TRX mRNAs encoding the smaller isoform (missing exon II) are expressed both maternally and zygotically, while the larger form is expressed only zygotically.

Zygotically expressed TRX mRNAs encoding the larger protein isoform are initially expressed at stage 4, before cellularization is complete. The pattern overlaps the expression domains of the BX-C genes Ubx, abd-A and Abd-B. Expression is higher in the ventral than the dorsal part of the embryo. By stage 5, strong staining is evident in a long ventral band corresponding to the prospective mesoderm. This pattern is transient and evolves into a broader expression domain encompassing the entire germ band during the extended germ band stage (Stassen, 1995). In terms of the five TRX mRNAs, transcript M is maternally expressed. Zygotic expression begins at stage 4 of the syncytial blastoderm, initially confined to the ventral region fated to become mesoderm and later developing into four pair-rule like stripes spreading dorsally. The predominant mRNA here is ME, expressed in the mesoderm. In terms of late expression the L RNA isoform is the main component and is expressed in the ventral cord (Sedkov, 1995).

Tissue-specific TAFs counteract Polycomb to turn on terminal differentiation


Male germ cells differentiate from adult stem cell precursors, first proliferating as spermatogonia, then converting to spermatocytes, which initiate a dramatic, cell type–specific transcription program. In Drosophila, five testis-specific TAF homologs (tTAFs) encoded by the can, sa, mia, nht, and rye genes are required for meiotic cell cycle progression and normal levels of expression in spermatocytes of target genes involved in postmeiotic spermatid differentiation. Requirement for the tTAFs is gene selective: Many genes are transcribed normally in tTAF mutant spermatocytes. Tissue-specific TAFs have also been implicated in gametogenesis and differentiation of specific cell types in mammals. In addition to action with TBP (TATA box–binding protein) in TFIID, certain TAFs associate with HAT (histone acetyltransferase) or Polycomb group (PcG) transcriptional regulatory complexes. To elucidate how tissue-specific TAFs can regulate gene-selective transcription programs during development, the mechanism of action of the Drosophila tTAFs was investigated in vivo (Chen, 2005).

The tTAF proteins were concentrated in a particular subcompartment of the nucleolus in primary spermatocytes. Expression of a functional green fluorescence protein (GFP)–tagged genomic sa rescuing transgene revealed that expression of Sa-GFP turns on specifically in male germ cells soon after initiation of spermatocyte differentiation and persists throughout the remainder of the primary spermatocyte stage, disappearing as cells entered the first meiotic division. Some Sa-GFP was detected associated with condensing chromatin. However, most Sa-GFP localized to the nucleolus, in a pattern complementary with Fibrillarin, which marks a fibrillar nucleolar subcompartment. Staining with antibodies against endogenous Sa, Can, Nht, or Mia proteins showed similar temporal expression and nucleolar localization in primary spermatocytes, consistent with collaborative function of the tTAFs. In contrast, the generally expressed sa homolog TAF8 and its binding partner TAF10b are excluded from the nucleolus (Chen, 2005).

Several components of the Polycomb Repression Complex 1 (PRC1) transcriptional regulator appear in the nucleolus in spermatocytes, coincident with tTAF expression and dependent on tTAF function. Polycomb (Pc) protein expresses from a Pc-GFP genomic transgene localized on chromatin, but in addition becomes concentrated in the nucleolus in primary spermatocytes. Both Pc-GFP and staining of endogenous protein with antibody against Pc (anti-Pc) revealed localization to the same nucleolar subcompartment as the one containing tTAFs. Recruitment of Pc to the nucleolus exactly coincides with onset of expression of the tTAFs after early G2 phase in spermatocytes. Relocalization of Pc depends on wild-type tTAF activity: Pc localizes to chromatin but is not concentrated in the nucleolus in tTAF mutant spermatocytes. Two other components of the PRC1 core complex, Polyhomeotic (Ph) and Drosophila Ring protein (dRing), also become concentrated in the nucleolus in primary spermatocytes dependent on tTAF function. Failure of PRC1 components to localize to the nucleolus in tTAF mutants is not caused by nucleolar loss because Fibrillarin staining appears normal in the mutants. H3K27me3 laid down by action of the PRC2 complex acts as a docking site for the Pc chromodomain to recruit PRC1 and block transcription initiation. H3K27me3 localizes on chromatin in spermatocytes, along with Pc. However, no H3K27me3 was detected in the nucleolus in spermatocytes, suggesting that PRC1 components may be recruited to the nucleolus by a different mechanism independent of chromatin (Chen, 2005).

The tTAFs are required for activation of robust transcription of several spermatid differentiation genes, whereas the PcG proteins are known to mediate transcriptional repression. Chromatin immunoprecipitation (ChIP) suggested that the tTAFs might allow robust transcription of spermatid differentiation genes in part by counteracting repression by Pc, perhaps causing dissociation of PRC1 from cis-acting control sequences at target genes (Chen, 2005).

ChIP from wild-type testes using anti-Sa revealed enrichment of tTAF binding at three different known target genes (fzo, Mst87F, and dj), compared with binding at intergenic regions 10 to 20 kb away or at a tTAF-independent gene expressed in the same cell type (cyclin A or sa itself), suggesting that the tTAFs are in occupancy at target genes. Real-time polymerase chain reaction (PCR) analysis revealed ~10-fold enrichment of Sa at a target (mst87F) compared with a non-target gene (sa) (Chen, 2005).

ChIP analysis also revealed that Pc protein binds to tTAF-dependent target genes in tTAF mutant testes, and that wild-type function of the tTAFs reduce Pc binding. ChIP with anti-Pc from can mutant testes preferentially precipitates the three tTAF target promoters, compared with intergenic regions or promoters from two different nontarget controls. Quantification by real-time PCR showed more than 50-fold enrichment of Pc at the target gene mst87F compared with the tTAF-independent control sa. In contrast, relative occupancy of Pc at the tTAF targets was not significantly different from that at the non-targets in wild-type testes (Chen, 2005).

The tTAFs may act near the promoter of target genes (fzo) to allow expression by directly or indirectly reducing nearby binding of PRC1. ChIP using primer pairs across the promoter region of fzo revealed that the tTAF enrich most strongly for sequences just upstream of the transcription start site. In contrast, Pc-containing protein complexes (in tTAF mutant testes) enrich for a broader distribution, including sequences near and downstream of the transcription start site, consistent with localization of Pc at Ultrabithorax (Ubx) locus in wing discs and on the hsp26 promoter in vivo (Chen, 2005).

Binding of the tTAFs at target promoters may allow expression through recruitment or activation of the Trithorax group (TrxG) transcriptional activation complex, which often acts in opposition to repression by PcG proteins. Trx, like its mammalian homolog MLL, creates an H3K4me3 epigenetic mark. ChIP from wild-type testes revealed H3K4me3 at or near the promoter regions of the three tTAF targets tested, as well as at nontargets. Analysis using primer pairs across the tTAF target fzo region revealed that H3K4me3 associated most strongly with sequences spanning the promoter. In contrast, ChIP with anti-H3K4me3 from can mutant testes did not enrich for the tTAF target promoters. Quantitative PCR revealed 36-fold enrichment of the promoter region of the tTAF-dependent mst87F gene by ChIP for H3K4me3 in wild-type compared with can mutant testes (Chen, 2005).

Consistent with the presence of H3K4me3 at target promoters in wild-type testes, trx function appears to be required for continued expression of two different kinds of tTAF-dependent targets. Boule triggers the G2/M transition in meiosis I by allowing translation of twine and requires tTAFs for protein accumulation, setting up a cross-regulatory mechanism so that meiotic cell cycle progression awaits expression of terminal differentiation genes. When temperature-sensitive trx1 flies grown at permissive temperature were shifted to nonpermissive temperature as adults, the Boule protein level in mutant testes substantially decreased over time at nonpermissive temperature compared with the level in wild-type flies shifted in parallel or trx1 flies held at permissive temperature. Likewise, analysis of mRNA levels by semiquantitative PCR revealed a ~40% decrease in transcript level for the tTAF target gene fzo, but not for the tTAF-independent gene cyclin A, in testes from trx1 mutant flies shifted to non-permissive temperature compared with the level in testes from similarly treated wild-type flies (Chen, 2005).

