The expression of the zeste gene varies through the life cycle of the fly. Its transcription is most abundant in maternal RNA, declines to very low levels during larval growth, but rises again in late third instar larvae and pupae. Nearly ubiquitous expression of zeste is found in late embryos and first instar larvae, but disappears almost completely except in brain and gonads by third instar larva. Shortly before pupation expression rises again in imaginal discs, Malpighian tubules, and salivary glands, and again becomes nearly ubiquitous in pupae. zeste continues to be expressed in adult brain and gonads. Wild-type salivary gland chromosomes contain about 60 strong bands of Zeste immunofluorescence at specific cytological locations. After heat induction of larvae containing the hs-zeste gene, many hundreds of bands appear. Such results suggest the involvement of zeste in the expression of a wide variety of genes at different developmental stages (Pirrotta, 1988).

Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos

Polycomb group (PcG) and trithorax group (trxG) proteins are conserved chromatin factors that regulate key developmental genes throughout development. In Drosophila, PcG and trxG factors bind to regulatory DNA elements called PcG and trxG response elements (PREs and TREs). Several DNA binding proteins have been suggested to recruit PcG proteins to PREs, but the DNA sequences necessary and sufficient to define PREs are largely unknown. This study used chromatin immunoprecipitation (ChIP) on chip assays to map the chromosomal distribution of Drosophila PcG proteins, the N- and C-terminal fragments of the Trithorax (TRX) protein and four candidate DNA-binding factors for PcG recruitment. In addition, histone modifications associated with PcG-dependent silencing and TRX-mediated activation were mapped. PcG proteins colocalize in large regions that may be defined as polycomb domains and colocalize with recruiters to form several hundreds of putative PREs. Strikingly, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding, suggesting that recruiter proteins have a dual function in activation as well as silencing. One major discriminant between activation and silencing is the strong binding of Pleiohomeotic (PHO) to silenced regions, whereas its homolog Pleiohomeotic-like (PHOL) binds preferentially to active promoters. In addition, the C-terminal fragment of TRX (TRX-C) showed high affinity to PcG binding sites, whereas the N-terminal fragment (TRX-N) bound mainly to active promoter regions trimethylated on H3K4. The results indicate that DNA binding proteins serve as platforms to assist PcG and trxG binding. Furthermore, several DNA sequence features discriminate between PcG- and TRX-N-bound regions, indicating that underlying DNA sequence contains critical information to drive PREs and TREs towards silencing or activation (Schuettengruber, 2008; tull text of article).

The genome-wide mapping of PcG factors, TRX, their associated histone marks, and potential PcG recruiter proteins in Drosophila embryos revealed several important features. First, similar to the PcG distribution in Drosophila cell lines, PcG proteins strongly colocalize and form large domains containing multiple binding sites. Second, the N-terminal and C-terminal fragments of TRX show different binding affinities to repressed and active chromatin. The N-terminal fragment of TRX has low affinity to PcG binding sites but is strongly bound to thousands of active promoter regions that are trimethylated on H3K4, whereas the C-terminal fragment of TRX only showed high binding affinity to PcG binding sites. Third, the majority of PcG recruiter binding sites are associated with H3K4me3 and TRX-N foci and not with PH binding. The binding ratio between the PHO protein and its homolog PHOL is a major predictive feature of PcG versus TRX recruitment. Finally, supervised and unsupervised sequence analysis methods led to the identification of sequence motifs that discriminate between most of the PcG and TRX binding sites, but these motifs are likely to be working jointly, and none of them seems to drive recruitment by itself (Schuettengruber, 2008).

To date, PREs have been only characterized in Drosophila. These elements are not defined by a conserved sequence, but include several conserved motifs, which are recognized by known DNA binding proteins like GAGA factor (GAF), Pipsqueak (PSQ), Pleiohomeotic and Pleiohomeotic-(like) (PHO and PHOL), dorsal switch protein (DSP1), Zeste, Grainyhead (GH), and SP1/KLF. The genomic profiles provide a comprehensive view on the potential role of these factors in the establishment of PcG domains (Schuettengruber, 2008).

