted for, facilitating identification of physical chromatin modules and their hierarchical pattern of folding. Next, it was possible to group physical domains into two clusters (annotated postfactum as active or inactive) based on their intrachromosomal contacts and to generally describe interdomain contacts as those within or between clusters. This supported and extended previous findings on the relationship between transcriptional activity and position within chromosome territories. Although the combined model explains much of the chromosome folding behavior, specific long-range chromatin interactions were still apparent. One group of functional long-range interactions that has already been investigated and is clearly visible in the Hi-C map associates PcG-regulated genes that co-occupy Polycomb bodies (Sexton, 2012).

In summary, this Hi-C study has provided a fundamental chromatin interaction map framework, providing the basis for mathematical models to assess the link between chromosome structure and function. The characterization of hierarchically folded discrete physical modules, which may be epigenetically defined, forms a hitherto unappreciated base from which more complicated chromosome topologies can arise. It is posited that this and future Hi-C datasets, combined with specific perturbation experiments, will inform more sophisticated mathematical models of chromosome folding, forming a foundation for new important insights into what shapes nuclear structure and how this in turn affects genome function (Sexton, 2012).

Regulation of Polycomb group genes Psc and Su(z)2 in Drosophila melanogaster

Certain Polycomb group (PcG) genes are themselves targets of PcG complexes. Two of these constitute the Drosophila Psc-Su(z)2 locus, a region whose chromatin is enriched for H3K27me3 and contains several putative Polycomb response elements (PREs) that bind PcG proteins. To understand how PcG mechanisms regulate this region, the repressive function of the PcG protein binding sites was analyzed using reporter gene constructs. It was found that at least two of these are functional PREs that can silence a reporter gene in a PcG-dependent manner. One of these two can also display anti-silencing activity, dependent on the context. A PcG protein binding site near the Psc promoter behaves not as a silencer but as a down-regulation module that is actually stimulated by the Pc gene product but not by other PcG products. Deletion of one of the PREs increases the expression level of Psc and Su(z)2 by twofold at late embryonic stages. Evidence is presented suggesting that the Psc-Su(z)2 locus is flanked by insulator elements that may protect neighboring genes from inappropriate silencing. Deletion of one of these regions results in extension of the domain of H3K27me3 into a region containing other genes, whose expression becomes silenced in the early embryo (Park, 2012).

Continued: Polycomb Targets of activity part 2/2

Polycomb: Biological Overview | Evolutionary Homologs | Protein interactions | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.