In summary, occupancy of tTAFs and Pc at target promoters appears to be mutually exclusive in wild-type and tTAF mutant spermatocytes, suggesting that the tTAFs may turn on target gene expression by counteracting repression by Polycomb, either directly or indirectly reducing Pc binding and allowing local action of Trx. Loss of function of Pc in marked clones of homozygous mutant cells does not restore terminal differentiation in a tTAF mutant background, suggesting that in addition to counteracting repression by Pc, tTAFs may also be required at the promoter region independent of Pc, possibly to recruit Trx or other cofactors for transcription activation. Transcriptional derepression by sequestration of PcG proteins has been observed during HIV-1 infection, when the viral Nef protein recruits the PRC2 component Eed to the plasma membrane. Likewise, the tTAFs may sequester Pc to the nucleolus. The tTAFs Nht, Can, and Mia are homologs of the generally expressed TAF4, TAF5, and TAF6, which are found as stoichiometric components of the PRC1 complex purified from fly embryos, raising the possibility that the tTAFs might associate with a population of Pc-, Ph-, and dRing-containing complexes in the nucleolus. If so, interactions in the nucleolus are likely to differ from interactions at the promoters of target genes, because the ChIP results indicate immunoprecipitation of tTAFs without Pc (Chen, 2005).

The PcG and TrxG proteins act to maintain cell fates set during embryogenesis throughout development. Emerging evidence indicates that PcG and TrxG complexes also play critical roles in decisions between proliferating precursor cell fates and terminal differentiation, for example, in the blood cell lineages. In particular, the mammalian PcG protein Bmi-1 promotes proliferation and blocks differentiation of normal and leukemic stem cells, and is required for establishment or maintenance of adult hematopoietic stem cells in mouse. Transcriptional silencing by PcG action may allow self-renewal and continued proliferation of precursor cells by blocking expression of terminal differentiation genes. This repression must be reversed to allow production of terminally differentiated cells, whereas failure may allow overproliferation of precursors and eventually cancer. Although central for both normal development and understanding the genesis of cancer, little is known about the mechanisms that reverse such epigenetic silencing to allow expression of the terminal differentiation program. These findings in the male germ line provide an example of how cell type– and stage–specific transcriptional regulatory machinery, turned on as part of the developmental program, might allow onset of terminal differentiation by counteracting repression by the PcG and highlight the importance of subnuclear localization in regulation of transcriptional regulation (Chen, 2005).

Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos



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).


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).



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

trithorax mutants show homeotic transformation of segmental structures to more anterior structures. Some alleles are lethal. These transformations are enhanced when only one copy of the BX-C is present, implying an interaction between trx and the genes of the BX-C. Loss of trx greatly reduces Ultrabithorax protein levels. In this case, Abdominal-A protein levels are also reduced, but to a lesser extent (Breen, 1991).

As a first step to characterizing specific developmental functions of Trx, phenotypes of 420 combinations of 21 trx alleles have been examined. Eclosing and pharate adults were examined for phenotypes associated with reduced expression of the trx homeotic target genes Scr of the ANT-C and Ubx, abdominal-A, and Abdominal-B of the BX-C. Allele-specific phenotypes were sought that would reveal different functional domains of Trx. Eight hypomorphic alleles were characterized, seven of which primarily affect imaginal development. These hypomorphic alleles are sufficient for embryogenesis but provide different levels of trx function at homeotic genes in imaginal cells. Results demonstrate that Trx is used in tissue-specific contexts at the target genes examined. Some trx genotypes appear to have almost no Scr function in T1 leg discs, no Ubx function in T3 leg discs, and greatly reduced function of the other genes examined in their respective imaginal tissues. Some trx genotypes exhibit additional phenotypes, some of which are also seen in trx- somatic clones. These latter phenotypes are similar to ones produced by mutations in elements of signal transduction pathways. It is suggested that the differential effects of trx mutations on different tissues and cells may be due in part to the differential regulation of Trx by cell-signaling mechanisms (Breen, 1999).

One of the hypomorphic alleles alters the N terminus of TRX, which severely impairs larval and imaginal growth. Hypomorphic alleles that alter different regions of Trx equivalently reduce function at affected genes, suggesting Trx interacts with common factors at different target genes. All hypomorphic alleles examined complement one another, suggesting cooperative Trx function at target genes. Comparative effects of hypomorphic genotypes support previous findings that Trx has tissue-specific interactions with other factors at each target gene. Some hypomorphic genotypes also produce phenotypes that suggest TRX may be a component of signal transduction pathways that provide tissue- and cell-specific levels of target gene transcription (Breen, 1999).

A model is presented for Trx function. Trx is recruited to polycomb response elements (PREs) of target genes. Once assembled, it acts with other trxG proteins to stimulate target gene transcription through chromatin remodeling as inferred by the SET domain it shares with other proteins known to alter chromatin. An interesting possibility has been suggested -- that Trx influences the level of target gene histone acetylation. It is not clear if Trx participates in the initial transcription of its target genes. In specific cells, it is necessary for detectable levels of target gene transcription. In others, it is needed only for enhanced target gene transcription so that the cells will have levels of target gene transcription similar to their parent cells. From this information, it is conceivable that Trx assembles in a lineage-dependent manner to act as a constitutive facilitator of other transcription factors. If transcription of a target gene is not initiated in a cell, PcG proteins bound to the gene's PRE chromatin form a silencing structure that supersedes colocalized Trx. A gene's PcG protein-silencing structure is then inherited by progeny cells. Phenotypic and gene expression analyses of two trxG genes, ash2 and mor, suggest that their proteins participate in downstream functions of developmental signaling pathways. Phenotypic results of this study allow that Trx activity may be modulated downstream of cell signaling to attain cell-specific levels of target gene transcription. This role of Trx is supported by findings that propose a similar role for HRX (aka MLL, ALL1, HTRX), the human homolog of Trx. The interaction of a dual-specificity phosphatase inhibitor with the SET domain of HRX suggests it is activated through signal transduction and later deactivated to promote differentiation. In this light, a model is presented in which TRX acts as a downstream mediator in multiple signal transduction pathways, including those signaled by morphogens, to elicit ligand concentration-dependent responses at target genes. In this role, Trx provides a dynamic response capacity to a variety of cell-signaling events (Breen, 1999).

The model is based on activities in PS7 of the visceral mesoderm where trx is needed for normal levels of Ubx expression. However, other ligands and their receptors may be substituted to account for the effects of trx mutations on other cell types. In the model, Trx is modified as a downstream substrate of signaling pathways, whose ligands may include the TGF-ß homolog, Decapentaplegic; the WNT-1 homolog, Wingless protein; Hedgehog protein, and ligands of the Drosophila Epidermal growth factor receptor such as Spitz protein. Signaling intermediates may include factors such as Mothers against dpp and Schnurri in the Dpp pathway. It is also possible that TRX is required for normal levels of expression of signaling pathway genes. Thus, trx mutants might develop hypomorphic signal transduction phenotypes. The expression of dpp in trx mutants does not support this possibility (T. R. Breen, unpublished results), though the expression of many other signaling element genes in trx mutants needs to be examined (Breen, 1999).

The snr1 gene is essential for viability and genetically interacts with brm and trithorax, suggesting cooperation in regulating homeotic gene transcription (Dingwall, 1995).

Mutations in the ash-1 and ash-2 genes cause a wide variety of homeotic transformations that are similar to the transformations caused by mutations in the trithorax gene. The genetic interaction between ash-1, ash-2 and trithorax provides substantional evidence that they are members of a functionally related set of genes (Shearn, 1989).