The presence of PHO at all PREs indicates that PHO is a crucial determinant of PcG-mediated silencing, consistent with earlier analysis on one particular PRE. On the other hand, PHOL and Zeste were bound at a small subset of PREs. Zeste was previously shown to be necessary for maintaining active chromatin states at the Fab-7 (Frontabdominal-7) PRE/TRE. Therefore, Zeste and PHOL may primarily assist transcription rather than PcG-mediated silencing. GAF and DSP1 resemble PHO as they bind to many (albeit less than PHO) PREs as well as to active promoters. Supervised DNA motif analysis indicated a higher density of GAF, DSP1, and PHO binding sites at PREs as compared to other bound regions at non-PH sites. This suggests that cooperative binding of these proteins may provide a platform for PcG protein binding. Moreover, GAF may act by inducing chromatin remodeling to remove nucleosomes, since the regions bound by PcG proteins show a characteristic dip in H3K27me3 signal that has been attributed to the absence of nucleosomes in those regions. These nucleosome depletion sites are the places wherein histone H3 to H3.3 replacement takes place. Indeed, several of the Zeste-bound regions and GAGA binding sequences were shown to localize to peaks of H3.3, suggesting the possibility that GAF may recruit PcG components to PHO-site-containing PREs as well as recruit TRX to promoters via nucleosome disruption (Schuettengruber, 2008).

In addition to an increased density of motifs for GAF, PHO, and PHOL, unsupervised spatial cluster analysis identified specific motifs that distinguish the PH sites from the K4me3 cluster. Although the identity of the factors binding to these motifs is unknown, this suggests that the DNA sequence of PREs contains much of the information needed to recruit PcG proteins and to define silent or active chromatin states. With this distinction, it may be possible to develop an algorithm to faithfully predict the genomic location of PREs. Earlier attempts to predict PREs in the fly genome have made progress toward this goal, but they are still far from reaching the required sensitivity and specificity. The use of a sequence analysis pipeline that is not dependent on prior knowledge was demonstrated here to generate new discriminative motifs with a potential predictive power. The unique genomic organization of PcG domains may suggest that the genome is using, not only local sequence (high-affinity transcription factor binding sites located at the binding peaks) information to determine PREs, but also integration of regional sequence information (stronger affinity on 5 kb surrounding PREs). Using such regional information to predict PREs may break the current specificity and sensitivity barriers (Schuettengruber, 2008).

ChIP on chip data showed that PHO binding comes in two distinct flavors. In one class of target sites, PHO binding coincides with PH sites within PC domains, whereas outside these domains, it is largely colocalized with PHOL, TRX-N, and H3K4me3 . PHOL binding was weaker at PH sites and was mainly present along with marks associated with gene activation. Quantitative ChIP assays revealed that PH, PHO, and PHOL were bound in PREs/TSS of their target genes in both ON and OFF states, but the ON state was marked by a decrease in PH binding and a corresponding increase in PHOL levels, whereas the OFF state was characterized by an increase in both PH and PHO binding levels (Schuettengruber, 2008).

