InteractiveFly: GeneBrief

bithoraxoid: Biological Overview | References

This overview will present two contradictory views of the noncoding RNAs of the bithoraxoid complex. Mazo's group (Petruk, 2006) maintain that transcription of noncoding RNAs is associated with repression, while Paro's group (Schmidtt, 2005), maintain that intragenic transcription through a polycomb group response element counteracts silencing (Schmitt, 2005). It is still too early to tell which group (or both) is correct; therefore, both studies will be presented in order to give a complete view of the two arguments. Also note that studies of gene repression in vertebrates indicate that ncRNA can mediate epigenetic silencing of a chromosomal domain in trans, suggesting that ncRNAs may interact with chromatin modification enzymes to regulate gene expression in trans (Rinn, 2007). Therefore, this third study, presented immediately below, gives a third viewpoint of the function of noncoding RNAs. Finally the study by Sanchez-Elsner (2006), presented here, shows that ncRNA can mediate recruitment of the TrxG protein Ash1 to a Tre to stimulates Ubx expression.

Rinn (2007) summarizes the contradictory results. Noncoding RNAs are emerging as regulatory molecules in specifying specialized chromatin domains, but the prevalence of different mechanism by which they act is not known. In Drosophila, transcription of ncRNAs was proposed to induce HOX gene expression by activation of cis regulatory elements (Schmitt, 2005) or by ncRNA-mediated recruitment of the TrxG protein Ash1 (Sanchez-Elsner, 2006). However, an alternative model, termed 'transcriptional interference', argues that ncRNA transcription prevents the expression of 3' located Hox genes (Petruk, 2006). These two classes of models make opposite predictions on the correlation between expression of 5' ncRNA and the 3' HOX gene. The finding of widespread position-specific ncRNAs that flank and are coordinately induced with neighboring human HOX genes is consistent with models of cis activation by ncRNA transcription. Only 10% of HOX ncRNAs demonstrate anti-correlated expression pattern with their cognate 3′ HOX genes, suggesting that transcriptional interference is not the main mode of ncRNA action, at least in the cell types studied. The results are also consistent with a recent analysis of HOX gene activation during teratocarcinoma cell differentiation, where transcription of certain 5' ncRNAs immediately preceded HOX gene activation (Sessa, 2006). Transcriptional interference (Petruk, 2006) may be a more prominent mechanism during embryonic development, where its role in Hox gene expression was documented in Drosophila (Rinn, 2007).

Transcription of bxd noncoding RNAs promoted by trithorax represses Ubx in cis by transcriptional interference

Much of the genome is transcribed into long non-coding RNAs (ncRNAs). Previous data have suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex prevent silencing of Ultrabithorax (Ubx), and recruit activating proteins of the trithorax group to their maintenance elements. This study found that, surprisingly, Ubx and several bxd ncRNAs are expressed in non-overlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. The data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 (containing Trx, Sbf1 and dCBP) binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. It is proposed that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription (Petruk, 2006).

The Hox genes of the bithorax complex (BX-C) have spatially restricted expression patterns that vary within and between segments and tissues. Transcription factors encoded by segmentation genes establish the patterns of the Hox genes Ubx, abd-A, and Abd-B of the BX-C in embryos. After the segmentation proteins decay, Hox expression patterns are maintained epigenetically by proteins of the trithorax group (trxG) and the Polycomb group (PcG). PcG genes maintain the silent state, whereas trxG genes maintain the active state of Hox genes. PcG and trxG proteins act through partially overlapping sets of response elements known as maintenance elements (Petruk, 2006).

One of the most startling discoveries of the genomic era has been that much of the genome is transcribed into non-coding RNAs (ncRNAs). Recent attention has focused on small interfering RNAs (siRNAs) and micro-RNAs (miRNAs) that modulate gene activity by an antisense mechanism termed RNA interference (RNAi), which interferes with mRNA stability or translation. However, the most abundant and least characterized class of ncRNAs are long and have mostly unknown functions (Petruk, 2006 and references therein).

The intergenic regions of the Hox genes in D. melanogaster produce many long ncRNAs that may regulate Hox gene coding sequences. Increasing attention has been directed to the role of transcription of MEs in the regulation of BX-C genes. Several ncRNAs are transcribed through a well-studied ME in the bxd regulatory region that lies between the Ubx and abd-A transcription units (Cumberledge, 1990: Lipshitz, 1987; Sanchez-Herrero, 1989). The bxd ME regulates Ubx (Chan, 1994; Muller, 1991; Simon, 1993). Transcription through bxd precedes activation of Ubx coding RNAs (hereafter referred to as 'Ubx RNA', or simply as 'Ubx'), suggesting that ncRNAs might regulate Ubx (Rank, 2002). Transcription patterns of ncRNAs appear similar to those of the neighboring Hox genes and are collinear with regulatory domains along the chromosome (Bae, 2002). A synthesis of genetic (Bender, 2002; Hogga, 2002; Rank, 2002) and transgenic studies (Schmitt, 2005) led to the idea that transcription of ncRNAs through MEs interferes with PcG-mediated silencing, perhaps by preventing recruitment of PcG proteins. A recent study suggests that transcription of MEs may recruit trxG proteins to maintenance elements (Sanchez-Elsner, 2006). If indeed transcription of MEs simultaneously prevents PcG binding and establishes trxG binding, this could be a key element of Hox regulation. Clearly, this model requires that intergenic bxd RNAs are expressed in the same cells as Ubx. However double-labeling of intergenic and coding RNAs at high resolution has not been performed, so this attractive model has not been rigorously tested (Petruk, 2006).

The trxG protein complex TAC1 plays important roles in maintaining expression of homeotic genes throughout embryogenesis (Petruk, 2001). Recent attention has focused on the role of trxG proteins in histone modifications and in altering nucleosome positioning. TAC1 contains three proteins, Trx, Sbf1 and dCBP, and thus acetylates histones and methylates histone H3 at Lys-4 (H3-K4), due to the enzymatic activities of dCBP and the SET domain of Trx, respectively (Petruk, 2001; Smith, 2004). How binding of Trx to MEs regulates expression of Hox genes is unclear. Because embryos have a mixture of cells expressing and not expressing each Hox gene, it has not been possible to determine precisely how trxG protein binding correlates with transcription of Ubx and bxd ncRNAs (Petruk, 2006).

This study shows that Ubx is repressed by bxd transcription. The bxd ncRNAs do not act by siRNA or miRNA-based mechanisms, but repress Ubx in cis through a transcription-dependent mechanism. Alternative association of TAC1 with either Ubx or bxd correlates with their transcription. TAC1 appears to be part of an interdependent network of general elongation factors that associate with active genes. It is suggested that a key role of TAC1 in establishing the mosaic pattern of Ubx expression is to promote elongation of bxd ncRNAs, which in turn represses expression of Ubx (Petruk, 2006).

An attractive notion has been that transcription of bxd ncRNAs, which precedes that of Ubx in embryos, facilitates correct spatial expression of Ubx. Previous studies showed that transcription through the ME could interfere with silencing, so it was proposed that bxd ncRNA transcription normally prevents recruitment of PcG proteins to the ME. However, the current experiments unambiguously demonstrate that Ubx and bxd ncRNAs are transcribed in different cells in embryos. The results also suggest that bxd ncRNAs do not facilitate Ubx expression in larval imaginal discs, as was recently proposed (Sanchez-Elsner, 2006). Instead, transcription of ncRNAs correlates with repression of Ubx. It is possible that the abnormal transcription induced in previous studies interfered with transcription of ncRNAs in the BX-C, rather than with ME function, a possibility that can be tested experimentally. It will be interesting to use the system of sorting Ubx+ and Ubx− nuclei to examine binding of PcG proteins in nuclei where bxd ncRNAs either are, or are not, transcribed (Petruk, 2006).

The experiments rule out trans-repression by bxd ncRNAs, and instead support repression of Ubx in cis by transcription of these RNAs per se. A likely mechanism of this repression is transcriptional interference, since it was shown that ncRNA transcription extends into the region just upstream of the Ubx initiation site, which may well disrupt protein-DNA interactions required for Ubx initiation. However, this does not rule out promoter competition, and both of these mechanisms may contribute to the observed effects. Previous genetic studies (Grimaud, 2006) and the results presented in this study show that bxd ncRNAs do not work by RNAi. An RNAi-based repression mechanism has been described for the miRNA produced by the iab-4 transcript, which directly interacts with the 3'-untranslated region of Ubx and prevents translation (Ronshaugen, 2005). These authors show that ectopic expression of iab-4 leads to homeotic phenotypes in the haltere, but do not show that loss of RNAi prevents this effect, nor has the effect of loss of function mutations of the iab-4 transcript been tested, so it remains to be seen if the iab-4 transcript is a bona fide miRNA (Petruk, 2006).

Since significant levels of bxd ncRNAs were not detected in imaginal discs, nor do they persist to late embryonic stages, they are unlikely be responsible for repression of Ubx throughout development. In fact, Papp (2006) report that Trx is bound to the bxd ME in both wing and haltere discs, which have low and high levels of Ubx expression, respectively. The difference between binding of Trx to the bxd ME in embryos and in discs (Papp, 2006) may be a consequence of the absence of transcription of bxd ncRNAs in discs and its presence in embryos, or to other uncharacterized differences between Ubx regulation in embryos and discs. Also, since Trx binds constitutively in some areas of the ME, Papp may have detected such binding in imaginal discs (Petruk, 2006).

Intergenic transcription also cannot explain repression of Ubx in the anterior of the embryo, where it is thought that hunchback and PcG genes set up and maintain the anterior boundary of Ubx expression. However, the pattern of bxd ncRNA transcription, which prefigures, in a complementary fashion, the mosaic pattern of Ubx expression within the parasegments of the embryonic trunk, appears to be essential for proper Ubx initiation. The Ubx pattern may then be maintained or modified at later embryonic stages through repression by other Hox proteins (i.e., abdA and AbdB) and by PcG genes. Thus, maintenance of Ubx expression likely requires multiple mechanisms that are employed at different developmental stages (Petruk, 2006).

The data support a role for Trx in transcriptional elongation as a mechanism for maintenance of a developmentally regulated gene. It has been argued that Trx does not have a direct role in activation of homeotic genes in Drosophila, but instead prevents repression of transcription by PcG proteins. However, the current data suggest that trx is required for recruitment of elongation factors and for efficient completion of transcripts. Therefore, maintenance of transcriptional activity by Trx may be a consequence of its role in elongation, and a block in elongation might lead to the establishment of PcG-mediated repression. Alternatively, Trx may be required only for normal levels of Hox gene expression, and not for maintenance of low levels of expression, a possibility consistent with at least some aspects of the trx mutant phenotype (Petruk, 2006).