The observation of a shared pathway in the function of a chromatin insulator and trithorax group (trxG) and Polycomb group (PcG) gene activation and silencing is suggestive of a common mechanism at work. If this is the case, mutations in trxG and PcG genes, known to be involved in activation and silencing, might also affect the ability of the insulators to interfere with enhancer-promoter interactions. To test this possibility, the effect of trxG and PcG mutations on the abdominal coloration of flies carrying the yellow2 mutation (affecting coloration) was measured using insertion of an insulator-containing gypsy retrotransposon. Males hemizygous for the y2 allele show brown abdominal pigmentation in the fifth and sixth abdominal segments, instead of the black pigmentation observed in wild-type males, due to the effect of the insulator on the upstream body cuticle enhancer. This insulator effect on the body enhancer is altered by hypomorphic mutations in mod(mdg4), which gives rise to a variegated phenotype resulting from different expression levels of the yellow gene in adjacent groups of cells. In some cuticle cells, the effect of the insulator is reversed, resulting in normal expression of the yellow gene; in other cells, the effect of the insulator on enhancer-promoter communication appears to be enhanced, further repressing yellow gene expression. To examine the effect of trxG mutations on insulator function, the partially nonfunctional insulator, renderend such by hypomorphic alleles of mod(mdg4), was tested. An examination was carried out of the consequence of mutations in trxG genes, such as trithorax, on the frequency and severity of a mod(mdg4) phenotype engendered by Mod(mdg4) action at the gypsy insulator (Gerasimova, 1998).

Both the penetrance and severity of a variegated phenotype due to insulator function are enhanced by mutations in trxG genes. trx mutation results in a decrease in the number of dark spots with respect to that observed in hypomorphilc mod(mdg4) males, with only a few spots visible in a light brown-colored background. A stronger effect can be seen when trx is combined with brahma or ash1. Mutations in polycomb cause the opposite result, reversing the effect of the insulator on enhancer-promoter interactions and resulting in a wild-type expression of the yellow gene in the body cuticle. These results indicate that mutations in trxG genes cause an enhancement of the variegated phenotype induced by mod(mdg4) mutations in the yellow gene, suggesting that decreased levels of these proteins enhance the inhibitory effect of the insulator on enhancer-promoter interactions. In contrast, mutations in Pc impair the ability of the insulator to inhibit enhancer-promoter interactions, restoring normal expression of the gene. The effects of trxG and PcG mutations on insulator function at the yellow gene are not a result of homeotic transformations in abdominal segments that cause changes in the pigmentation of the cuticle, since these effects are not observed in flies carrying a wild-type copy of the yellow gene. In addition, the same effect can be observed with other gypsy-induced mutations such as scute-1 and cut-6. Flies of the genotype ct6; brm+ trx+ mod(mdg4)T16/brm2 trxB11 mod(mdg4)+ display a much stronger cut phenotype than ct6; mod(mdg4)T16/mod(mdg4)+ individuals, suggesting that the effect of TrxG and PcG proteins on gypsy insulator function is general and does not depend on the nature of the affected gene. A similar result was obtained with the sc1 mutation. The effects of trxG and PcG mutations on insulator function suggest that the proteins encoded by these genes might be structural components of the gypsy insulator or they might regulate its function (Gerasimova, 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 proteins encoded by two groups of conserved genes, the Polycomb and trithorax groups, have been proposed to maintain, at the level of chromatin structure, the expression pattern of homeotic genes during Drosophila development. To identify new members of the trithorax group, a collection of deficiencies was screened for intergenic noncomplementation with a mutation in ash1, a trithorax group gene. Five of the noncomplementing deletions uncover genes previously classified as members of the Polycomb group. This evidence suggests that there are actually three groups of genes that maintain the expression pattern of homeotic genes during Drosophila development. The products of the third group appear to be required to maintain chromatin in both transcriptionally inactive and active states. Six of the noncomplementing deficiencies uncover previously unidentified trithorax group genes. One of these deficiencies removes 25D2-3 to 26B2-5. Within this region, there are two allelic, lethal, P-insertion mutations that identify one of these new trithorax group genes. The gene has been called little imaginal discs based on the phenotype of mutant larvae. The protein encoded by the little imaginal discs gene is the Drosophila homolog of human retinoblastoma binding protein 2 (Gilde, 2000).

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

Six of the deficiencies uncovered genes that were previously classified in the Polycomb group. They were so classified, because they either enhanced the Polycomb mutant phenotype or caused a phenotype like Polycomb mutants. This result was quite unexpected because the antagonism between trithorax and Polycomb group genes suggested that loss of function of Polycomb group genes should suppress trithorax mutant phenotypes, while these deficiencies showed an enhancement of trithorax group mutant phenotypes. Nevertheless it is likely that the Polycomb group genes uncovered by these deficiencies are responsible for the observed intergenic noncomplementation with ash1RE418. It was thought possible that the observed intergenic noncomplementation is specific for ash1 mutations rather than general for mutations in trithorax group genes. This possibility was excluded for four of the five genes by showing that E(Pc)1, Psc1, Su(z)21, AsxXF23, Asx3, and Asx13 also show intergenic noncomplementation with trxb11 and/or brm2 and increase the penetrance of two different double mutants: ash1VF101 trxb11 and brm2 trxe2. It has also been reported that Asx mutations show intergenic noncomplementation with mutations in trithorax group genes. In some of these cases, the different mutant alleles tested give inconsistent results. For example, both ScmD1 and Scmm56 show intergenic noncomplementation with ash1VV183 and enhance the phenotype of the ash1VF101 trxb11 double mutant, whereas Scm302 does not enhance the phenotype of ash1VV183 and suppresses the phenotype of ash1VF101 trxb11. It is supposed that this difference is due to differences in the specific alterations of the Scm protein caused by these mutations (Gilde, 2000).

Until now the antagonism of function between the products of Polycomb group genes and trithorax group genes has been demonstrated unidirectionally by the suppression of Polycomb group mutant phenotypes by mutations in trithorax group genes. Advantage was taken of the intergenic noncomplementation of mutations in trithorax group genes to assay suppression of trithorax group mutant phenotypes by mutations in genes previously classified as Polycomb group genes. Among ash1VF101;trxb11 and brm2;trxe2 double heterozygotes, 52% and 35%, respectively, of adult flies express transformations of the third thoracic segment to the second thoracic segment. Most mutations in seven of the genes that have been classified as members of the Polycomb group (Polycomb, polyhomeotic, pleiohomeotic, Polycomb-like, multi sex combs, extra sex combs, and Super sex combs) suppress the penetrance of these transformations, in both of these double heterozygotes. Moreover, most mutations in these genes do not show intergenic noncomplementation with mutations in any of the three trithorax group genes that have been tested. It is suggested that these genes represent the Polycomb group defined here as genes in which loss-of-function mutations enhance the dominant phenotype caused by Polycomb mutations and suppress the phenotype caused by heterozygosity for double mutations in trithorax group genes such as ash1VF101;trxb11 and brm2;trxe2 (Gilde, 2000).

The zeste (z) gene encodes a transcription factor that binds DNA in a sequence-specific manner. The z1 mutation causes reduced white gene transcription. Mutations in three genes identified as dominant modifiers of the zeste-white interaction, Enhancer of zeste, Suppressor of zeste-2, and Sex comb on midleg, can also cause phenotypes like mutations in Polycomb group genes. Here it is shown that mutations in these three genes also behave as mutations in trithorax group genes: they show intergenic noncomplementation with mutations in trithorax group genes and/or increase the penetrance of ash1VF101;trxb11 and/or brm2;trxe2 heterozygotes. Moreover, mutations in three other genes identified as suppressors of the zeste-white interaction, Suppressor of zeste-4, Suppressor of zeste-6, and Suppressor of zeste-7, may show intergenic noncomplementation with mutations in trithorax group genes and/or increase the penetrance of ash1VF101;trxb11 heterozygotes. The biochemical mechanism by which mutations in these genes modify the zeste-white interaction is not known. However, it is thought to be significant that many of the genes identified as Suppressors of zeste behave as if they are both trithorax and Polycomb group genes; that Enhancer of Polycomb is a suppressor of zeste, and that sex combs extra is an enhancer of zeste (Gilde, 2000).