Chromatin at the Ubx TSS, the bx PRE, and the bxd PRE (the same primers were used in the current study) by comparing haltere/third leg imaginal discs (ON state) with wing imaginal discs (OFF state). A 50% reduction was found of PH binding levels at the bx PRE, a minor decrease at bxd, and no change in the Ubx TSS. ChIP experiments demonstrated a 50% decrease in PH levels at bx PRE and at the Ubx TSS and a minor decrease at bxd PRE when comparing haltere/third leg imaginal discs to eye imaginal discs. A slight decrease was observed in the levels of PHO in haltere/third leg disc (ON state) as compared to eye imaginal discs (OFF state) at the bx and bxd PRE, whereas another study did not see differences in the levels of PHO. The most likely explanation for these discrepancies is that the peripodal membrane cells of the wing imaginal discs express Ubx, whereas all cells silence this gene in eye imaginal discs (Schuettengruber, 2008). pho1 mutant eye discs, the absence of PHO causes derepression of the homeotic genes Ubx and Antp. However, the expression levels in pho1 mutants are still much weaker compared to tissues where these genes are normally expressed. This low degree of activation could be explained by compensatory binding of PHOL to the PHO sites in order to maintain PcG-mediated silencing, even if the PHOL-dependent rescue function is incomplete as pho1 mutants die as pharate adults. PHO and PHOL have indeed been described as redundant in their role in PcG-mediated silencing since they bind to the same DNA sequence motif in vitro. However, out of the 1,757 places wherein both PHO and PHOL were significantly bound, only 807 shared the same local maxima. Another 559 (32%) peaks were within 250 bp of each other. This suggests that, in vivo, these two proteins prefer slightly different sequences, with PHO more strongly attracted to PREs, whereas PHOL binds better to promoters. Moreover, PHO interacts directly with PC and PH, as well as with the PRC2 components E(z) and Esc, whereas PHOL only interacts with Esc in yeast two-hybrid assays. Stronger interactions between PHO and PcG components may stabilize PHO binding at PREs, favoring it over the binding of PHOL. It is thus possible that the primary function of PHOL is as a transcription cofactor, and that its recruitment to PREs is subsidiary to PHO (Schuettengruber, 2008).

This study reports the genome-wide distribution of TRX. This protein has been proposed to counteract PcG-mediated silencing. It has been demonstrated that TRX colocalizes with Polymerase II and elongation factors in Drosophila polytene chromosomes. They it was showm that PcG and TRX proteins bind to a PRE mutually exclusively in salivary gland chromosomes. In contrast, other studies found binding of TRX at discrete sites at PREs and promoter regions of HOX genes, and suggested that TRX coexists with PRC1 components at silent genes. This study postulated that these differences might be explained by the use of different TRX antibodies, one against the N-terminal domain and one against the C-terminal domain of TRX. Notably, the TRX protein is proteolytically cleaved into an N-terminal and a C-terminal domain, but the fate of the two moieties after cleavage has never been addressed in vivo (Schuettengruber, 2008).

Genome-wide mapping studies using the same antibody against the N-terminal fragment (TRX-N) as used previously, showed that the binding affinity of the N-terminal fragment to PREs is rather weak, whereas TRX-N binds thousands of promoter regions trimethylated on H3K4, indicating a general role of TRX-N in gene activation. In contrast, ChIP on chip profiling using an antibody against the C-terminal TRX fragment showed high binding levels at PRE/TREs, whereas binding to promoter regions (where the TRX N-terminal fragment is strongly bound) is rather weak. The strong quantitative correlation between the binding intensities of PH and TRX-C suggests that TRX-C can indeed bind to silent PcG target genes. These data are confirmed by the colocalization of PH and TRX-C at inactive Hox genes in salivary gland polytene chromosomes and in diploid cell nuclei (as seen in a combination of DNA fluorescent in situ hybridization (FISH) and immunostaining; unpublished data). Thus, PcG silencing may involve locking the C-terminal portion of TRX in an inactive state that perturbs transcription activation events. The fact that TRX is recognized by two different antibodies that recognize PREs (H3K4me3-depleted regions) or TSSs suggests that these antibodies reflect the activity state of the protein and thus represent a powerful tool to study the switching of genes between silencing and activation (Schuettengruber, 2008).

Similar to mapping studies in Drosophila cell lines, H3K27me3 also forms large domains in Drosophila embryos. These large PcG domains could provide the basis of a robust epigenetic memory to maintain gene expression states during mitosis. As previously suggested, stably bound PcG complexes at PREs may loop out and form transient contacts with neighboring chromatin, which become trimethylated on H3K27. H3K27me3 might then attract the chromodomain of the PC protein, which may be occasionally trapped at these remote sites by cross-linking mediated by the chromodomain of PC. Alternatively, PcG subcomplexes missing some of the subunits might spread from the PRE into flanking genomic regions containing H3K27me3 histones (Schuettengruber, 2008).