This work strongly supports a general role for Trx and TAC1 in transcription, and agrees with previous findings that TAC1 relocates from other genes to the transcribed region of hsp70 following induction of the cellular stress response (Smith, 2004). The histone methyltransferase activity of Set1, the SET domain protein homologous to Trx, has a role in transcription, and MLL was suggested to play a similar role in mammals. It is suggested that this role is in transcriptional elongation, because Trx and elongation factors are co-ordinately recruited, because Trx binds downstream of the promoter more strongly to the 5’ than the 3’ end, and because transcripts extending to the 3’ end are more strongly affected by trx mutations, for both Ubx and bxd ncRNAs (Petruk, 2006).

TAC1 is also present at the promoter, and this is unaffected by mutations in elongation factors. Therefore, association of TAC1 with the promoter likely precedes the recruitment of elongation factors. Thus, TAC1 may play several distinct roles, one in initiation, another during the recruitment of the elongation complex and perhaps a third during subsequent elongation, where its ability to modify histones may be required for effective completion of long transcripts (Petruk, 2006).

This work provides the first direct evidence of the involvement of long ncRNAs in regulation of homeotic genes of Drosophila. Repression of Ubx is apparently mediated by expression of several intergenic ncRNAs in different germ layers of Ubx-expressing parasegments. TAC1 may be required for efficient read-through by Pol II into the region upstream of the Ubx initiation site, and as a result, for efficient repression of Ubx. Therefore, a direct link is proposed between elongation facilitated by the TAC1 epigenetic complex and repression of Ubx by intergenic transcription. A goal for the future will be to determine if other homeotic genes of Drosophila, and of other organisms, are also regulated by long ncRNAs whose expression is regulated by TAC1 proteins (Petruk, 2006).

Intergenic transcription through a polycomb group response element counteracts silencing

Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, this study shows that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex that were tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system (Schmitt, 2005).

A previous study has shown that in wild-type Drosophila embryos, the characterized PREs of the BX-C are transcribed in a spatially and temporally regulated manner which reflects the pattern of activity of their associated genes (Rank, 2002). The analysis of the transgenic system used in this study indicates that transcription through the Fab-7 element until the beginning of the first-instar larval stage is sufficient for the stable activation of this PRE. Since these data were obtained in a transgenic background, it was next asked whether endogenous PRE sequences are transcribed only during early development or whether transcription persists until later stages (Schmitt, 2005).

In the brain of third-instar larvae, the homeotic genes are expressed in a characteristic pattern along the anterior-posterior axis, following the principle of spatial colinearity. If transcription through PREs is required for the maintenance of epigenetic activation, it would be expected that the bxd, Mcp, and Fab-7 elements would also be transcribed in this tissue. By in situ hybridizations, RNAs spanning the bxd, Mcp, and Fab-7 sequences were detected in a pattern that reflects the expression of the target genes Ubx and AbdB, respectively. PRE transcripts were detected only in the sense direction with respect to the orientation of the coding genes. In contrast to the mRNAs, the noncoding RNAs showed a punctate staining in the brain tissue, suggesting that they are localized to the nuclei. Fluorescent in situ hybridizations in combination with DAPI staining showed the Fab-7 transcripts in the brain of third-instar larvae to appear as single or doublet spots within the cell nuclei. In line with previous studies, in which noncoding transcripts through infra-abdominal (iab) regions of the BX-C were found to be restricted to the nucleus in embryos, the Fab-7, Mcp, and bxd RNAs were detected at discrete loci within the nucleus at this stage. It is expected that these signals represent nascent, noncoding transcripts, suggesting that they may be degraded following their synthesis. To determine the relative amounts of RNA accounting for these signals, the expression levels of total and nascent AbdB mRNA were compared with the level of noncoding Fab-7 RNA in SF4 tissue culture cells by real-time RT-PCR. The results from this analysis showed that the level of Fab-7 RNA is approximately sixfold less abundant than the amount of AbdB target mRNA (Schmitt, 2005).

The finding that PRE transcripts can be detected in the brain of third-instar larvae suggests that in the context of the endogenous BX-C, continued transcription through PREs may be required for the stable maintenance of the epigenetically activated state (Schmitt, 2005).

Apart from the homeotic genes, the PcG/trxG memory system is known to regulate many more targets. It was thus asked whether PcG-regulated gene transcription is accompanied by transcription through the associated PREs at loci other than the BX-C. To test this, RNA probes were designed directed against the genetically characterized engrailed (en) PRE, as well as against three predicted PREs that potentially regulate the genes slouch (slou), spalt major (salm), and tailless (tll). The genetically characterized PRE of the en gene and the predicted PREs of the salm and tll genes are localized in the respective promoter regions, whereas the predicted PRE of the slou gene lies 3 kb upstream of the transcriptional start site. In situ hybridizations showed that the en PRE as well as the predicted PREs of the slou, salm, and tll genes are transcribed in embryos. The transcription through these sequences shows the same spatial regulation as that of the mRNAs of the known (en) and predicted target genes (slou, salm, and tll). For the en, slou, and salm PREs, transcripts of both sense and antisense strands were detected, whereas transcripts of the tll PRE were only detected in the antisense orientation. In addition, the putative PRE transcripts display a nuclear localization similar to that of the bxd, Mcp, and Fab-7 RNAs. Three of the transcribed PRE sequences lie in the promoter regions of their associated genes. Interestingly, the antisense strand of the predicted tll PRE is also transcribed in the optic lobes of the brain in third-instar larvae, where the tll mRNA is also expressed. In contrast, no transcription was detected through the en and salm PREs at this stage, although the mRNAs for both genes are transcribed at high levels (Schmitt, 2005).

These results suggest that the activation of a gene does not result in spurious transcription through its promoter region, but rather that the transcription observed in embryos is involved in triggering the epigenetic activation of the known and predicted PREs tested. The fact that transcripts were detected only through the tll PRE and not through the en and salm PREs later in development suggests that the maintenance of the activated state may be regulated by different mechanisms in different tissues. Alternatively, the en and salm genes might be controlled by more than one PRE, which are differentially deployed in different tissues and would thus show a differential pattern of epigenetic activation and noncoding transcription (Schmitt, 2005).

This work has present evidence that transcription through a PRE prevents silencing and switches the maintenance mode of the element to activity. These results with the transgene system indicate that noncoding transcription is not a consequence of gene activation but is causally related to the prevention of silencing. The process of transcription appears to be central to this mechanism, while the RNAs produced seem to play a minor role, if they do have a function at all (Schmitt, 2005).

Intergenic transcription in the BX-C has a profound phenotypic effect on the expression of the Hox genes. Results with transgenes suggest that the spatially and temporally regulated transcription of noncoding RNAs in the BX-C induces a remodeling of the chromatin, thereby preventing PcG-mediated silencing. The consequence of this is the segment-specific activation of the Hox genes. This is probably especially important in large gene complexes where PREs are located at long distances from the promoters they regulate. In a transgenic assay, it has been shown that the activation of a minimal 219-bp Fab-7 PRE is not accompanied by transcription through the element. However, in this case the minimal PRE was juxtaposed to the promoter, probably benefiting from the open chromatin environment induced by the bound transcription factors. Other results (Rank, 2002), however, indicate that an 870-bp large Fab-7 PRE, under similar conditions but containing more PcG protein-binding sites, cannot be activated anymore. This suggests that over a certain threshold level of silencing, imposed by the stability of the silencing complexes, chromatin remodeling by transcription is required to remove PcG complexes in order to counteract their silencing activity in a mitotically heritable fashion (Schmitt, 2005).

With a transgenic reporter system, this study showed that the constitutive transcription through the Fab-7 PRE from the actin5c promoter results in the stable epigenetic activation of this element. This raises the question of how this could be achieved mechanistically. As has been proposed for the developmental regulation of globin gene expression and the regulation of VDJ_H-recombination in mice, the opening of the chromatin structure at a transcribed PRE may be induced by the passing of the transcriptional machinery through the regulatory sequence. It has been shown that the elongating RNA polymerase II complex is associated with the SWI/SNF remodeling complex and a histone acetyl transferase activity. Such enzymatic activities linked with the transcription machinery may catalyze the epigenetic modification of chromatin (Schmitt, 2005).

In this respect, it is interesting to note that most of the trxG mutants were initially uncovered as suppressors of the Pc phenotype, and that the combination of PcG with trxG mutations can restore the typical phenotype of the single mutations to wild type. The molecular mechanism behind this antagonism is not clear. Using clonal analysis, it as been reported that the trxG proteins Ash1 and Trx do not function as coactivators of Hox gene expression, but that they are required as anti-repressors to prevent PcG-induced silencing. In contrast, the Brahma complex containing the trxG proteins Brm, Osa, and Mor was shown to act as a coactivator of transcription, and a subset of trxG genes encode components of the Mediator complex. The finding that transcription through Fab-7 induces the epigenetic activation of this PRE may explain how trxG complexes involved in more general transcriptional processes antagonize the establishment of PcG silencing (Schmitt, 2005).

Interestingly, in budding yeast, intergenic transcription through a promoter has been shown to prevent the binding of a transcriptional activator to its target sequences. It is possible that, in a similar fashion, the transcription through PREs may lead to the displacement of repressive PcG complexes from the chromatin and/or the prevention of PcG recruitment to the PRE in the first place. Enzymatic activities carried by the RNA polymerase II complex may subsequently or in addition modify histones at PREs with positive epigenetic marks like acetyl or methyl moieties. Interestingly, it was recently shown that the sequential induction of HoxB gene expression in mouse embryonic stem (ES) cells by retinoic acid correlates with the orchestrated looping out of this locus from chromosome territories. This indicates that, in addition to locus-wide changes in the chromatin structure such as histone modifications, the transcriptional activity of genes may be regulated by an additional order of complexity. It is possible that, in addition to inducing 'small-scale' changes in the chromatin structure, transcription through PREs may lead to a subnuclear relocation of the target gene locus from a repressive into a transcriptionally permissive environment (Schmitt, 2005).

Removal or inhibition of binding of PcG silencing complexes to PREs by tissue-specific transcription appears to be an attractive mechanism to counteract the constant pressure of the repressive system acting by default. However, with such a solution, the problem of epigenetic maintenance is simply moved to another level, openging the question of what prevents the intergenic transcription from being silenced by the PcG. The simple answer that promoters of noncoding transcripts are not sensitive to PcG/PRE silencing is probably not valid. Noncoding transcripts of the BX-C are activated by the same set of early segmentation genes as the Hox protein encoding mRNAs. As such, their subsequent regulation might be subjected to the same regiment of factors as the protein-encoding transcripts. However, the problem of transcriptional memory might be reduced to the problem of how to inhibit PcG silencing in particular cells/tissues, while the rest will be down-regulated by default (Schmitt, 2005).