It is proposed that the six genes (previously classified as Polycomb group genes) belong in a distinct group; in these genes, loss-of-function or antimorphic mutations show intergenic noncomplementation with mutations in trithorax group genes and increase the penetrance caused by double heterozygosis of mutations in trithorax group genes. It is proposed that this group be called the ETP (Enhancers of trithorax and Polycomb mutations) group. Loss-of-function mutations in this group of genes not only enhance the dominant phenotype caused by Polycomb mutations, as do mutations in Polycomb group genes but they also enhance the phenotype caused by heterozygosity for double mutations in trithorax group genes, such as ash1VF101;trxb11 and brm2;trxe2, as do mutations in trithorax group genes (Gilde, 2000).

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

The 133 deficiencies examined collectively uncover ~70% of the genome. Of these, only 6 exhibit intergenic noncomplementation with mutations in all 3 of the trithorax group genes tested and do not uncover previously identified trithorax group genes. Either there must be only a small number (i.e., closer to 10 than to 100) of genes in the entire genome in which mutations fail to complement mutations in the trithorax group genes tested or only deficiencies that uncover 2 or more such genes the were detected in this assay. Four of the deficiencies fail to complement mutations in all 3 trithorax group genes but do not suppress the Polycomb mutant phenotype. Perhaps these deficiencies uncover genes whose products act downstream of the homeotic selector genes, for example, as cofactors necessary for the activity or stability of homeotic selector gene products (Gilde, 2000).

Two of these six deficiencies suppressed the Polycomb mutant phenotype and did not uncover a known trithorax group gene. One of these six deficiencies, Df(2L)cl-h3 (25D2-3;26B2-5), uncovers two different trithorax group genes. The distal gene is within 25D4;25E1. It may be identical to E(var)2-25E, which was recovered in a screen for enhancers of position-effect variegation. Several of the mutations recovered in that screen have proven to be allelic to trithorax group genes. The proximal gene is within 25F4-4;26B2-5. Three lines of evidence have been presented, indicating that the allelic mutations l(2)10424 (now known as lid1) and l(2)k06801 (now known as lid2) represent P-element insertion mutations within this proximal gene, which has been named little imaginal discs. (1) Both alleles are lethal in combination with deficiencies that remove 25F4-4;26B2-5. (2) lid2 enhances the phenotype of ash1, brahma, and trithorax mutations and suppresses the phenotype of a Polycomb deletion. (3) Precise revertants of lid1 are homozygous viable and fail to enhance the phenotype of ash1, brahma, or trithorax mutations and fail to suppress the phenotype of a Polycomb deficiency (Gilde, 2000).

Despite the fact that lid mutations satisfy the criteria used for mutations in trithorax group genes, no homeotic transformations were observed in homozygous or trans-heterozygous mutant embryos or larvae. Instead, a small disc phenotype was observed. Certain allelic combinations of ash1 mutations also cause a small disc phenotype. The few lid mutants that survived the pupal stage expressed bristle phenotypes, similar to mutations in the trithorax group gene ash2. Therefore, lid mutations do cause phenotypes similar to those caused by mutations in other trithorax group genes. The failure to detect a high frequency of homeotic transformations in the two lid mutants is interpreted as a consequence of the nature of the mutations caused by the P-element insertions in these alleles (Gilde, 2000).

The predicted lid gene product is extremely similar to the human retinoblastoma binding protein 2 gene product (RBP-2). RBP-2 was discovered in a screen for proteins that interact with the pocket domain of the retinoblastoma protein (pRB). The full-length sequence of RBP-2 was later determined and found to contain nuclear localization motifs as well as sequence motifs characteristic of transcriptional regulators. RBP-2 has been shown to physically interact with mammalian TATA-binding protein as well as with p107 and Rb (also known as p110). No information is available about the molecular mechanism of LID function. However, given the similarity of LID to RBP-2 and the binding of RBP-2 to pRB there are several intriguing possibilities (Gilde, 2000).

The role of pRB in cell cycle regulation and proliferation is mediated, at least in part, by its interaction with the transcription factor E2F. It interacts physically with E2F to repress transcription and cell cycle progression. Overexpression of RBP-2 in cultured cells was shown to overcome the pRB-mediated suppression of E2F activity. A Drosophila mutant of E2F, E(var)3-95E, was discovered as a dominant enhancer of variegation. E2F is necessary for proliferation and differentiation in the Drosophila eye and interacts genetically with a Drosophila homolog of Rb: RBF. The finding that lid mutations cause defects in imaginal disc cell proliferation may be due to the loss of negative regulation of RBF leading to increased E2F repression of cyclin E (Gilde, 2000).

Histone acetylation has profound effects on transcriptional regulation and both global and local chromatin structure. The Rb protein has recently been found to physically associate with a histone deacetylase, HDAC1, and to repress transcription. The function of LID could be to counteract the repressive activity that histone deacetylation has on chromatin. Two multiprotein complexes from yeast, ADA and SAGA, function as nucleosome acetyltransferases, with GCN5 as the catalytic subunit; GCN5 mutations display synthetic lethality with SWI/SNF mutations. This is especially interesting, since brahma is a Drosophila homolog of yeast SWI2/SNF2, and lid interacts genetically with brahma. Further evidence for an association of trithorax group gene products and pRB is that by both two-hybrid and coimmunoprecipitation studies, Hbrm and Brg1, two human homologs of brahma, are associated with pRB family members. The balance between acetylation and deacetylation is clearly implicated in the function of trithorax group genes. Though the role RBP-2 plays in chromatin regulation is not known, the fact that it could be involved in the inactivation or relocation of a histone deacetylase fits well with how it is thought that trithorax group genes help to maintain an open chromatin conformation (Gilde, 2000).

In addition to the connections of pRB with E2F, cyclin E, and the cell cycle and to the connections of pRB with histone deacetylation and repression of transcription, there is a connection of pRB with the nuclear matrix and nuclear matrix-associated proteins. p110Rb is associated with the nuclear matrix in a cell cycle-dependent manner. Many p110Rb-associated factors have been previously found to be associated with the nuclear matrix, including SV40 large T antigen, adenovirus E1a, human papilloma E7 protein, lamin A, p84, and NRP/B. One model posits that functions within the nucleus occur at specific sites, and this functional compartmentalization of the nucleus is accomplished by localizing the machinery for each task to a specific site. For example, a hypothetical scenario consistent with this model would be that once activated, a homeotic selector gene may be bound by one or more trithorax group protein complexes that maintain the activated state by creating a site on the nuclear matrix for the transcription machinery itself and for proteins involved in acetylation and/or nucleosome remodeling and/or phosphorylation that are necessary for optimal expression. In this context, the change in subnuclear localization of the modifier of mdg-4 gene product may be relevant. Modifier of mdg4, also known as E(var)3-93D, is a trithorax group gene. Loss-of-function mutations enhance the phenotype of ash1;trithorax and brahma;trithorax double mutations and suppress the phenotype of Polycomb mutations. The product of this gene, MOD, is normally associated with the nuclear matrix. However, the subnuclear localization of MOD is dramatically altered in both trithorax group and Polycomb group mutant backgrounds. In trithorax group mutants MOD is primarily cytoplasmic; in Polycomb group mutants MOD is present in the central region of the nucleus rather than the nuclear matrix. Many of the models for the organization of higher order chromatin structures are based on associations with nuclear matrix components. It will be interesting to determine the subnuclear localization of LID and observe whether there are changes in this localization during the cell cycle and/or in trithorax group and Polycomb group mutant backgrounds (Gilde, 2000).