Although genome-wide PcG profiles in Drosophila embryos correlate well with profiles from Drosophila cell lines, it has recently been shown that PcG protein binding profiles are partially remodeled during development. Comparison of PcG target genes showed that 40% of the targets are unique. The fact that a consistent number of targets are only found in one or two of the samples indicates tissue specific PcG occupancy. Thus, although PcG proteins have been often invoked as epigenetic gatekeepers of cellular memory processes, they may be involved as well in dynamic gene regulation during fly development, similar to their function in mammalian cells (Schuettengruber, 2008).

Effects of Mutation or Deletion

The original zeste mutant (in both males and females) has a distinctly dilute eye color that grows brown with age. Transvection is the effect of regulatory elements of one copy of a target gene on the expression of a second copy of the gene on a homologously paired chromosome. Such transvection effects were first observed in the Ultrabithorax (Ubx) gene. The effect is dependent on zeste (Kaufman, 1972).

Mutations in zeste do dot affect the cis-regulation of endogenous Ubx, but expression of small Ubx promoter constructs are strongly dependent on zeste. This difference is due to redundant cis-regulatory elements in the Ubx gene, which presumably contain binding sites for factors that overlap in function with Zeste. (Laney, 1996).

Flies doubly heterozygous for GAGA (synonym: trithorax like) and Ubx exhibit larger halteres than flies mutant for Ubx alone, and, with incomplete penetrance and variable expressivity, show homeotic transformations of the haltere and postnotum into wing. When zeste mutations are crossed into this double heterozygotic background, a similar range of phenotypes is observed. However, the fraction of animals displaying the enhanced Ubx phenotype is increased 2 to 19 fold, depending on the GAGA allele used. This increase in penetrance is observed with two different zeste alleles. Therefore, mutations in zeste increase the likelihood that limiting amouts of GAGA factor and UBX will lead to reduced expression of Ubx and to homeotic transformation of haltere into wing (Laney, 1996).

The zeste1 (z1) mutation of the zeste gene produces a mutant yellow eye color instead of the wild-type red. Genetic and molecular data suggest that z1 achieves this change by altering expression of the wild-type white gene in a manner that exhibits transvection effects. There exist suppressor and enhancer mutations that modify the z1 eye color. A study has been made of those belonging to the Suppressor 2 of zeste complex [Su(z)2-C]. The Su(z)2-C consists of at least three subregions called Psc (Posterior sex combs), Su(z)2 and Su(z)2D (Distal). The products of these subregions are proposed to act at the level of chromatin. Complementation analyses predict that the products are functionally similar and interacting. The alleles of Psc define two overlapping phenotypic classes, the hopeful and hapless. The distinctions between these two classes and the intragenic complementation seen among some of the Psc alleles are consistent with a multidomain structure for the product of Psc (Wu, 1995).

The DNA-binding protein encoded by the zeste gene of Drosophila activates transcription and mediates interchromosomal interactions such as transvection. The mutant protein encoded by the zeste1 (z1) allele retains the ability to support transvection, but represses white. Similar to transvection, repression requires Zeste-Zeste protein interactions and a second copy of white, either on the homologous chromosome or adjacent on the same chromosome. Two pseudorevertants of z1 (z1-35 and z1-42) were characterized, as well as another zeste mutation (z78c) that represses white. The z1 lesion alters a lysine residue located between the N-terminal DNA-binding domain and the C-terminal hydrophobic repeats involved in Zeste self-interactions. The z78c mutation alters a histidine near the site of the z1 lesion. Both z1 pseudorevertants retain the z1 lesion and alter different prolines in a proline-rich region located between the z1 lesion and the self-interaction domain. The pseudorevertants retain the ability to self-interact, but fail to repress white or support transvection at Ultrabithorax. To account for these observations and evidence indicating that Zeste affects gene expression through Polycomb group (Pc-G) protein complexes that epigenetically maintain chromatin states, it is suggested that the regions affected by the z1, z78c, and pseudorevertant lesions mediate interactions between Zeste and the Pc-G maintenance complexes. Because the DNA-binding domain of Zeste is unaffected by the z1-35 and z1-42 mutations, the data indicate that both repression of white and transvection require interactions other than Zeste-DNA and Zeste-Zeste interactions, and that these other interactions involve the proline-rich region. It is postulated that the proline-rich region interacts with other transcription factors to mediate repression and activation and that the region affected by the z1 and z78c lesions regulates these interactions (Rosen, 1998).