With the processive transcription as the central issue, the cell cycle might become an important factor for the maintenance of active transcription. In Drosophila, PcG proteins dissociate from the chromosomes at mitosis. Thus, if after mitosis intergenic transcription started before PcG proteins rebind to the PREs, the chromatin would be turned into an active mode, and would thus be protected from PcG-mediated silencing until the next round of cell division. At this point, it remains an open question whether continuous transcription of noncoding RNAs is required throughout the cell cycle to prevent the PcG complexes from rebinding, or whether the initial setting of positive epigenetic marks by the early transcription process is sufficient to prevent silencing during interphase. This proposed mechanism further reduces the problem of how transcriptional activity is maintained to the problem of how only a positive epigenetic mark is maintained during DNA replication and mitosis. Here, recent advances in studies of histone variants propose some attractive candidate marks. In particular, the histone variant H3.3 associated with the transcription of active genes could be envisaged as a possible positive signal that is locally maintained and propagated at cell division. As has been suggested before, targeted deposition of the H3.3 variant at sites of active transcription may serve to remove repressive epigenetic marks such as methylation. It was shown that the establishment of stable PcG silencing complexes not only requires a sequence component, but is also accompanied by the methylation of K9 and K27 of histone H3. In contrast, positive marks, which have been shown to be mainly associated with H3.3 compared to H3, would be specifically enriched at a transcribed PRE and transmitted through mitosis. After cell division, these epigenetic marks may then in turn provide a platform for noncoding transcription through the PRE early in the cell cycle, which itself may re-establish the full active chromatin environment, unsuitable for PcG protein binding and silencing. Additionally, the reported result that an activated PRE is still maintained over a certain period after transcription has ceased (by removal of the promoter by the inducible Cre recombinase, suggests that this positive epigenetic signal is quite stable and is only diluted out by multiple cell divisions (Schmitt, 2005).

In summary, it is proposed that transcriptional maintenance during development by the PcG/trxG system is primarily a process of preventing PcG silencing to occur at those target genes that need to be maintained active in a defined cell lineage. The advantage of this mode of action is that a positive epigenetic mark, surviving DNA replication and mitosis, is sufficient to ensure stable and heritable maintenance of gene expression patterns, as the silenced mode is created by default. As such, transcription of intergenic sequences would serve as an anti-silencing mechanism that would continually counteract the initiation of this PcG-mediated silencing. In the future, it will be important to pursue the involvement of the various trxG components in the establishment and maintenance of regulatory transcription mechanisms and to analyze the link to cell cycle control and the identity and propagation of the positive epigenetic marks required to sustain active transcription (Schmitt, 2005).

Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs

Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. This study characterized the transcriptional landscape of the four human HOX loci at five base pair resolution in eleven anatomic sites, and identified 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes, possess unique sequence motifs, and their expression demarcate broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. A 2.2 kilobase ncRNA was identified residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states (Rinn, 2007).

By analyzing the transcriptional and epigenetic landscape of the HOX loci at high resolution in cells with many distinct positional identities, a panoramic view was obtained of multiple layers of regulation involved in maintenance of site-specific gene expression. The HOX loci are demarcated by broad chromosomal domains of transcriptional accessibility, marked by extensive occupancy of RNA polymerase II and H3K4 dimethylation and, in a mutually exclusive fashion, by occupancy of PRC2 and H3K27me3. The active, PolII-occupied chromosomal domains are further punctuated by discrete regions of transcription of protein-coding HOX genes and a large number of long ncRNAs. These results confirm the existence of broad chromosomal domains of histone modifications and occupancy of HMTases over the Hox loci, and extend on those observation in several important ways (Rinn, 2007).

First, by comparing the epigenetic landscape of cells with distinct positional identities, it was showm that the broad chromatin domains can be programmed with precisely the same boundary but with diametrically opposite histone modifications and consequences on gene expression. The data thus functionally pinpoint the locations of chromatin boundary elements in the HOX loci, the existence of some of which have been predicted by genetic experiments. One such boundary element appears to reside between HOXA7 and HOXA9. This genomic location is also the switching point in the expression of HOXA genes between anatomically proximal versus distal patterns and is the boundary of different ancestral origins of HOX genes, raising the possibility that boundary elements are features demarcating the ends of ancient transcribed regions. Second, the ability to monitor 11 different HOX transcriptomes in the context of the same cell type conferred the unique ability to characterize changes in ncRNA regulation that reflect their position in the human body. This unbiased analysis identified more than 30 kb of new transcriptional activity, revealed ncRNAs conserved in evolution, mapped their anatomic patterns of expression, and uncovered enriched ncRNA sequence motifs correlated with their expression pattern -- insights which could not be gleamed from examination of EST sequences alone. The finding of a long ncRNA that acts in trans to repress HOX genes in a distant locus is mainly due to the ability afforded by the tiling array to comprehensively examine the consequence of any perturbation over all HOX loci. The expansion of a handful of Hox-encoded ncRNAs in Drosophila to hundreds of ncRNAs in human HOX loci suggests increasingly important and diverse roles for these regulatory RNAs (Rinn, 2007).

An important limitation of the tiling array approach is that while improved identification of transcribed regions is obtained, the data does not address the connectivity of these regions. The precise start, end, patterns of splicing, and regions of double-stranded overlap between ncRNAs will need to be addressed by detailed molecular studies in the future (Rinn, 2007).

The results uncovered a new mechanism whereby transcription of ncRNA dictates transcriptional silencing of a distant chromosomal domain. The four HOX loci demonstrate complex cross regulation and compensation during development. For instance, deletion of the entire HOXC locus exhibits a milder phenotype than deletion of individual HOXC genes, suggesting that there is negative feedback within the locus. Multiple 5' HOX genes, including HOXC genes, are expressed in developing limbs, and deletion of multiple HOXA and HOXD genes are required to unveil limb patterning defects. The results suggest that deletion of the 5' HOXC locus, which encompass HOTAIR, may lead to transcriptional induction of the homologous 5' HOXD genes, thereby restoring the total dosage of HOX transcription factors. How HOX ncRNAs may contribute to cross-regulation among HOX genes should be addressed in future studies (Rinn, 2007).

HOTAIR ncRNA is involved in Polycomb Repressive Complex 2-mediated silencing of chromatin. Because many HMTase complexes lack DNA binding domains but possess RNA binding motifs, it has been postulated that ncRNAs may guide specific histone modification activities to discrete chromatin loci. This study has shown that HOTAIR ncRNA binds PRC2 and is required for robust H3K27 trimethylation and transcriptional silencing of the HOXD locus. HOTAIR may therefore be one of the long sought after RNAs that interface the Polycomb complex with target chromatin. A potentially attractive model of epigenetic control is the programming of active or silencing histone modifications by specific noncoding RNAs. Just as transcription of certain ncRNA can facilitate H3K4 methylation and activate transcription of the downstream Hox genes (Sanchez-Elsner, 2006; Schmitt, 2005), distant transcription of other ncRNAs may target the H3K27 HMTase PRC2 to specific genomic sites, leading to silencing of transcription and establishment of facultative heterochromatin. In this view, extensive transcription of ncRNAs is both functionally involved in the demarcation of active and silent domains of chromatin as well as being a consequence of such chromatin domains (Rinn, 2007).

Several lines of evidence suggest that HOTAIR functions as a bona fide long ncRNA to mediate transcriptional silencing. First, full length HOTAIR is detected in vivo and in primary cells, but not small RNAs derived from HOTAIR indicative of miRNA or siRNA production. Second, depletion of full length HOTAIR led to loss of HOXD silencing and H3K27 trimethyation by PRC2, and third, endogenous or in vitro transcribed full length HOTAIR ncRNA physically associated with PRC2. While these results do not rule out the possibility that RNA interference pathways may be subsequently involved in PcG function, they support the notion that the long ncRNA form of HOTAIR is functional. The role of HOTAIR is reminiscent of XIST, another long ncRNA shown to be involved in transcriptional silencing of the inactive X chromosome. An important difference between HOTAIR and XIST is the strictly cis-acting nature of XIST. HOTAIR is the first example of a long ncRNA that can act in trans to regulate a chromatin domain. While a trans repressive role for HOTAIR was observed, the data do not permit ruling out a cis-repressive role in the HOXC locus. siRNA-mediated depletion of HOTAIR was substantial but incomplete; further, the proximity between the site of HOTAIR transcription and the neighboring HOXC locus may ensure significant exposure to HOTAIR even if the total pool of HOTAIR in the cell were depleted. The precise location of HOTAIR at the boundary of a silent chromatin domain in the HOXC locus makes a cis-repressive role a tantalizing possibility. Judicious gene targeting of HOTAIR may be required to address its role in cis-regulation of chromatin (Rinn, 2007).

The discovery of a long ncRNA that can mediate epigenetic silencing of a chromosomal domain in trans has several important implications. First, ncRNA guidance of PRC2-mediated epigenetic silencing may operate more globally than just in the HOX loci, and it is possible that other ncRNAs may interact with chromatin modification enzymes to regulate gene expression in trans. Second, PcG proteins are important for stem cell pluripotency and cancer development; these PcG activities may also be guided by stem cell or cancer-specific ncRNAs. Third, Suz12 contains a zinc finger domain, a structural motif that can bind RNA, and EZH2 and EED both have in vitro RNA binding activity. The interaction between HOTAIR and PRC2 may also be indirect and mediated by additional factors. Detailed studies of HOTAIR and PRC2 subunits are required to elucidate the structural features that establish the PRC2 interaction with HOTAIR. As is illustrated in this study, high throughput approaches for the discovery and characterization of ncRNAs may aid in dissecting the functional roles of ncRNAs in these diverse and important biological processes (Rinn, 2007).

Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax

Homeotic genes contain cis-regulatory trithorax response elements (TREs) that are targeted by epigenetic activators and transcribed in a tissue-specific manner. The transcripts of three TREs located in the Drosophila homeotic gene Ultrabithorax mediate transcription activation by recruiting the epigenetic regulator Ash1 to the template TREs. TRE transcription coincides with Ubx transcription and recruitment of Ash1 to TREs in Drosophila. The SET domain of Ash1 binds all three TRE transcripts, with each TRE transcript hybridizing with and recruiting Ash1 only to the corresponding TRE in chromatin. Transgenic transcription of TRE transcripts restores recruitment of Ash1 to Ubx TREs and restores Ubx expression in Drosophila cells and tissues that lack endogenous TRE transcripts. Small interfering RNA-induced degradation of TRE transcripts attenuates Ash1 recruitment to TREs and Ubx expression, which suggests that noncoding TRE transcripts play an important role in epigenetic activation of gene expression (Sanchez-Elsner, 2006).