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


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

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

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

Alternative epigenetic chromatin states of polycomb target genes

Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (Trx) and Ash1. This study has analyzed the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins Trx and Ash1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, it was shown that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in Ash1, the N-terminal, but not C-terminal moiety of Trx and H3K27ac. Ash1/Trx N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a 'balanced' state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a 'void' state, lacking transcription, H3K27ac, or binding of Trx or Ash1. It is concluded that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, Trx, and activators. N-ter Trx plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to Trx/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the 'bivalent' chromatin state of embryonic stem cells, and gene expression in development (Schwartz, 2010).

Key to the current understanding of PcG mechanisms is the fact that, while PcG proteins are present in most kinds of cells, the decision whether or not to repress a target gene depends crucially on whether that gene had been repressed in the previous cell cycle. This effect is responsible for the epigenetic maintenance of the repressed state and associated chromatin modifications. Similarly, through the action of Trx and Ash1, a PcG target gene that had not been repressed tends not to become repressed in the subsequent cell cycle and remains susceptible to transcriptional activators. By comparing PcG/TrxG and transcriptional landscapes in three lines of Drosophila cultured cells it was found that the full repertoire of chromatin states that PcG target genes can assume is not limited to the repressed state dominated by PcG mechanisms and the transcriptionally active state governed by TrxG proteins but in addition includes transcriptionally active 'balanced' states subjected to simultaneous or at least rapidly alternating control by both PcG and TrxG proteins, and a transcriptionally inactive 'void' state lacking both PcG and TrxG control. Thus, although PcG mechanisms first achieved fame for producing stable long-term silenced states in Drosophila homeotic genes, it is clear that, in the general case, PcG states are not necessarily stable nor long-term (Schwartz, 2010).

The results establish clearly that in robust PcG target regions (i.e. Class I PcG target regions) PcG and TrxG regulation are tightly coupled. Considering the role of MLL1 in the regulation of HOX genes and the similarity between PcG complexes in flies and mammals, it is expected that the same holds true for mammalian cells. It is possible that PcG and Trx recruitment to PRE/TREs share some DNA-binding proteins or DNA motifs. It will be important to determine whether PcG and Trx bind simultaneously or alternate over time. The nature of the Trx complex that binds to PRE/TREs remains enigmatic. To date the only Trx complex characterized biochemically is TAC1, purified from Drosophila embryos (Petruk, 2001). It is said to contain uncleaved full length Trx, anti-phosphatase Sbf1 and histone acetyltransferase dCBP. This study could detect no uncleaved Trx in the nuclei of cultured cells indicating that the Trx bound at PRE/TREs of repressed genes does not represent TAC1. Proteolytically cleaved human orthologs of Trx, MLL1 and MLL2 have been purified as part of complexes similar in composition to the yeast COMPASS4. Although the PRE/TRE binds both parts of the cleaved Trx, it lacks some COMPASS components and lacks H3K4 trimethylation, suggesting that it involves a different complex whose composition is yet to be characterized (Schwartz, 2010).

Consistent with genetic evidence, the presence of Ash1 and Trx at PcG target regions is linked to their transcriptional activity. However the two proteins show important differences in their behavior: binding of Ash1 is limited to transcriptionally active (fully derepressed or balanced) PcG targets and is not detected at completely repressed target loci. Trx is more complex. Both N-ter and C-ter parts of the protein associate with PREs regardless of the transcriptional status of their target genes and bind in the vicinity of TSS specifically when a target gene is transcriptionally active. In addition, the N-ter moiety of Trx together with Ash1 forms broad domains that encompass transcriptionally active PcG target genes. The different behavior of N-ter and C-ter parts of Trx may account for the discrepancy between reports of the co-localization of Trx and PcG proteins at many chromosomal sites and reports claiming that Trx binds exclusively to transcriptionally active target genes. The different accounts are due to the use of anti-Trx antibodies specific to different parts of the protein (Schwartz, 2010).

Whether C-ter or N-ter specific antibodies were used, the number of Trx bound regions detected in these experiments is small compared to the number of active genes. This argues against a general role for Trx in transcription and is consistent with the limited number of regions detected on polytene chromosomes by various antibodies directed against C-ter or N-ter Trx. In marked contrast to these observations, Schuettengruber (2009) has recently reported exclusive association of N-ter but not C-ter moiety of Trx with TSS of most active transcription units in the chromatin of embryonic cells. The same report also asserted that in embryonic cells Trx C-ter but not Trx N-ter is bound at PREs. Remarkably the Trx N-ter specific antibody used by Schuettengruber is reportedly the same as one of the two used in experiments from this lab. While the possibility cannot be excluded that the behavior of the N-ter moiety of Trx in embryos is totally different from that in cultured or salivary gland cells, it is suspected that more likely the preparation of the antibody used by Schuettengruber cross-reacted with some general transcription factor particularly abundant in embryonic cells. This emphasizes the importance, even the necessity, of using two or more independent antibodies to verify genome-wide ChIP results (Schwartz, 2010).

The RNAi knockdown experiments show that the broad binding of Ash1 and Trx N-ter within transcriptionally active PcG target regions is interdependent. This is consistent with the reported dissociation of Ash1 from polytene chromosomes of the salivary gland cells subjected to Trx RNAi and the severe reduction of Trx N-ter binding to polytene chromosomes of ash1 mutant larvae. Despite interdependence in binding there is no compelling evidence that Ash1 and Trx N-ter are in the same protein complex. Although an interaction between Trx and Ash1 has been reported, it was said to require the intact SET domain of Trx, which is absent from its N-ter moiety. This study has not found that Trx N-ter and Ash1 co-precipitate from nuclear extracts, strengthening the impression that the two peptides do not interact directly (Schwartz, 2010).

A histone mark associated with Ash1/Trx N-ter domains is H3K27ac. Acetylation of H3K27 can antagonize PcG activity by competing with the placement of the H3K27me3 mark, which in our model is needed for effective contact of the PRE complex with the promoter, as well as for stable PcG binding. In fact, targeting a histone H3 acetylase to a PRE is sufficient to prevent the epigenetic maintenance of repression indicates that in Drosophila the HAT responsible for bulk acetylation of H3K27 is CREB-binding protein (dCBP), encoded by the nejire gene. Direct association of both Trx and Ash1 with dCBP has been previously reported. Consistent with this, the Trx knock-down experiment, which also impairs Ash1 binding, shows that either or both proteins promote acetylation of K27 in the chromatin of active PcG targets. It also shows, however, that the level of H3K27ac is not directly related to the amount of Ash1 or Trx N-ter bound. It is possible that a small amount of Trx and/or Ash1 remaining on the chromosomes after RNAi depletion is sufficient to target enough HAT activity to maintain nearly normal levels of H3K27ac. Alternatively H3K27 acetylation may be produced by a process that is not mechanistically linked to Ash1 or Trx but is promoted by the two. A global reduction of immunostaining of polytene chromosomes with anti-H3K27ac antibodies and a global elevation of immunostaining with anti-H3K27me3 antibodies in trx mutant larvae was recently reported. The current study did not detect any global changes in either H3K27ac or H3K27me3 levels. It is possible that the effects are generally weak and could only be detected on polytene chromosomes that consist of thousands of chromatin fibers bundled together. It is noted, however, that the reported changes of H3K27 acetylation and trimethylation levels involved numerous chromosomal sites, most of which, according to current data, do not stably bind PcG or TrxG, suggesting that in these cases the effect of trx mutation may have been indirect (Schwartz, 2010).

The microarray data show that narrower peaks of H3K27ac are also found near numerous active promoters, at genes not known to be PcG targets. A role of H3K27ac at these promoters may be to antagonize dimethylation of H3K27, which is abundantly distributed throughout the genome and may have a general negative effect on transcription (Schwartz, 2010).