In vivo analysis of Drosophila SU(Z)12 function

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Suppressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions the binding pattern to polytene chromosomes was investigated and the localization to other proteins was compared. SU(Z)12 was found to bind to about 90 specific eukaryotic sites, however, not the white locus. Staining was found at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, it was shown that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, the binding pattern of Heterochromatin Protein 1 (HP1) was analyzed and it was found that SU(Z)12 and HP1 do not co-localize (Chen, 2008).

In Drosophila, Polycomb group (PcG) proteins are negative regulators of homeotic gene expression and play an important role in maintaining silenced states during development of the fly. The regulatory regions of homeotic genes, such as Ultrabithorax (Ubx) and Abdominal-B (Abd-B), contain specific cis-regulatory elements needed for PcG complexes to mediate this silencing effect. Several such Polycomb response elements (PREs) have been identified, not only in the Antennapedia (AntpC) and Bithorax complexes (BxC) but also in the regulatory regions of many genes mainly encoding transcription factors. PREs appear to be complex DNA elements targeted by several proteins, not only PcG but also trithorax group (TrxG) proteins, proposed to have an opposing effect to PcG, i.e. to maintain an active state of homeotic genes. Recently, a genome-wide analysis of PcG targets in Drosophila identified over 200 genes that are simultanously bound by the three PcG proteins Polycomb (PC), Enhancer of zeste (E(Z)) and Posterior sex combs (PSC). However, none of these proteins bind directly to DNA but are recruited either by DNA-binding proteins bound to PREs or by specifically modified histones. The Frontabdominal-7 (Fab-7) region of the Abd-B locus, e.g., the iab-7 PRE, which is approximately 230 bp in length, contains three DNA motifs that are recognized by five proteins: Zeste (Z), GAGA factor (GAGA), Pipsqueak (PSQ), Pleiohomeotic (PHO) and Pho-like (PHO-L). Interestingly, both PcG repressors and TrxG activators appear to bind at PREs in both the repressed and the active state. Instead, transcription of non-coding RNA through the PREs has been shown to be important for initiation of the correct transcriptional status of homeotic genes during development. Earlier work suggested that PRE transcription counteracts PcG-dependent silencing of Hox genes, while more recent work show that non-coding RNA is not present in cells expressing Ubx, indicating that transcription through PREs promotes gene silencing. The silencing ability of a given PRE is, however, also strongly dependent on the genomic context, homologous pairing or proximity to other PRE sequences (Chen, 2008).

The TrxG gene zeste encodes a non-essential transcription factor, which binds to DNA not only within PREs but also in regulatory regions of many genes, e.g. the white locus. The neomorphic zeste 1 (z 1 ) mutation causes an amino acid exchange, which renders the mutated protein extremely sticky. This results in transcriptional repression of paired white + (w + ) genes in z 1 mutants. Thus, in this zeste- white interaction, the X-linked white gene, either paired in trans (e.g., in females) or in cis (e.g. in males carrying a tandem duplication of white), will be repressed, resulting in a yellow eye colour. Whenever unpaired, the white gene is not repressed by z 1 , resulting in wild-type eye pigmentation. The zeste gene is also implicated to cause transvection, i.e., the ability of regulatory elements on one chromosome to affect the expression of the homologous gene in a somatically paired chromosome. A third type of pairing sensitive effects by z 1 is found in transgenic lines, where PRE-induced silencing of a mini-white gene often is pairing dependent. However, homologous pairing is not always required for PRE-induced silencing of w + expression and in such cases the z 1 allele has no influence on the repression (Chen, 2008 and references therein).