The identity of cells in metazoan organisms is established during development and mitotically propagated throughout the entire life cycle. Phylogenetically highly conserved protein families of epigenetic regulators determine the fate of developing cells by establishing and maintaining mitotically stable gene expression programs. In Drosophila, members of the trithorax group (trxG) of epigenetic regulators maintain active transcription states, whereas members of the Polycomb group (PcG) maintain repressed transcription states. Many epigenetic regulators control gene expression by establishing transcriptional competent or silent chromatin structures. Several epigenetic activators [Trx, trithorax-related (Trr)] and repressors (Enhancer of zeste) are lysine-specific histone methyltransferases (HMTs) and contain a SET domain, the catalytic hallmark motif of HMTs. Methylation of lysine residues in histones H3 and H4 has been correlated with epigenetic activation [Lys⁴ in H3 (H3-K4)] and repression [Lys⁹ and Lys²⁷ in H3 (H3-K9)] (Sanchez-Elsner, 2006).

The epigenetic activator Absent small and homeotic discs(Ash1) promotes transcriptional activation by trimethylating H3-K4, H3-K9, and Lys²⁰ in H4 (H4-K20). Ash1 maintains activated transcription states in larval imaginal discs that give rise to the appendages in the adult fly. For example, Ash1 is essential for the expression of the homeotic gene Ultrabithorax (Ubx) in third-leg and haltere imaginal discs, and Ubx expression coincides with Ash1-mediated histone methylation (Sanchez-Elsner, 2006).

PcG and trxG regulators are recruited to specific chromosomal elements that are present in the cis-regulatory region of target genes. The same element can act as an activating or a silencing module. In the repressed state, the elements represent Polycomb response elements (PREs) and facilitate the recruitment of PcG proteins. In the activated state, the DNA-elements function as trithorax response elements (TREs) and recruit trxG proteins. Transcription of noncoding RNAs (ncRNAs) from TRE/PRE elements switches silent PREs into TREs, which indicates that TRE/PRE transcription plays an important role in epigenetic activation. How transcription of TREs culminates in the recruitment of trxG regulators is unknown. This study addressd the question of how epigenetic regulators without known DNA binding capabilities, such as Ash1, recognize and bind target genes in chromatin (Sanchez-Elsner, 2006).

The coincidence of the tissue-specific transcription and trans-regulatory activity patterns of TREs and trxG proteins, respectively, suggests that not only TRE/PRE transcription but also the resulting ncRNAs might play a role in epigenetic activation. This study analyzed the role of ncRNAs transcribed from three Ubx TRE/PREs. The Ubx locus contains a cluster of three characterized TRE/PREs (TRE1 to TRE3; see TRE1 to TRE3 in the UCSC genome browser) within the boundaries of the chromosomal memory element (CME) bxd that is located 22 kb upstream of the Ubx promoter (Sanchez-Elsner, 2006).

To correlate the transcriptional activity of Ubx with bxd transcription in Drosophila, RACE was used to detect bxd transcripts in third-leg discs. Three capped, polyadenylated bxd transcripts transcribed by RNA polymerase II (tre1, tre2, tre3) were detected in third-leg and haltere discs (Sanchez-Elsner, 2006).

The RT-PCR was used to determine whether the presence of the three TRE transcripts coincides with Ubx transcription. RNA was isolated from third-leg discs and haltere imaginal discs (haltere discs), which both transcribe Ubx, and from wing imaginal discs (wing discs) and embryonic Drosophila Schneider 2 (S2) cells that do not transcribe Ubx. Transcripts from Ubx and all three TREs were detected in third-leg and haltere discs, whereas Ubx and TRE transcripts were not detected in S2 cells and wing discs (Sanchez-Elsner, 2006).

To investigate whether Ash1 is recruited to transcriptionally active Ubx TREs, in vivo cross-linked chromatin immunoprecipitation (XChIP) was used to detect Ash1 at the Ubx TREs in third-leg, haltere, and wing discs and in S2 cells, all of which express ash1. Ash1 was detected at all three TREs in third-leg and haltere discs. In addition, the characteristic Ash1 histone methylation pattern was detectable in all three TREs and the transcriptionally active Ubx promoter in third-leg discs. Ash1 was not detected at the TREs of the transcriptionally inactive Ubx locus in wing discs and S2 cells, which do not transcribe TREs (Sanchez-Elsner, 2006).

The recruitment of Ash1 to Ubx was compared in wild-type and homozygous mutant ash1²² third-leg discs by XChIP. The ash1²² mutant is recessive lethal and expresses a truncated protein that lacks the SET domain and trans-activation activity. Ash1 and the characteristic Ash1 histone methylation pattern were detected at the transcriptionally active Ubx locus in wild-type discs but not in ash1²² mutant discs; this finding indicates that recruitment of Ash1 and Ash1-mediated histone methylation coincides with activation of Ubx expression in third-leg discs. TRE transcription was monitored in the wild-type and ash1²² mutant third-leg discs by RT-PCR. TRE transcripts were detected at comparable levels in wild-type and mutant discs, which indicates that Ash1 is not a major regulator of TRE transcription in imaginal discs (Sanchez-Elsner, 2006).

The association of Ash1 with TREs in cells producing TRE transcripts suggests that TRE transcription or TRE transcripts nucleate recruitment of Ash1 to Ubx TREs. SET-domain proteins can bind single-stranded RNA and DNA in vitro, and ncRNA has been implicated in protein recruitment in gene dosage compensation. In vitro protein-RNA binding assays were used to assess whether Ash1 associates with TRE transcripts. Ash1SET, which consists of amino acids 1001 to 1619, retained TRE1(+), TRE2(+), and TRE3(+) but not the H3-K9-specific HMT Medusa (Mdu). In contrast, Ash1, Ash1DeltaN, and Mdu did not bind the antisense RNA of the Ubx TREs. Ash1DeltaN did not interact with the N-element in tre2, which corresponds to the DNA spacer separating TRE-2 and TRE-3 (Sanchez-Elsner, 2006).

In competition experiments, unlabeled TRE transcripts could outcompete the interaction of Ash1 with the corresponding TRE transcript. In contrast, double-stranded TRE transcripts, double-stranded DNA TRE sequences, and DNA-RNA hybrids consisting of TRE transcripts and TREs failed to disrupt the interaction; these findings suggest that Ash1 associates with single-stranded TRE transcripts (Sanchez-Elsner, 2006).

To delineate the RNA-binding motif of Ash1, the interaction of truncated ash1 proteins with TRE transcripts was investigated. In addition to Ash1SET, Ash1DeltaN (amino acids 1001 to 2218), which contains the Ash1 SET module, and Ash1N (amino acids 1 to 1001) and Ash1C (amino acids 1619 to 2218), which both lack the SET domain and cysteine-rich regions, were tested. Ash1DeltaN and Ash1SET, but not Ash1N and Ash1C, retained TRE transcripts, indicating that the SET domain of Ash1 binds TRE transcripts in vitro (Sanchez-Elsner, 2006).

XChIP was used to investigate whether Ash1 associates with TRE transcripts in vivo. Ash1 coprecipitated with TRE transcripts but not control transcripts from mock-treated chromatin. Ash1 binds TRE transcripts in ribonuclease (RNase) III-treated chromatin, indicating that double-stranded RNA (dsRNA) motifs within TRE transcripts do not mediate the association of TRE transcripts with Ash1. In contrast, Ash1 did not interact with TRE transcripts from RNase A- and RNase H-treated chromatin, indicating that single-stranded RNA (ssRNA) is important for the association of Ash1 with TRE transcripts. The disruption of the association between Ash1 and TRE transcripts by RNase H (which degrades DNA-RNA hybrids) in chromatin suggests that TRE transcripts hybridize with DNA in chromatin (Sanchez-Elsner, 2006).

Is the association of Ash1 with TREs dependent on RNA? XChIP to compare the interaction of Ash1 and TRE in mock- and RNase-treated chromatin. Antibodies to Ash1 precipitated all three TREs, but not the spacer DNAs, from mock-treated and RNase III-treated chromatin, indicating that dsRNA does not contribute to the interaction of Ash1 with TREs. In contrast, treating chromatin with RNase H or RNase A attenuated the association of Ash1 with TREs, indicating that the association of Ash1 with the Ubx TREs is RNA-dependent. The disruption of the interaction of Ash1 with TREs in chromatin by RNase H and RNase A raises the possibility that ssRNA motifs in RNA-DNA hybrids play a role in the recruitment of Ash1 to TREs (Sanchez-Elsner, 2006).

To verify that the observed attenuation of Ash1-TRE interactions is based on specific rather than general disruption of protein-DNA interactions in RNase-treated chromatin, the recruitment of the general transcription factor TFIID to target genes was investigated in mock- and RNase-treated chromatin. The TATA-binding protein (TBP) subunit of TFIID interacts with the TATA box in eukaryotic promoters. PCR detected the interaction of TBP with the promoter of Ubx and string, whose transcription requires TFIID activity. TBP interacted with both promoters in mock-treated and RNase A-, RNase H-, and RNase III-treated chromatin, indicating that RNase treatment did not attenuate TBP-promoter interactions and protein-gene interactions in general (Sanchez-Elsner, 2006).

To test whether the detected association of Ash1 with TREs and TRE transcripts occurs in chromatin or is the result of fortuitous interactions generated in chemically cross-linked chromatin, the association of Ash1 with TRE transcripts and TREs in native chromatin was investigated with the use of native chromatin immunoprecipitation (NChIP). Ash1 bound all three TREs and TRE transcripts in mock- and RNase III-treated chromatin but not in RNase H- or RNase A-treated chromatin, indicating that Ash1 coimmunoprecipitates with TREs and TRE transcripts in native chromatin. An association of Ash1 with the N portion of the TRE2(+) transcript, as observed in cross-linked chromatin, was not detectable in native chromatin; this result indicates that, as in vitro, Ash1 binds the RNA corresponding to TRE-2 but not the N region of the TRE2(+) transcript (Sanchez-Elsner, 2006).

Collectively, these data indicate that the recruitment of Ash1 to the TREs of Ubx is mediated by RNA and suggests the existence of a trimeric protein-nucleic acid complex in chromatin, consisting of Ash1, TREs, and TRE transcripts (Sanchez-Elsner, 2006).