In Drosophila cultured cells most PcG target genes are either completely repressed or fully derepressed or entirely devoid of both PcG and TrxG regulation. However in about 5% of cases, exemplified by the Psc-Su(z)2 or inv-en loci, binding of PcG complexes does not result in complete transcriptional silencing and can coexist with binding of Ash1/Trx N-ter. Whether a PcG target gene is capable of and will assume a 'balanced' state may depend on the nature of the PRE, its binding complexes, and the promoter of the target gene. More likely, however, the major determinants are the nature and concentration of activators and repressors that act in concert with PcG/TrxG. Consistent with this idea, in imaginal disc cells, which are controlled by much more complex regulatory networks than cultured cells, simultaneous presence of both PcG and TrxG proteins at the transcriptionally active PcG target genes appears to be more common. Interestingly a common feature of the 'balanced' chromatin state in both cultured and imaginal disc cells is the confinement of Ash1/Trx N-ter binding to the regions immediately around the promoters. This is taken as a hint that the formation of a broad Ash1/Trx N-ter domain starts in the vicinity of the TSS (Schwartz, 2010).

Much has been said about the 'bivalent' state, i.e. containing both PcG repressive marks and transcriptional activity marks, as characteristic of genes in mammalian embryonic stem cells. In Drosophila, in cases such as those of the Psc-Su(z)2 or inv-en loci, the balanced action of PcG and TrxG results in chromatin states similar in appearance to the 'bivalent' state. It is supposed that, like the 'balanced' chromatin state of Drosophila PcG targets, the 'bivalent' domains of embryonic stem cells would be associated with both PcG proteins and mammalian orthologs of Trx and Ash1 (Schwartz, 2010).

PcG target genes may also assume a state lacking both PcG and TrxG proteins. The fact that in several instances the same locus resides in this 'void' chromatin state in cultured cells of completely different origin argues against it being a product of genomic aberrations. Several experimental observations also indicate that the 'void' state is not a peculiarity of cultured cells. Thus, in salivary glands the hh gene lacks PcG binding and H3K27me3 but remains transcriptionally inactive, as in D23 cells. More recently a comparison of embryonic and imaginal disc cells showed that in many cases lack of PC binding was not accompanied by transcriptional activity. The void chromatin state might be simply interpreted as a derepressed region that is transcriptionally inactive because the needed activator is absent. However, this does not explain why in this state Trx is also absent from the PRE, implying that neither PcG nor Trx binding is the default state of the PRE or that some specific condition prevents the recruitment of both. No other known repressive marks such as H3K9 methylation has been detected at these sites (Schwartz, 2010).

In line with the finding that the lack of PcG repression in the 'void' chromatin state does not automatically lead to the activation of a target gene is the observation that PC knock-down elicits a very specific genomic response. Remarkably Sex combs reduced (Scr), Antennapedia (Antp) and Abd-B, the HOX genes whose derepression in heterozygous +/Pc- flies gives the famous Polycomb phenotype, are also among the genes most sensitive to PC knockdown in BG3 cells. The sensitivity of these genes cannot be explained by intrinsically poor recruitment of PcG proteins as Abd-B and Scr are controlled by multiple strong PREs capable of robust repression when placed next to a reporter gene. It is supposed that the reason for the differential sensitivity to PC levels lies in the availability of the corresponding transcriptional activators. It is propose that in the BG3 cells the transcriptional activators of sensitive PcG target genes are present but at levels insufficient to override repression under normal conditions. The knockdown of PC lowers the threshold required for derepression. It is suggested that a general role of PcG and TrxG mechanisms is to modulate the constraints on the levels of transcriptional activators required to switch the expression of PcG target genes. This concept helps to explain why, despite the implication of the PcG system in the control of all morphogenetic pathways, the reduction of PcG levels during differentiation of mammalian cell lineages or tissue regeneration in flies results in the execution of very specific genomic programs (Schwartz, 2010).


Agger, K., Cloos, P. A., Christensen, J., Pasini, D., Rose, S., Rappsilber, J., Issaeva, I., Canaani, E., Salcini, A. E. and Helin, K. (2007). UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 449: 731-734. PubMed Citation: 17713478

Ahmad, K. and Henikoff, S. (2002). The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9: 1191-1200. PubMed Citation: 12086617

Alvarez-Venegas, R., et al. (2003). ATX-1, an Arabidopsis homolog of Trithorax, activates flower homeotic genes. Curr. Biol. 13: 627-637. 12699618

Arakawa, H., et al. (1998). Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene. Proc. Natl. Acad. Sci. 95(8): 4573-4578. PubMed Citation: 9539779

Ardehali, M. B., et al. (2011). Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription. EMBO J. 30: 2817-28. PubMed Citation: 21694722

Beisel, C., et al. (2007). Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins. Proc. Natl. Acad. Sci. 104(42): 16615-16620. Medline abstract: 17921257

Bieli, D., Kanca, O., Requena, D., Hamaratoglu, F., Gohl, D., Schedl, P., Affolter, M., Slattery, M., Muller, M. and Estella, C. (2015). Establishment of a developmental compartment requires interactions between three synergistic cis-regulatory modules. PLoS Genet 11: e1005376. PubMed ID: 26468882

Breen, T. R. and Harte, P. J. (1991). Molecular characterization of the trithorax gene, a positive regulator of homeotic gene expression in Drosophila. Mech. Dev. 35(2): 113-27. PubMed Citation: 1684714

Breen, T. R. and Harte, P. J. (1993). Trithorax regulates multiple homeotic genes in the bithorax and Antennapedia complexes and exerts different tissue-specific, parasegment-specific and promoter-specific effects on each. Development 117: 119-34. PubMed Citation: 7900984

Breen, T. R., Chinwalla, V. and Harte, P. J. (1995). Trithorax is required to maintain engrailed expression in a subset of engrailed-expressing cells. Mech Dev 52: 89-98. PubMed ID: 7577678

Breen, T. R. (1999). Mutant alleles of the Drosophila trithorax gene produce common and unusual homeotic and other developmental phenotypes. Genetics 152(1): 319-344. PubMed ID: 10224264

Broeker, P. L., et al. (1996). The mixed lineage leukemia (MLL) protein involved in 11q23 translocations contains a domain that binds cruciform DNA and scaffold attachment region (SAR) DNA. Curr. Top. Microbiol. Immunol. 211: 259-268. PubMed ID: 8585957

Caldas, C., et al. (1998). Isolation and characterization of a pufferfish MLL (mixed lineage leukemia)-like gene (fMll) reveals evolutionary conservation in vertebrate genes related to Drosophila trithorax. Oncogene 16(25): 3233-3241. PubMed ID: 96818219681821

Cao, F., Chen, Y., Cierpicki, T., Liu, Y., Basrur, V., Lei, M. and Dou, Y. (2010). An Ash2L/RbBP5 heterodimer stimulates the MLL1 methyltransferase activity through coordinated substrate interactions with the MLL1 SET domain. PLoS One 5: e14102. PubMed ID: 21124902

Capotosti, F., Hsieh, J. J. and Herr, W. (2007). Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways. Mol. Cell. Biol. 27(20): 7063-72. Medline abstract: 17698583

Carrington, E. A. and Jones, R. S. (1996). The Drosophila Enhancer of zeste gene encodes a chromosomal protein: examination of wild-type and mutant protein distribution. Development 122: 4073-4083. PubMed ID: 9012527

Cavalli, G. and Paro, R. (1999). Epigenetic inheritance of active chromatin after removal of the main transactivator. Science 286(5441): 955-8. PubMed ID: 10542150

Chan, C-S., Rastelli, L. and Pirrotta, V. (1994). A Polycomb response element of the Ubx gene that determines an epigenetically inherited state of repression. EMBO J 11: 2553-64. PubMed ID: 7912192

Chang, Y. L., et al. (1995). Functional reconstruction of trans regulation of the Ultrabithorax promoter by the products of two antagonistic genes, trithorax and Polycomb. Mol Cell Biol 15: 6601-6612. PubMed ID: 8524225