The biochemical characterization of Drosophila PcG proteins suggests that there are at least two distinct multi-protein complexes, each containing several PcG proteins. The Polycomb repressive complex 1 (PRC1) contains PC, PSC, polyhomeotic (PH) and Sex combs extra (SCE or dRING) and some additional accessory proteins such as Z, Sex comb on midleg (SCM), and general transcription factors. A second complex (PRC2) consists of E(Z), Extra sex combs (ESC), Suppressor of zeste-12 (SU(Z)12) and NURF55 (Chromatin associated factor-1 subunit, CAF-1), as well as some accessory proteins; Polycomb-like (PCL), RPD3 and SIR2. The core proteins E(Z), ESC and SU(Z)12 are conserved both in mammals and plants. The E(Z) protein contains a SET domain which specifically methylates lysine residues of histone 3. The SU(Z)12 protein contains a zinc finger and a well conserved region called the VEFS box, which has been shown to bind to EZH2 protein (the human E(Z) homolog) and Heterochromatin protein 1α (HP1α) in mammalian cells). Recently, in vitro binding between mammalian SUZ12 and MEP50 was reported. MEP50 binds selectively to histone H2A and interacts with the arginine methyltransferase PRMT5 and H2A, mediating transcriptional repression of target genes. So far the SU(Z)12 protein has not been ascribed any specific function in the PRC2 complex, apart from increasing the ability of the E(Z) protein to tri-methylate the lysine 27 residue of histone H3 (3meK27 H3) in vitro and forming, with NURF55, the minimal nucleosome-binding module of PRC2. 3meK27 H3 is a target of the chromodomain of the PC protein in the PRC1 complex. These two main silencing complexes together with their accessory molecules seem to inhibit transcription by preventing nucleosome remodeling and, by binding to promoter regions, block the transcription initiation machinery. The precise molecular mechanisms are, however, still poorly understood (Chen, 2008).

The first Su(z)12 mutant allele was first identified as a suppressor of the zeste- white interaction and was also shown to be important for correct maintenance of the silenced state of the Ubx gene during development and to suppress position-effect variegation (PEV)). The biochemical studies of the PcG complexes have definitely linked SU(Z)12 to PcG-mediated gene silencing; however, the involvement in position effect variegation and in regulation of the white gene expression in combination with z 1 is less well investigated. To elucidate whether Su(z)12 directly regulates the white gene expression genetic interaction studies were used with Su(z)12 alleles and it could be revealed that the dominant derepression of white expression caused by Su(z)12 mutants is dependent on the repressive action of the z 1 allele, but in order to observe this derepression either the white gene has to be paired (in females) or contain insertions of transposable elements at the white gene (in males). Another way to find out whether SU(Z)12 has other functions, apart from gene silencing via PREs, is to investigate protein binding and nuclear localization and compare with binding patterns of the other PRC2 subunits. Therefore, SU(Z)12 binding to polytene chromosomes was analyzed. It was found that SU(Z)12 binds to at about 90 specific loci, however, not detectably at the white locus. Neither is any increase of 3meK27 H3 binding found at the white locus in repressed strains, indicating that other repressive mechanisms are acting at white. Results with HP1 suggest that heterochromatin silencing may play a role. Moreover, it is concluded that there is a complete co-localization between SU(Z)12 and E(Z) and that the overlap with other PcG proteins is high, indicating that SU(Z)12 in salivary gland tissue always is in complex with PRC2. However, there was a surprisingly low degree of co-localization with 3meK27 H3. In order to rule out the action of other H3-K27 methylases, somatic Su(z)12 knock-out clones were generated and it was concluded that SU(Z)12 is essential for function of the PRC2 complex in tri-methylation of lysine 27 in histone H3 in vivo (Chen, 2008).