Ash1 is detectable at about 150 loci on Drosophila polytene chromosomes. To assess whether RNA facilitates Ash1 recruitment to target loci other than Ubx, the interaction was compared of Ash1 with target loci on mock- and RNase-treated chromosome squashes. Compared to mock-treated chromosomes, RNase treatment attenuated the association of Ash1 with the majority of the target loci. This result suggests that RNA plays an important role in the recruitment of Ash1 to target genes in chromatin (Sanchez-Elsner, 2006).

To assess whether TRE transcripts associate with chromatin, whether Ash1 coprecipitates TRE transcripts from chromatin-free nuclear extract was investigated. Ash1 bound TRE transcripts in chromatin but not chromatin-free nuclear extract, indicating that TRE transcripts are preferentially associated with chromatin in the cell (Sanchez-Elsner, 2006).

XChIP was used to determine whether the association of Ash1 with TRE transcripts precedes the recruitment of Ash1 to TREs in chromatin, or vice versa. In vivo cross-linked chromatin was isolated from wild-type and ash1²² mutant third-leg discs, sheared, and immunoprecipitated with antibodies to dimethylated H3-K9 present at the TREs of the transcriptionally active and inactive Ubx locus in third-leg discs. The antibody to dimethylated H3-K9 coprecipitated with TREs and TRE transcripts from the chromatin of wild-type and ash1²² third-leg discs, indicating that TRE transcripts are retained at Ubx TREs before recruitment of Ash1 (Sanchez-Elsner, 2006).

To dissect the role of TRE transcripts in Ubx transcription, it was asked whether transiently transcribed TRE transcripts could restore the recruitment of Ash1 to Ubx TREs and Ubx expression in S2 cells, which express Ash1 but lack endogenous TRE transcripts. S2 cells were transiently transfected with plasmids transcribing sense or antisense TRE transcripts. In PCR assays, Ubx transcription was undetectable in S2 cells transiently transcribing antisense TRE transcripts or mdu. In contrast, Ubx transcription was activated by one TRE transcript and cooperatively activated by multiple TRE transcripts (Sanchez-Elsner, 2006).

XChIP was used to determine whether activation of Ubx transcription by transient TRE transcripts coincides with the recruitment of Ash1 to TREs. In vivo cross-linked chromatin was isolated from wild-type S2 cells and cells transiently transcribing one or multiple TRE transcripts and control RNAs, and it was then immunoprecipitated with antibodies to Ash1 and the Ash1 histone methylation pattern. Ash1 was not detected at the TREs of transcriptionally silent Ubx in cells transcribing mdu or antisense TRE RNAs. In contrast, Ash1 and the Ash1 histone methylation pattern were detected at the Ubx TREs in cells transcribing TRE1(+), TRE2(+), and/or TRE3(+). Each of the three TRE transcripts facilitated the association of Ash1 only with the corresponding template TRE but not with other TREs (Sanchez-Elsner, 2006).

To verify the specificity of the described recruitment, whether TRE transcripts facilitate recruitment of Ash1 to CMEs containing TREs/PREs and genes other than Ubx was investigated. In XChIP assays, Ash1 was not detected at Drosophila genes and the CMEs MCP and Fab7 in S2 cells transcribing TRE1(+), TRE2(+), or TRE3(+). Thus, TRE transcripts facilitate Ash1 recruitment specifically to the corresponding TRE template DNA (Sanchez-Elsner, 2006).

NChIP and XChIP were used to assess whether transiently transcribed TRE transcripts associate with TREs and Ash1 in chromatin. Native chromatin was isolated from wild-type S2 cells and S2 cells transiently cotranscribing all three sense or antisense TRE transcripts. Ash1 did not associate with TRE transcripts and TREs in cross-linked and native chromatin from S2 cells transcribing mdu. In contrast, Ash1 interacted with TREs and TRE transcripts in S2 cells cotranscribing TRE1(+), TRE2(+), and TRE3(+) (Sanchez-Elsner, 2006).

The association of Ash1 with TREs and TRE transcripts was attenuated by RNase A and RNase H but not RNase III. RNase treatment did not abolish the association of TBP with the Ubx promoter. These results indicate that Ash1 associates with TRE transcripts and TREs in vivo and that TRE transcripts mediate the association of Ash1 with TREs in trans (Sanchez-Elsner, 2006).

To test this hypothesis, RNA interference (RNAi) was used to assess whether degradation of TRE transcripts attenuates recruitment of Ash1 to Ubx TREs and Ubx expression in third-leg discs. In vitro cultivated third-leg discs were incubated with small interfering RNAs (siRNAs) targeting all three TRE transcripts or with control siRNA. RT-PCR and XChIP assays indicated that siRNA-mediated degradation of TRE transcripts attenuates Ubx transcription and the interaction of Ash1 with TREs (Sanchez-Elsner, 2006).

Next, The binary Gal4/UAS system was used to determine whether ectopic transcription of TRE transcripts restores recruitment of Ash1 to Ubx TREs and Ubx transcription. Effector flies carrying a heat-inducible driver (hsp70Gal4) were crossed with reporter flies carrying Gal4-dependent reporter genes (UAS-TRE) consisting of Gal4-responsive UAS DNA sites and a promoter driving the transcription of sense and antisense TRE transcripts. Heat treatment of second-instar larvae resulted in ectopic transcription of TRE transcripts in all imaginal discs of third-instar larvae. Ectopic transcription of each TRE transcript nucleated ectopic transcription of Ubx in wing imaginal discs and facilitated the recruitment of Ash1 to the corresponding Ubx TREs. It is noteworthy that ectopic TRE transcription in second-instar larvae caused lethality in pupae. In contrast, ectopic Ubx expression was not observed in discs prepared from heat-treated parental strains and discs transcribing antisense TRE transcripts (Sanchez-Elsner, 2006).

Transcription of antisense TRE transcripts attenuated endogenous transcription of Ubx in wing discs isolated from young third-instar larvae, which suggests that ectopic transcription of antisense RNA interferes with the TRE transcript-mediated recruitment of Ash1 to Ubx TREs. In summary, these data provide evidence that noncoding TRE transcripts facilitate activation of Ubx expression by recruiting Ash1 to the Ubx TREs in the fly (Sanchez-Elsner, 2006).

Recent studies have shown that noncoding RNAs play an important role in the recruitment of proteins in several epigenetic phenomena. siRNAs have been lined to heterochromatin formation and transcriptional silencing of transgenes and transposons. siRNAs facilitate the recruitment of HMTs and DNA methyltransferases to chromatin. In Schizosaccharomyces pombe, heterochromatic silencing involves the RNA-induced initiator of transcriptional gene silencing complex (RITS), which contains an siRNA component that is essential for the recruitment of RITS to heterochromatic loci. The inability of RNase III, the key enzyme of the RNAi machinery, to degrade TRE transcripts into siRNAs and the interaction of Ash1 with full-length TRE transcripts in chromatin strongly argues against the involvement of siRNAs in the described RNA-dependent recruitment of Ash1 to chromatin (Sanchez-Elsner, 2006 and references therein).

Long ncRNAs are key players in imprinting and gene dosage compensation. In Drosophila, gene dosage compensation is achieved by a global twofold up-regulation of transcription from the male X chromosome and depends on the activity of the dosage compensation complex (DCC) that contains male-specific proteins and two ncRNAs, RNA on X 1 (rox1) and RNA on X 2 (rox2). Both RNAs are transcribed by single-copy genes that, as well as several other X chromosome regions, serve as chromatin entry sites for the DCC on paternal X chromosomes. Rox1 and Rox2 facilitate the assembly and recruitment of the DCC to chromatin entry sites. In mammals, spreading of Xist RNA culminates in X chromosome inactivation. Current models propose that the association between ncRNAs and chromatin involves their interaction with proteins, nascent transcripts at template DNA, or the template DNA. The observed attenuation of the association between TRE transcripts and TREs by RNase H suggests that TRE transcripts are retained at TREs through hybridization with the corresponding template DNA. Because none of the known DNA repair systems targets DNA-RNA hybrids, RNA-DNA hybrids represent stable molecular entities that, in general, may anchor ncRNAs at corresponding DNA templates in chromatin (Sanchez-Elsner, 2006 and references therein).

The three TRE transcripts of Ubx do not share common sequence motifs. This is not surprising, because the functionally redundant rox RNAs and functionally identical regions in Xist, which are required for chromatin localization and protein recruitment, lack identifiable sequence motifs. Because many RNA-protein interactions are facilitated by RNA secondary structures, the interaction of Ash1 with TRE transcripts might be mediated by secondary RNA structures rather than sequence motifs. In addition, the specificity of RNA-protein interactions is often generated by induced-fit mechanisms that involve complex, extensive conformational changes in both proteins and the target RNA generating a specific interaction surface (Sanchez-Elsner, 2006).

rox1 and rox2 RNAs transcribed from autosomes can localize to and mediate gene dosage compensation on the male X chromosome, indicating that the chromatin entry of rox RNAs does not depend on transcription of chromatin entry sites in cis. Thus, the association of transiently transcribed TRE transcripts with TREs in S2 cells suggests that TREs function as chromatin entry sites for the corresponding TRE transcripts in trans and cis, and that the transcription and chromatin entry site activities of TREs are functionally separated. Cumulatively, these results support a model in which RNAs transcribed from the TREs of Ubx are retained at TREs through DNA-RNA interactions and provide a RNA scaffold that is bound by Ash1 (Sanchez-Elsner, 2006).

Recruitment of Drosophila Polycomb-group proteins by Polycomblike, a component of a novel protein complex in larvae

Polycomb-group (PcG) proteins are highly conserved epigenetic transcriptional repressors that play central roles in numerous examples of developmental gene regulation. Four PcG repressor complexes have been purified from Drosophila embryos: PRC1, PRC2, Pcl-PRC2 and PhoRC. Previous studies described a hierarchical recruitment pathway of PcG proteins at the bxd Polycomb Response Element (PRE) of the Ultrabithorax (Ubx) gene in larval wing imaginal discs. The DNA-binding proteins Pho and/or Phol are required for target site binding by PRC2, which in turn is required for chromosome binding by PRC1. This study identified a novel larval complex that contains the PcG protein Polycomblike (Pcl) that is distinct from PRC1 and PRC2 and which is also dependent on Pho and/or Phol for binding to the bxd PRE in wing imaginal discs. RNAi-mediated depletion of Pcl in larvae disrupts chromosome binding by E(z), a core component of PRC2, but Pcl does not require E(z) for chromosome binding. These results place the Pcl complex (PCLC) downstream of Pho and/or Phol and upstream of PRC2 and PRC1 in the recruitment hierarchy (Savla, 2008).