Chen, X., Hiller, M., Sancak, Y. and Fuller. M. T. (2005). Tissue-specific TAFs counteract Polycomb to turn on terminal differentiation. Science 310: 869-872. 16272126

Chinwalla, V., Jane, E. P. and Harte, P. J. (1995). The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites. EMBO J 14: 2056-2065. PubMed ID: 7744011

Cui, X., et al. (1998). Association of SET domain and myotubularin-related proteins modulates growth control. Nat. Genet. 18(4): 331-337. PubMed ID: 9537414

De Vivo, I., et al. (1998). Growth stimulation of primary B cell precursors by the anti-phosphatase sbf1. Proc. Natl. Acad. Sci. 95(16): 9471-9476. PubMed ID: 9689104

Dingwall, K. D., et al. (1995). The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex. Mol Biol Cell 6: 777-791. PubMed ID: 7579694

Dou, Y., et al. (2005). Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell 121: 873-885. 15960975

Dou, Y. L., Milne, T. A., Ruthenburg, A. J., Lee, S., Lee, J. W., Verdine, G. L., Allis, C. D. and Roeder, R. G. (2006). Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat. Struct. Mol. Biol. 13: 713-719. Medline abstract: 16878130

Ernst, P., Wang, J., Huang, M., Goodman, R. H. and Korsmeyer, S. J. (2001). MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein. Mol. Cell. Biol. 21: 2249-2258. PubMed Citation: 11259575

Feller, C., Prestel, M., Hartmann, H., Straub, T., Soding, J. and Becker, P. B. (2012). The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset. Nucleic Acids Res 40: 1509-1522. PubMed ID: 22039099

FitzGerald, K. T. and Diaz, M. O. (1999). MLL2: A new mammalian member of the trx/MLL family of genes. Genomics 59(2): 187-92. 10409430

Francis, N. J., Follmer, N. E., Simon, M. D., Aghia, G. and Butler, J. D. (2009). Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro. Cell 137: 110-122. PubMed ID: 19303136

Gerasimova, T. I. and Corces, V. G. (1998). Polycomb and trithorax group proteins mediate the function of a chromatin insulator. Cell 92(4): 511-521.


Gindhart, J. G. and Kaufman, J. C. (1995). Identification of Polycomb and trithorax group responsive elements in the regulatory region of the Drosophila homeotic gene Sex combs reduced. Genetics 139: 797-814

Goodrich, J., et al. (1997). A polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386: 44-51.

Guccione, E., et al. (2007). Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive. Nature 449: 933-937. Medline abstract: 17898714


Isono, K., et al. (2005). Mammalian Polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1. Genes Dev. 19: 536-541. 15741318

Jones, R. S. and Gelbart, W. M. (1993). The Drosophila Polycomb-group gene Enhancer of zeste contains a region with sequence similarity to trithorax. Mol Cell Biol 13: 6357-66

Kassis, J. A. (1994). Unusual properties of regulatory DNA from the Drosophila engrailed gene: three "pairing-sensitive" sites within a 1.6-kb region. Genetics 136: 1025-38

Katsani, K. R., et al. (2001). A homeotic mutation in the trithorax SET domain impedes histone binding. Genes Dev. 15: 2197-2202. 11544176

Kim, T. and Buratowski, S. (2009). Dimethylation of H3K4 by Set1 recruits the Set3 histone deacetylase complex to 5' transcribed regions. Cell 137(2): 259-72. PubMed Citation: 19379692

Kinstrie, R., Lochhead, P. A., Sibbet, G., Morrice, N. and Cleghon, V. (2006). dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX. Biochem J. 398(1): 45-54. PubMed Citation: 16671894

Koken, M. H., Saib, A. and de The, H. (1995). A C4HC3 zinc finger motif. C R Acad Sci III 318: 733-739

Komori, H., Xiao, Q., Janssens, D. H., Dou, Y. and Lee, C. Y. (2014). Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor Buttonhead. Elife 3 [Epub ahead of print]. PubMed ID: 25285447

Kuzin, B., Tillib, S., Sedkov, Y., Mizrokhi, L. and Mazo, A. (1994). The Drosophila trithorax gene encodes a chromosomal protein and directly regulates the region-specific homeotic gene forkhead. Genes-Dev. 8(20): 2478-90

Lee, M. G., Villa, R., Trojer, P., Norman, J., Yan, K. P., Reinberg, D., Di Croce, L. and Shiekhattar, R. (2007). Demethylation of H3K27 regulates Polycomb recruitment and H2A ubiquitination. Science 318: 447-450. PubMed Citation: 17761849

Liu, H., Cheng, E. H., Hsieh, J. J. (2007). Bimodal degradation of MLL by SCFSkp2 and APCCdc20 assures cell cycle execution: a critical regulatory circuit lost in leukemogenic MLL fusions. Genes Dev. 21(19): 2385-98. Medline abstract: 17908926

Mazo, A. M., et al. (1990). The trithorax gene, a trans-acting regulator of the bithorax complex in Drosophila, encodes a protein with zinc-binding domains. Proc. Natl. Acad. Sci. 87(6): 2112-6. PubMed Citation: 2107543

Milne, T. A., et al. (2002). MLL targets SET domain methyltransferase activity to hox gene promoters. Mol. Cell 10: 1107-1117. 12453418

Milne, T. A., et al. (2005). Menin and MLL cooperatively regulate expression of cyclin-dependent kinase inhibitors. Proc. Natl. Acad. Sci. 102: 749-754. PubMed citation: 15640349

Mohan, M., et al. (2011). The COMPASS family of H3K4 methylases in Drosophila. Mol. Cell. Biol. 31(21): 4310-8. PubMed Citation: 21875999

Mozer, B. A. and Dawid, I. B. (1989). Cloning and molecular characterization of the trithorax locus of Drosophila melanogaster. Proc. Natl. Acad. Sci. 86(10): 3738-42. 89264498

Muyrers-Chen, I., Rozovskaia, T., Lee, N., Kersey, J. H., Nakamura, T., Canaani, E., and Paro, R. (2004). Expression of leukemic MLL fusion proteins in Drosophila affects cell cycle control and chromosome morphology. Oncogene 23: 8639-8648. PubMed citation: 15378024

Nakamura, T., et al. (2000). huASH1 protein, a putative transcription factor encoded by a human homologue of the Drosophila ash1 gene, localizes to both nuclei and cell-cell tight junctions. Proc. Natl. Acad. Sci. 97: 7284-7289

Nakamura, T., et al. (2002). ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol. Cell 10: 1119-1128. 12453419

Nislow, C., Ray, E. and Pillus, L. (1997). SET1, A yeast member of the Trithorax family, functions in transcriptional silencing and diverse cellular processes. Mol. Biol. Cell 8(12): 2421-2436

Novotny, E., et al. (2009). In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9. Mech. Dev. 126(7): 517-22. PubMed Citation: 19393316

Oh, H., Slattery, M., Ma, L., Crofts, A., White, K. P., Mann, R. S. and Irvine, K. D. (2013). Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes. Cell Rep 3: 309-318. PubMed ID: 23395637

Orlando, V. and Paro, R. (1995). Chromatin multiprotein complexes involved in the maintenance of transcription patterns. Curr. Opin. Genet. Dev. 5: 174-179

Papp, B. and Muller, J. (2006). Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 20: 2041-2054. PubMed Citation: 16882982

Patel, A., et al. (2013). Automethylation activities within the Mixed Lineage Leukemia-1 (MLL1) core complex reveal evidence supporting a 'two-active site' model for multiple histone H3 lysine 4 methylation. J. Biol. Chem. [Epub ahead of print]. PubMed ID: 24235145

Patel, S. R., Kim, D., Levitan, I. and Dressler, G. R. (2007). The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4 methyltransferase complex. Dev. Cell 13(4): 580-92. PubMed citation: 17925232