Regulation of the white gene by zeste is complex; first, the Z1 mutant protein can form large protein aggregates that cooperatively bind to several ZBS within the regulatory region of white, second, Z protein is involved in recruiting both silencing and activating maintenance complexes (when bound to PREs) and finally the white mutants studied here contain insertions of various mobile elements, which might inflict heterochromatic properties to the white gene or interfere with dosage compensation in males. Using genetic interaction studies, this study found that Su(z)12 + is required for the Z1 mediated repression of white, but no physical binding of SU(Z)12 protein at the white locus is found in polytene chromosomes. Neither is any increased 3meK27 H3 binding there. One explanation for the derepression of white expression by Su(z)12 mutants could be that there are tissue specific differences in PcG silencing, and that in eye discs the white gene is silenced by PcG, but not in salivary glands. This is not likely, since there is no PRE at the white locus, only a set of ZBS, which is not sufficient to recruit silencing complexes. Another alternative is thus that the derepression observed in Su(z)12 mutants are secondary effects. Possibly, lower levels of SU(Z)12 can down-regulate other PcG proteins, which is accompanied by a simultaneous depletion of Z1 proteins, relieving white repression. Alternatively, Z1 can recruit still other silencing mechanisms to the white locus (Chen, 2008).

Su(z)12 mutant derepression is observed in homozygous z 1 females but only in a few white alleles in males. Insertions of mobile elements at the white locus in these mutants might superimpose heterochromatic silencing, repressing transcription further. However, these specific transposon insertions on their own accord do not visibly repress white transcription. Interestingly, a novel weak binding of HP1 at the white locus in polytene chromosomes was found in one of the strains that are derepressed by Su(z)12 mutations, suggesting that the inserted transposable element could recruit heterochromatic silencing proteins, adding to the silencing already present. The loss of one copy of the Su(var)205 gene slightly relieves this silencing. It is concluded that the z 1 repression in conjunction with transposable element insertions results in a yellow eye colour, allowing detection of the derepression caused by Su(z)12 mutants. This shows that a combination of repressive mechanisms is acting at the white gene in the studied mutants, but that PcG and heterochromatin silencing are not the main ones (Chen, 2008).

It was not possible to show any co-localization between SU(Z)12 and HP1 proteins on Drosophila polytene chromosomes (except at telomeres and the chromocenter), which is also confirmed by the binding pattern of HP1 on polytene chromosomes. This is in contrast to the in vitro findings with the mammalian counterparts, where the region between the Zinc finger and the VEFS box in human SU(Z)12 protein directly binds to mammalian HP1α in vitro. The human HP1α protein is showing a somewhat higher homology to the Drosophila HP1b protein sequence than to HP1 encoded by Su(var)205. Therefore, Drosophila HP1 may not be the functional homologue of HP1α, which could explain why no co-localization was found with SU(Z)12 and HP1 in Drosophila. It would be interesting to further study the role of HP1b in gene silencing (Chen, 2008).

Müller and coworkers reported that SU(Z)12 is essential for the binding of PRC2 to nucleosomes, and that SU(Z)12 and NURF55 together constitute the minimal nucleosome-binding complex. These two subunits alone show a better binding to nucleosomes in vitro than a complex also containing E(Z). This study has found that SU(Z)12 and E(Z) always co-localize to polytene chromosomes, and that NURF55 is also present at these sites. Since a FLAG-tagged ESC protein also completely co-localizes with E(Z) this indicates that the complete core PRC2 complex is present at these sites and is functional, and that only NURF55 can bind to chromatin without the other subunits. E(Z) and ESC have also been found to bind to the chromocenter so probably the role of SU(Z)12 as suppressor of PEV is still connected to the function of the PRC2 complex. This refutes the hypothesis that SU(Z)12 has functions outside of the PRC2 complex (Chen, 2008).