Drosophila Polycomb-group (PcG) genes were originally identified as negative regulators of Hox genes. PcG-mediated silencing in Drosophila occurs in essentially two broadly defined stages: assumption of transcriptional repression responsibilities from gene-specific transcription factors in early embryos, followed by maintenance of the silenced state through many cycles of cell division beginning in mid-late-stage embryos and continuing throughout the remainder of development (Savla, 2008).

Although much of the genetic analysis of PcG functions and studies of the mechanisms by which PcG proteins are targeted to specific genomic sites have focused on their activities in larval tissues, in vitro biochemical analyses have focused on PcG complexes isolated from embryos: PRC1, PRC2 and PhoRC. PRC1 possesses multiple chromatin modifying activities in vitro suggesting that it, among PcG complexes, might be most directly responsible for preventing transcription. The primary functions of PhoRC and PRC2 appear to be to recruit and/or stabilize target site binding by PRC1, and potentially other PcG proteins. PhoRC includes the DNA-binding PcG protein Pleiohomeotic (Pho), which binds to sites within Polycomb Response Elements (PREs) that serve as docking platforms for PcG proteins. Pho directly interacts with components of both PRC1 and PRC2, and is required for recruitment of both complexes. The E(z) subunit of PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), facilitating recruitment of PRC1 (Savla, 2008).

A variant of PRC2 has recently been described that includes the PcG protein Polycomblike (Pcl). On the basis of gel filtration analysis of native complexes in embryo nuclear extracts and the stoichiometry of the purified Pcl-PRC2 complex, it appears that the majority of embryonic Pcl is present in Pcl-PRC2, but that the other PRC2 core subunits, E(z), Su(z)12, Esc and NURF55 (also known as Caf1 - FlyBase), predominantly are in a complex(es) lacking Pcl. It has been proposed that inclusion of Pcl in PRC2 is required for high levels of H3K27me3 in vivo, although the in vitro histone methyltransferase activity of Pcl-PRC2 is indistinguishable from that of PRC2 lacking Pcl. In this study, a larval Pcl-containing complex is identified that is distinct from PRC2 and PRC1 and shown to be required for chromosome binding by these PcG complexes (Savla, 2008).

In order to examine potential differences between embryonic and larval stage PcG complexes, larval nuclear extracts were fractionated over a Superose 6 gel filtration column, and western blots of the fractions were probed with anti-E(z) and anti-Pcl antibodies. Larval E(z)-containing complexes have a relative mass of ~500 to 600 kDa, similar to that of embryonic PRC2 complexes that lack Pcl. However, Pcl was undetectable in E(z)-containing fractions and appeared to be in a complex with a relative mass of ~1500 kDa. This is different from the fractionation profile of Pcl from embryo extracts, in which it co-fractionates with E(z) in native complexes with relative mass estimates in the range of ~650 kDa to 1000 kDa, suggesting that, unlike its association with a subset of PRC2 complexes in embryos, Pcl functions as a component of a distinct complex in larvae, which will be referred to as the Pcl-Complex (PCLC) (Savla, 2008).

In order to further investigate the relationship of Pcl with other PcG proteins and its role in PcG-mediated silencing in larvae, chromatin immunoprecipitation (ChIP) assays were performed on wing imaginal discs. The PcG maintains the transcriptional silence of the Hox gene Ultrabithorax (Ubx) in the epithelial cells of wing discs. Other PcG proteins, including the DNA-binding proteins Pho and Phol and components of the PRC1 and PRC2 complexes, have previously been shown to be present at the major PRE in the Ubx cis-regulatory bxd region in this tissue. Consistent with a previous report, Pcl also was detected at the bxd PRE, and appears to largely colocalize with E(z) and Phol (Savla, 2008).

A hierarchical relationship among PcG proteins at the bxd PRE in which Pho and/or Phol are required, but are not necessarily sufficient, for recruitment of PRC2, which in turn facilitates recruitment of PRC1. In order to determine how Pcl might fit into this recruitment pathway, ChIP assays were performed on E(z) mutant wing imaginal discs. E(z)⁶¹ is a temperature-sensitive allele that displays nearly wild-type activity at 18°C, but strongly reduced activity at 29°C. Following shift from 18°C to 29°C, bxd PRE binding by E(z)⁶¹ protein is rapidly lost and along with it the detection of H3K27me3 and Pc in this region. ChIP assays of wing discs dissected from E(z)⁶¹ larvae 24 hours following shift from 18° to 29°C confirmed loss of E(z) from the PRE, but revealed no effect on Pcl and Phol binding to PRE fragments 3 and 4, but a slight decrease of both proteins at the PRE 2 fragment. It is speculated that Pcl and Phol signals at this proximal edge of the PRE are partly due to protein-protein cross-links, which might be reduced in the absence of PRC2. Retention of Pcl at the PRE in the absence of E(z) and by extension absence of PRC1, which requires PRC2 for binding to this region, confirms that Pcl is not a stable subunit of larval versions of either PRC1 or PRC2 and is consistent with its inclusion in a distinct complex (Savla, 2008).

Flies that are homozygous for null Pcl alleles die as embryos and no conditional Pcl alleles exist, precluding reciprocal experiments on Pcl mutant larvae. Therefore, transgenic fly lines were generated that contain inserts of a pWIZ-Pcl construct, which expresses Pcl shRNA under the control of Gal4, permitting inducible RNAi-mediated knockdown of Pcl in combination with Gal4 drivers. Individuals that contain both pWIZ-Pcl and P{GAL4-da.G32}, which constitutively expresses Gal4, died as early pupae and exhibited dramatically reduced levels of Pcl in wing imaginal discs. E(z) levels were not affected. ChIP assays of these Pcl-depleted wing discs confirmed reduced Pcl levels at the bxd PRE and revealed commensurate loss of E(z). Thus, although Pcl does not require PRC2 for PRE binding, Pcl, presumably functioning as a subunit of PCLC, is needed for stable binding of PRC2 to the bxd PRE. Phol remains at the PRE in the absence of Pcl (Savla, 2008).

In order to determine whether Pcl (like components of PRC1 and PRC2) requires Pho and/or Phol for PRE binding, ChIP assays were performed using wing imaginal discs from phol^81A; pho¹ larvae. Consistent with their role in recruiting other PcG proteins, Pcl was lost from the bxd PRE in the absence of Pho and Phol. These observations at the bxd PRE also appear to generally apply to PcG-binding sites throughout the genome (Savla, 2008).

These results demonstrate the existence of a distinct Pcl protein complex in larvae that is required for recruitment of PRC2 to chromosomal target sites and/or to stabilize its binding. As previously described, E(z), as a core subunit of PRC2, is required for target site binding by PRC1. Therefore, Pcl is indirectly required for chromosome binding by PRC1 as well, although direct interaction with PRC1 cannot be ruled out, similar to the way in which Pho may contribute to target site binding by PRC1 by interacting both with PRC2 subunits and with Pc, a core subunit of PRC1 (Savla, 2008 and references therein).

In vitro histone methyltransferase assays of Pcl-PRC2 show that its activity and specificity for methylation of H3K27 are essentially indistinguishable from that of PRC2 complexes lacking Pcl. ChIP analysis of Pcl mutant embryos has shown that Pcl does not seem to be required for target site binding by other PRC2 subunits, but that it may be needed for high levels of trimethylation of H3K27. One explanation for these observations is that the contribution of Pcl to Pcl-PRC2 in embryos might be to mediate interaction with other proteins that are yet to be identified. In larvae, Pcl exists as a subunit of a distinct complex. Given the ability of Pcl to directly interact with several PRC2 subunits, colocalization of Pcl and E(z) at the PRE, and dependence of E(z) on Pcl for binding to the bxd PRE and other genomic sites, it is likely that PCLC is closely associated with PRC2 at target sites in larvae. In both embryos and larvae, some of the activities attributed to Pcl might, upon further inspection, be due to the activities of other Pcl-associated proteins, the close apposition of which with PRC2 and other PcG complexes may be mediated by Pcl. The differential deployment of Pcl as a subunit of PRC2 and as a subunit of PCLC at distinct developmental stages is intriguing and might reflect the different molecular activities needed for establishment of silencing in embryos and maintenance of the silenced state in larval tissues. A more detailed understanding of the mechanisms by which Pcl contributes to PcG silencing will require identification of the other proteins contained within the larval PCLC complex and the potential biochemical activities of the complex (Savla, 2008).

Trithorax-like interaction with Pipsqueak

Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE of Ultrabithorax contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, DNA binding activities have been identified that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)(3) repeats for MHS-70 binding in vitro. Mutation of d(GA)(3) repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)(3) repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)(3) repeats, the Trl/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, indicating that Trl/GAGA factor and Psq are enhancers of Polycomb that have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of the bxd PRE by d(GA)(n) repeat binding activities mediates PcG-dependent silencing (Hodgson, 2001).

The results of the sequence-specific analysis suggest that d(GA)_n-specific binding factors are present in a complex defined by electrophoretic mobility, termed complex 2. Therefore, antibodies directed against two nuclear factors that bind d(GA)_n sequences, Trl/GAF and Psq, were tested in binding reactions with a bxd fragment termed MHS-70. In the presence of increasing amounts of antibody to Trl/GAF, the mobility of complex 2 was significantly retarded, migrating close to the sample well. Antibodies to Psq caused a modest but detectable retardation of complex 2. Neither of these antibodies alters the mobility of a second complex, complex 1. These results show that complex 2 contains detectable levels of Trl/GAF and Psq. The significantly reduced mobility of complex 2 in the presence of anti-TRL/GAF antibody presumably results from the ability to induce multimeric aggregates of DNA-TRL/GAF complexes. To show that the complexes formed by MHS-70 and the competing oligomers are equivalent, the formation of complex 2 with synthetic oligomers was tested with antibodies to Trl/GAF, Ph, and Psq. Antibodies to Ph, Trl/GAF, and Psq supershift complex 2 in synthetic oligomer binding reactions (Hodgson, 2001).

Polyhomeotic proximal (Php), Trl/GAF, and Psq have multiple isoforms. To determine which of these isoforms are potential components of complex 2, Western analysis of the Separose AS, BR0.6 and Q0.15 fractions was undertaken. Q0.15 is enriched for isoforms of Trl/GAF P67 plus Trl/GAF P54, Php105 plus Php64, and Psq P70. Taken together with the antibody supershift analysis, these results show that the distinct isoforms of Trl/GAF, Php and Psq coelute with complex 2 and suggest that these isoforms constitute potential subunits of complex 2 binding activities. It has been shown that the full-length isoform of PhP, Php-170, coimmunoprecipitates with the PcG proteins Pc, Psc, Su(z)2, and Scm. Western analyses of the three fractions described above show that there are no detectable levels of Pc, Su(z)2, Psc, or Sex Combs on Midleg (Scm) in Q0.15, indicating that these PcG proteins do not coelute with complex 1 and 2 binding activities. These results suggest that the complex 2 activity is a novel PcG activity containing distinct isoforms of PHP, TRL/GAF, and Psq (Hodgson, 2001).