Petruk, S., Sedkov, Y., Smith, S., Tillib, S., Kraevski, V., Nakamura, T., Canaani, E., Croce, C.M., and Mazo, A. (2001). Trithorax and dCBP acting in a complex to maintain expression of a homeotic gene. Science 294: 1331-1335. 11701926

Petruk, S., et al. (2006). Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference. Cell 127(6): 1209-21. Medline abstract: 17174895

Petruk, S., et al. (2008). Association of trxG and PcG proteins with the bxd maintenance element depends on transcriptional activity. Development 135: 2383-2390. PubMed Citation: 18550707

Petruk, S., Sedkov, Y., Johnston, D. M., Hodgson, J. W., Black, K. L., Kovermann, S. K., Beck, S., Canaani, E., Brock, H. W. and Mazo, A. (2012). TrxG and PcG proteins but not methylated histones remain associated with DNA through replication. Cell 150: 922-933. PubMed ID: 22921915

Prasad, R., et al. (1993). Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation. Cancer Res. 53(23): 5624-5628

Radman-Livaja, M., Verzijlbergen, K. F., Weiner, A., van Welsem, T., Friedman, N., Rando, O. J. and van Leeuwen, F. (2011). Patterns and mechanisms of ancestral histone protein inheritance in budding yeast. PLoS Biol 9: e1001075. PubMed ID: 21666805

Rozenblatt-Rosen, O., et al. (1998). The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex. Proc. Natl. Acad. Sci. 95(8): 4152-4157

Rozovskaia, T., et al. (1999). Trithorax and ASH1 interact directly and associate with the Trithorax group-responsive bxd region of the Ultrabithorax promoter. Mol. Cell. Biol. 19: 6441-6447

Rudenko, A., Bennett, D. and Alphey, L. (2003). Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila. EMBO Reports 4: 59-63. 12524522

Rudenko, A., Bennett, D. and Alphey, L. (2004). PP1β9C interacts with Trithorax in Drosophila wing development. Dev Dyn 231: 336-341. PubMed ID: 15366010

Schuettengruber B., et al. (2009). Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos. PLOS Biology 7: 0146-0146. PubMed Citation: 19143474

Schwartz, Y. B., Kahn, T. G., Stenberg, P., Ohno, K., Bourgon, R. and Pirrotta, V. (2010). Alternative epigenetic chromatin states of polycomb target genes. PLoS Genet. 6(1): e1000805. PubMed Citation: 20062800

Schweinsberg, S E. and Schedl, P. (2004). Developmental modulation of Fab-7 boundary function. Development 131: 4743-4749. 15329342

Sedkov, Y., et al. (1994). The bithorax complex is regulated by trithorax earlier in Drosophila embryogenesis than is the Antennapedia complex, correlating with a bithorax-like expression pattern of distinct early trithorax transcripts. Development 120: 1907-17. PubMed Citation: 7924996

Sedkov, Y., et al. (1999). Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein. Mech. Dev. 82(1-2): 171-179. PubMed Citation: 10354481

Sharif, J., Muto, M., Takebayashi, S., Suetake, I., Iwamatsu, A., Endo, T. A., Shinga, J., Mizutani-Koseki, Y., Toyoda, T., Okamura, K., Tajima, S., Mitsuya, K., Okano, M. and Koseki, H. (2007). The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450: 908-912. PubMed ID: 17994007

Shearn, A. (1989). The ash-1, ash-2 and trithorax genes of Drosophila melanogaster are functionally related. Genetics 121: 517-25. PubMed Citation: 2497049

Siebold, A. P., et al. (2009). Polycomb repressive complex 2 and Trithorax modulate Drosophila longevity and stress resistance. Proc. Natl. Acad. Sci. 107(1): 169-74. PubMed Citation: 20018689

Sierra, J., Yoshida, T., Joazeiro, C. A. and Jones, K. A. (2006). The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20(5): 586-600. 16510874

Simon, J. (1995). Locking in stable states of gene expression: transcriptional control during Drosophila development. Curr. Opin. Cell Biol. 7: 376-385. PubMed Citation: 7662368


Smith, E. R., Lee, M. G., Winter, B., Droz, N. M., Eissenberg, J. C., Shiekhattar, R. and Shilatifard, A. (2008). Drosophila UTX is a histone H3 Lys27 demethylase that colocalizes with the elongating form of RNA polymerase II. Mol. Cell. Biol. 28: 1041-1046. PubMed Citation: 18039863

Smith, S. T., Petruk, S., Sedkov, Y., Cho, E., Tillib, S., Canaani, E. and Mazo, A. (2004). Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat. Cell Biol. 6: 162-7. 14730313

Stassen, M. J., et al (1995). The Drosophila trithorax proteins contain a novel variant of the nuclear receptor type DNA binding domain and an ancient conserved motif found in other chromosomal proteins. Mech Dev 52: 209-223. PubMed Citation: 8541210

Takeda, S., et al. (2006). Proteolysis of MLL family proteins is essential for taspase1-orchestrated cell cycle progression. Genes Dev. 20: 2397-2409. PubMed citation: 16951254

Thummel, C. S. (1989). The Drosophila E74 promoter contains essential sequences downstream from the start site of transcription. Genes Dev 3 (6): 782-792. 89306615

Tie, F., et al. (2009). CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing. Development 136(18): 3131-41. PubMed Citation: 19700617

Tie, F., Banerjee, R., Saiakhova, A. R., Howard, B., Monteith, K. E., Scacheri, P. C., Cosgrove, M. S. and Harte, P. J. (2014). Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing. Development 141: 1129-1139. PubMed ID: 24550119

Tillib, S., et al. (1995). Conservation of structure and expression of the trithorax gene between Drosophila virilis and Drosophila melanogaster. Mech. Dev. 53: 113-22. PubMed Citation: 8555104

Tillib, S., et al. (1999). Trithorax- and Polycomb-group response elements within an Ultrabithorax transcription maintenance unit consist of closely situated but separable sequences. Mol. Cell. Biol. 19: 5189-5202. PubMed Citation: 10373568

Tiong, S. Y., Nash, D. and Bender, W. (1995). Dorsal wing, a locus that affects dorsoventral wing patterning in Drosophila. Development 121: 1649-1656. PubMed Citation: 7600982

Vaquero, A., et al. (2000). Functional mapping of the GAGA factor assigns its transcriptional activity to the C-terminal glutamine-rich domain. J. Biol. Chem. 275: 19461-19468. 10764754

Wong, P., et al. (2007). Meis1 is an essential and rate-limiting regulator of MLL leukemia stem cell potential. Genes Dev. 21(21): 2762-74. PubMed citation: 17942707

Xia, Z. B., et al. (2005). The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression. Proc. Natl. Acad. Sci. 102: 14028-14033. PubMed citation: 16169901

Xiao, H., et al. (2001). Dual functions of largest NURF subunit NURF301 in nucleosome sliding and transcription factor interactions. Molec. Cell 8: 531-543. 11583616

Yan, J., Chen, S. A., Local, A., Liu, T., Qiu, Y., Dorighi, K. M., Preissl, S., Rivera, C. M., Wang, C., Ye, Z., Ge, K., Hu, M., Wysocka, J. and Ren, B. (2018). Histone H3 lysine 4 monomethylation modulates long-range chromatin interactions at enhancers. Cell Res. PubMed ID: 29313530

Yano, T., et al (1997). Nuclear punctate distribution of ALL-1 is conferred by distinct elements at the N terminus of the protein. Proc. Natl. Acad. Sci. 94(14): 7286-7291. PubMed Citation: 9207083

Yokoyama, A., et al. (2007). Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol. Cell. Biol. 24(13): 5639-49. Medline abstract: 15199122

Zhang, K., et al. (2005). The set 1 methyltransferase opposes ipl 1 aurora kinase functions in chromosome segregation. Cell 122(5): 723-34. 16143104


date revised: 8 February 2018


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