It was surprising to find that there are SU(Z)12 binding sites that show no or very weak 3meK27 H3 binding on polytene chromosomes and that there are sites that contain high levels of 3meK27 H3 where PRC2 proteins are not present. Furthermore, it was unexpected to find high levels of 3meK27 H3 in puffs, which contain actively transcribing genes. Most 3meK27 H3 is otherwise seen in interbands, which are considered to contain regulatory regions for transcriptional activation of condensed chromatin in adjacent bands. ChIP analyses have revealed that 3meK27 H3 is binding to large domains covering entire genes that are silenced, while PRC2 proteins bind to more restricted regions. In the genome wide analysis of PcG targets performed by Pirrotta and co-workers, there is a very high co-localization between PcG proteins and 3meK27 H3, and also good correlation between 3meK27 H3 and transcriptional silence. However, 3 of the 149 binding sites reported lack either PcG binding or H3 methylation mark. Probably there are more examples since the report only includes sites where all four or three of the proteins (PC, E(Z), PSC and 3meK27 H3) bind simultaneously with a twofold enrichment. Furthermore, they use cell cultures for their analysis, which might give different results compared to differentiated tissues like salivary glands. The result obtain in the current study could also be caused by physical disruption of sub-nuclear compartments (like PcG bodies) where PRC2 complexes and chromosome regions with high levels of trimethylated histones normally reside. Yet another alternative could be that a dynamic reorganization of chromatin in polytene chromosomes occurs, leading to translocation of PcG proteins. It has been shown that PcG chromosome-association profiles can change during development. Further options could be that there are other yet-unidentified HMTs that can induce 3meK27 H3 formation, or that SU(Z)12, E(Z) and ESC are subunits in larval complexes with other functions than to methylate H3 residues (Chen, 2008).

Somatic knock-out of SU(Z)12 in wing discs results in a complete abolishment of 3meK27 H3, showing the vital role of SU(Z)12 for the function of the PRC2 complex. This is in agreement with the results reported by Cao (2004) for mammalian cell lines. The Su(z)12 knock-out clones are generally very small in size, indicating a role for SU(Z)12 in cell proliferation. Twin spots show an increase in histone methylation compared to heterozygous mutant cells. Clones over-expressing SU(Z)12+ also exhibit high level of H3 methylation compared to normal cells. This implies that SU(Z)12 is rate limiting and that SU(Z)12 over-expression facilitates assembly of PRC2 subunits, stabilizes existing PRC2 complexes or in some way augments HMT activity of these. It is known that most PcG proteins bind to some PcG genes, e g. Psc/Su(z)2 and ph (Schwartz, 2006) and this study also sees binding of SU(Z)12 at both these loci. Therefore, an alternative explanation could be that increased SU(Z)12 levels induces positive feedback, activating transcription of genes encoding PRC2 subunits. Indeed, examples of such stimulatory effects between PcG genes have been found. esc and E(z) mutants significantly decrease Pc, Psc and ph transcription levels indicating a stimulatory effect. Similarly, ASX, E(PC) and PCL positively regulates Psc transcription, while PH, PC and PSC negatively regulate Psc (Chen, 2008).

In humans the Suz12 locus is a region of frequent translocation in endometrial stromal sarcomas, which generates a fusion protein between JAZF1 and SUZ12. This suggests that over-expression of SUZ12 fusion protein has a causal role in the pathogenesis of this tumour (Koontz, 2001). Furthermore, SU(Z)12 is up-regulated in various other tumours (Kirmizis, 2003). The finding that over-expression of SU(Z)12 increases the HMT activity, possibly by inducing a positive feedback regulation of the other PRC2 genes, emphasize the importance of maintaining a balance between activity and silencing of e.g. tumour suppressor genes (Chen, 2008).


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zeste: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 February 2011

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