Trl null or hypomorphic alleles (Trl^13C, Trl⁶², and Trl⁸⁵) do not affect maintenance of silencing in vivo by bxd5.1 UbxlacZ. Similarly, embryos mutant for psq^RF13 (deletion of psq) and Df(2R)psq-lolaDelta18 (deletion of psq, termed psq^lola hereafter) show wild-type bxd5.1 UbxlacZ silencing. One potential reason for these results is that maternally deposited Trl or psq protein or mRNA rescue the effects of absence of zygotic proteins on embryonic silencing (Hodgson, 2001).

Genetic interactions between PcG genes are monitored by the enhancement of PcG mutations, providing a sensitive genetic assay for genes required in PcG-mediated silencing. Therefore, the ability of Trl and Psq mutations to enhance the extra sex combs phenotype of ph was tested. Trl enhances the extra sex combs phenotype of Pc. Similarly, Trl⁶² enhances the extra sex combs phenotype of ph² and ph⁴⁰⁹. The effects of psq^lola-Delta18 and psq²⁴⁰³ on enhancement of ph² and ph⁴⁰⁹ were tested. There is strong enhancement of the expressivity of the extra sex combs phenotype. These results are consistent with a role for Trl/GAF and Psq in PcG-mediated silencing of homeotic loci and indicate that Trl/GAF and Psq are enhancers of PC that have sequence-specific DNA binding activity (Hodgson, 2001).

pipsqueak encodes a factor essential for sequence-specific targeting of a polycomb group protein complex

To facilitate the biochemical study of the Pc-G complex, a Drosophila S2 cell line PC-FH was established that can express Pc protein with both FLAG epitope and hexahistidine tags at its C terminus. These modifications do not appear to affect the activity of Pc, since similar constructs can repress the Ubx reporter gene in cultured cells and partially rescue Pc mutants in transgenic flies. The tagged PC is under the control of a metallothionein promoter, which allows protein induction by the copper ion in a range up to 0.7 mM. A suboptimal concentration of CuSO₄ (i.e., 0.1 mM) was chosen for induction, since it appears to provide sufficient amounts of tagged protein complexes for purification. Nuclear extracts prepared from induced cells were fractionated by 10% to 40% (NH₄)₂SO₄ precipitation to enrich for large protein complexes. The extracts were then passed through a FLAG antibody column (i.e., M2 resin) and eluted with the FLAG peptide. Approximately 200-fold purification was obtained by affinity chromatography compared with the crude extracts. Many proteins appeared to be specifically coeluted with the Pc protein. Although the region corresponding to the size of Pc proteins appears to be heavily stained, the relative abundance of Pc proteins has been exaggerated by the presence of several proteins of similar size that can be better separated in high-resolution gels. An earlier study has shown that a specific subset of Pc-G proteins, including Psc, Ph, and HDAC1 are copurified, indicating the presence of multimeric Pc-G complexes in these fractions. Since this is the first Drosophila Pc-G complex shown to contain both histone modification activity and DNA binding activity and since homeotic genes are its best characterized targets, this complex is refered to as CHRASCH (chromatin-associated silencing complex for homeotics) to distinguish it from commonly referred Pc-G complexes or complexes characterized by other workers (Huang, 2002).

Since a functional Pc-G complex must act specifically on its response element (i.e., PRE), whether CHRASCH can bind such sequences in vitro was examined. PRE from several homeotic genes have been mapped, including the one from the upstream bxd region of Ubx. An ~440-bp fragment (B-151) from this region recapitulates transcriptional regulation by either Pc or trx in cultured cells. DNA fragments encompassing this region can also confer pairing-sensitive repression and are enriched for Pc proteins in chromatin immunoprecipitation experiments. B-151 therefore contains physiologically relevant binding sites for the Pc-G complex. Using two subfragments from B-151 as probes for EMSAs, CHRASCH was found to bind strongly to the bxd-b fragment, resulting in several slow-migrating bands. These bands presumably reflect the binding to a reiterated motif in the bxd-b fragment. The binding of CHRASCH to the bxd-b fragment appears to be specific, since it can be completely competed out by the addition of bxd-b but not by bxd-a or nonspecific vector sequences. A much weaker but specific binding of CHRASCH to bxd-a was also detected. The observation that bxd-b can compete for binding to bxd-a but that bxd-a can not compete effectively for binding to bxd-b suggests that these fragments might contain similar binding sequences, albeit with a lower affinity in the bxd-a fragment. Due to the difficulty in studying a weak binding activity with certainty, subsequent studies focused on bxd-b (Huang, 2002).

Four partially overlapping fragments from B-151 were used in competition assays to further map the binding sites of CHRASCH. Only the bxd-3 and bxd-4 fragments compete effectively for the binding to CHRASCH. In addition, bxd-4 appears to compete better than bxd-3. Therefore, it was deduced that the right half of B-151 must contain the primary binding sites for CHRASCH. Interestingly, transgenes containing small deletions in this region, but not in the left half of B-151, fail to silence the reporter gene effectively and can no longer respond to mutations in several Pc-G, indicating that this region is indeed relevant for Pc-G-mediated silencing. Three major sequence motifs can be identified in B-151. The first motif (C/T)GAG(C/T)G is the consensus binding site of the Zeste protein. Both the left and right halves of B-151 contain one Zeste binding site. The second motif ATGGC represents the binding site of a newly characterized member of Pc-G, pho, which encodes the Drosophila homolog of YY1. bxd-3 and bxd-4 each contain one copy of an almost identical YY1 site. The third motif is a (GA)_n repeat, which represents the consensus binding site of GAF encoded by Trithorax-like (Trl). Trl was originally identified as a member of trx-G; however, some recent studies suggest that it may also share some characteristics with Pc-G. While both bxd-3 and bxd-4 contain 2 separate clusters of this motif, one cluster in bxd-4 is much further extended (GAGAGAGGGAGAG versus GAGAG). Since bxd-4 has been shown to be more effective in competition assays, it is likely that the (GA)_n motif is most critical for CHRASCH binding. This possibility is further supported by the observation that CHRASCH binding to bxd-b is completely competed out by a fragment containing multiple (GA)_n repeats but not by the one containing multiple Zeste repeats or an oligonucleotide containing a YY1 binding site. Therefore, it is concluded that the binding sequences of CHRASCH consist primarily of the (GA)_n motif (Huang, 2002).

The association of the (GA)_n binding activity with CHRASCH is further confirmed by the following observations. When the DNA binding activity was examined in peptide-eluted fractions, it was found that fractions 6 and 8 had the strongest binding activities. These fractions also contained the highest amounts of PC and other associated proteins. Thus, the binding protein coelutes with CHRASCH in the immunoaffinity chromatography. In addition, it was found the DNA-protein complexes formed on bxd-b can be slightly supershifted by a preincubation with an affinity-purified PC antibody but not with a nonspecific IgG. The small supershift might be expected for a large complex in a gel composed of 3.5% polyacrylamide. Taken together, these results indicate that the (GA)_n binding protein is physically associated with CHRASCH (Huang, 2002).

Since chromatin immunoprecipitation experiments have shown that Trithorax-like is enriched in the region encompassing B-151 and its vicinity, whether the binding protein of CHRASCH is related to Trithorax-like was examined. The results are not consistent with this notion. As expected, a purified recombinant Trithorax-like binds specifically to the bxd-b fragment. In addition, the binding activity of Trithorax-like is drastically stimulated by zinc ions over a wide range, resulting in a further retardation of the DNA-protein complexes in the EMSA. By contrast, the binding activity of CHRASCH is not significantly affected at intermediate concentrations of zinc ions and becomes completely inactivated at a high concentration (i.e., 0.5 mM). The differential effects of zinc ions on DNA binding properties argue that different DNA binding proteins are involved in the binding. Furthermore, a Trithorax-like-specific antiserum was used to examine the protein preparations. Trithorax-like antiserum detected multiple bands corresponding to two major classes of isoforms, Trithorax-like-519 and -581, from the column input, but no specific Trl immunoreactivity was found in the eluate (Huang, 2002).

To further characterize the DNA binding protein of CHRASCH, a cross-linking method was used to specifically label this protein. An oligonucleotide probe was designed that allowed specific incorporation of both radioactive dCTP and a photoactivated cross-linking dTTP analogue (i.e., AB-dUTP) into the nucleotide sequences that correspond to the extended (GA)_n motif in bxd-4. Following DNA binding and UV irradiation, the cross-linked nucleoprotein complex was digested extensively with both DNase I and micrococcal nuclease to remove excessive DNA sequences. The indirectly labeled proteins were then resolved on an SDS-9% polyacrylamide gel. A major band of ~85 kDa was identified for the purified recombinant Trithorax-like, whereas a different pattern was observed for CHRASCH, which consists of a doublet of ~130 kDa and a doublet of ~70 kDa. Taken together, these results provide strong evidence that the DNA binding activity of CHRASCH is not contributed by Trithorax-like but by a novel factor(s) (Huang, 2002).

A (GA)_n-binding protein has been identified in Apis mellifera by screening an expression library with (GA)_n repeats. The Drosophila homolog of this protein was found to be encoded by pipsqueak (psq), identified originally by its grandchildless phenotype and subsequently by its effect on eye development. By differential transcriptional and translational initiation, psq produces two major mRNAs containing open reading frames for 1,065 (PSQ-A) and 630 to 646 amino acids (PSQ-B). These two isoforms share a common C-terminal PSQ domain capable of binding to the (GA)_n motif. The size similarity between one of the cross-linked proteins and PSQ-A prompted a test of whether Psq proteins copurify with CHRASCH. Using an antibody that reacts with both PSQ-A and B (Horowitz, 1996), it was found that PSQ-A and much less PSQ-B are clearly detectable in CHRASCH sample. For a preparation of FLAG-tagged TATA-box binding protein (TBP), however, a large amount of PSQ-A was found in the input but not in the eluted fraction. These results demonstrate that PSQ-A and CHRASCH have indeed been copurified. To further examine the association between Pc and Psq in vivo, coimmunoprecipitation was performed with embryonic nuclear extracts. Pc was precipitated by the Psq antibody. Although it is not clear whether the smaller ~70-kDa proteins detected in the cross-linking experiments represent degradation products of Psq or other unrelated proteins, these results strongly suggest that Psq-A may play a major role in DNA binding (Huang, 2002).

batman interacts with Polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila development. lola like (lolal), also known as batman (ban), functions in both activation and repression of homeotic genes, including the repression of Sex combs reduced. The 127-amino acid Lolal protein consists almost exclusively of a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. This domain is involved in the interaction between Lolal and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Lolal codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Lolal, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7 polycomb response element mediated pairing dependent silencing of mini-white transgene. lolal was also identified as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts geneticially with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism for how Trl plays a role in maintaining the repressed state of target genes involving Lolal, which may function as a mediator to recruit PcG complexes (Faucheux, 2003; Mishra, 2003).

Batman is thus the second BTB/POZ protein that has been shown to participate in a Trl-containing complex involved in homeotic gene regulation, the other being Psq. Psq, like Batman, binds to the bxd MHS-70 PRE fragment. Psq colocalizes and coimmunoprecipitates with Trl, an interaction that depends on the BTB/POZ domains of the two proteins. In addition, Psq shares functions with Trl and Batman, such as its requirement for the activation of Ubx, as well as for the repression of Scr. However, among the three proteins, Batman appears to display unique functional features since it does not contain a DNA binding domain and since ban mutant phenotypes include the extra sexcomb phenotype that is characteristic of PcG proteins. It is thus likely that understanding the possible function of d(GA)n binding complexes in tethering PcG or trxG complexes to PREs will require deciphering of the triangular interactions of the three BTB/POZ proteins Trl, Psq, and Batman (Faucheux, 2003).

Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins

Polycomb group (PcG) and trithorax group (trxG) proteins act as antagonistic regulators to maintain transcriptional OFF and ON states of HOX and other target genes. To study the molecular basis of PcG/trxG control, the chromatin of the HOX gene Ultrabithorax (Ubx) was analyzed in Ubx^OFF and Ubx^ONcells purified from developing Drosophila. PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all constitutively bound to Polycomb response elements (PREs) in the OFF and ON state. In contrast, the trxG protein Ash1 is only bound in the ON state; not at PREs but downstream of the transcription start site. In the OFF state, extensive trimethylation was found at H3-K27, H3-K9, and H4-K20 across the entire Ubx gene; i.e., throughout the upstream control, promoter, and coding region. In the ON state, the upstream control region is also trimethylated at H3-K27, H3-K9, and H4-K20, but all three modifications are absent in the promoter and 5' coding region. These analyses of mutants that lack the PcG histone methyltransferase (HMTase) E(z) or the trxG HMTase Ash1 provide strong evidence that differential histone lysine trimethylation at the promoter and in the coding region confers transcriptional ON and OFF states of Ubx. In particular, the results suggest that PRE-tethered PcG protein complexes act over long distances to generate Pc-repressed chromatin that is trimethylated at H3-K27, H3-K9, and H4-K20, but that the trxG HMTase Ash1 selectively prevents this trimethylation in the promoter and coding region in the ON state (Papp, 2006; Full text of article).

In wild-type animals, Ubx is expressed in all cells of the haltere and third-leg imaginal discs, but it is stably repressed in cells of the wing imaginal disc. Haltere/third-leg discs and wing discs thus represent uniform populations of Ubx^ON and Ubx^OFFcells, respectively. Batches of sonicated Ubx^ON and Ubx^OFF chromatin were prepared from pools that typically contained 1500 fixed and hand-dissected imaginal discs. This procedure allowed performance multiple immunoprecpitation reactions with different antibodies from the same chromatin preparation. Furthermore, with each antibody immunoprecipitations were performed from at least three independently prepared batches of chromatin. Then the abundance of specific DNA sequences was measured in the immunoprecipitates by real-time quantitative PCR (qPCR) at the following genomic locations. In the Ubx gene, 17 different regions were analyzed. Some of these fragments map to well-characterized Ubx cis-regulatory sequences. In particular, F1 is located at the imaginal disc enhancer PBX that activates expression in the haltere and third-leg discs, F4 and F5 are located within the core of the bxd PRE (Chan, 1994; Fritsch, 1999), F7 is located within the BXD enhancer that activates expression in the embryo but is inactive in discs (Müller, 1991; Castelli-Gair, 1992), F9 encompasses the Ubx transcription start site, F13 corresponds to the bx PRE, and F17 is located in the Ubx 3' untranslated region. Regions located outside of the Ubx locus served as controls (Papp, 2006).

Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and dSfmbt, a novel PcG protein that selectively binds to histone H3 and H4 tail peptides that are mono- or dimethylated at H3-K9 or H4-K20 (H3-K9me1/2 and H4-K20me1/2, respectively). PRC1 contains the PcG proteins Ph, Psc, Sce/Ring, and Pc. PRC1 inhibits nucleosome remodeling and transcription in in-vitro assays and its subunit Pc specifically binds to trimethylated K27 in histone H3 (H3-K27me3). PRC2 contains the PcG proteins E(z), Su(z)12, and Esc as well as Nurf55, and this complex functions as a histone methyltransferase (HMTase) that specifically methylates K27 in histone H3 (H3-K27) in nucleosomes (Papp, 2006).

This study used quantitative X-ChIP analysis to examine the chromatin of the HOX gene Ubx in its ON and OFF state in developing Drosophila larvae. Previous genetic studies had established that all of the PcG and trxG proteins analyzed in this study are critically needed to maintain Ubx OFF and ON states in the very same imaginal disc cells in which their binding to Ubx was analyzed in this study. The following conclusions can be drawn from the analyses reported in this study. (1) The PcG protein complexes PhoRC, PRC1, and PRC2 and the Trx protein are all highly localized at PREs, but they are all constitutively bound at comparable levels in the OFF and ON state. (2) The trxG protein Ash1 is bound only in the ON state, where it is specifically localized ~1 kb downstream of the transcription start site. (3) In the OFF state, PRC2 and other unknown HMTases trimethylate H3-K27, H3-K9, and H4-K20 over an extended 100-kb domain that spans the whole Ubx gene. (4) In the ON state, comparable H3-K27, H3-K9, and H4-K20 trimethylation is restricted to the upstream control regions and Ash1 selectively prevents this trimethylation in the promoter and coding region. (5) Repressed Ubx chromatin is extensively tri- but not di- or monomethylated at H3-K27, H3-K9, and H4-K20. (6) Trimethylation of H3-K27, H3-K9, and H4-K20 at imaginal disc enhancers in the upstream control region does not impair the function of these enhancers in the ON state. (7) TBP and Spt5 are bound at the Ubx transcription start site in the ON and OFF state, but Kis is only bound in the ON state. This suggests that in the OFF state, transcription is blocked at a late step of transcriptional initiation, prior to the transition to elongation. A schematic representation of PcG and trxG protein complex binding and histone methylation at the Ubx gene in the OFF and ON state is presented (Papp, 2006).

Unexpectedly, ChIP analysis by qPCR used in this study and in a similar study by the laboratory of Vincent Pirrotta (V. Pirrotta, pers. comm. to Papp, 2006) reveals that the relationship between PcG and trxG proteins and histone methylation is quite different from the currently held views. Specifically, X-ChIP studies have reported that H3-K27 trimethylation is localized at PREs and this led to the model that recruitment of PRC1 to PREs occurs through H3-K27me3 (i.e., via the Pc chromodomain). In contrast, the current study and that by Vincent Pirrotta found H3-K27 trimethylation to be present across the whole inactive Ubx gene, both in wing discs and in S2 cells (V. Pirrotta, pers. comm. to Papp, 2006). No specific enrichment of H3-K27 trimethylation at PREs has been detected; rather, a reduction of H3-K27me3 signals is observed at PREs, consistent with the reduced signals of H3 that are detected at these sites. Consistent with these results, genome-wide analyses of PcG protein binding and H3-K27me3 profiles in S2 cells revealed that, at most PcG-binding sites in the genome, PcG proteins are tightly localized, whereas H3-K27 trimethylation is typically present across an extended domain that often spans the whole coding region. How could the differences between this study and the earlier studies be explained? It should be noted that in contrast to the qPCR analysis used in the current study, previous studies all relied on nonquantitative end-point PCR after 36 or more cycles to assess the X-ChIP results. It is possible that these experimental differences account for the discrepancies (Papp, 2006).

PhoRC, PRC1, and PRC2 are all tightly localized at PREs but they are all constitutively bound at the inactive and active Ubx gene. This suggests that recruitment of PcG complexes to PREs occurs by default. Although all three complexes are bound at comparable levels to the bxd PRE in the inactive and active state and PhoRC is also bound at comparable levels at the bx PRE, it should be pointed out that the levels of PRC1 and PRC2 binding at the bx PRE are about twofold reduced in the active Ubx gene compared with the inactive Ubx gene. Even though there is still high-level binding of PRC1 and PRC2 at the bx PRE, it cannot be excluded that the observed reduction in binding helps to prevent default PcG repression of the active Ubx gene. It is possible that transcription through the bx PRE reduces PRC1 and PRC2 binding at this PRE. Transcription through PREs has been proposed to serve as an 'anti-silencing' mechanism that prevents default silencing of active genes by PREs (Papp, 2006),

The highly localized binding of all three PcG protein complexes at PREs, together with earlier studies on PRE targeting of PcG protein complexes supports the idea that not only PhoRC but also PRC1 and PRC2 are targeted to PRE DNA through interactions with Pho and/or other sequence-specific DNA-binding proteins. In the case of trxG proteins, the binding modes are more diverse. In particular, recruitment of Trx protein to PREs and to the promoter is also constitutive in both states but recruitment of Ash1 to the coding region is clearly observed only at the active Ubx gene. At present, it is not known how Trx or Ash1 are targeted to these sites. It is possible that a transcription-coupled process recruits Ash1 to the position 1 kb downstream of the transcription start site (Papp, 2006).

In contrast to the localized and constitutive binding of PcG protein complexes and the Trx protein, it was found that the patterns of histone trimethylation are very distinct in the active and inactive Ubx gene. The results also suggest that the locally bound PcG and trxG HMTases act across different distances to methylate chromatin. For example, H3-K4 trimethylation is confined to the first kilobase of the Ubx coding region where Ash1 and Trx are bound, whereas H3-K27 trimethylation is present across an extended 100-kb domain of chromatin that spans the whole Ubx gene. This suggests that PRE-tethered PRC2 is able to trimethylate H3-K27 in nucleosomes that are as far as 30 kb away from the bxd or bx PREs. Unexpectedly, it was found that the H3-K9me3 and H4-K20me3 profiles closely match the H3-K27me3 profile. At present it is not known which HMTases are responsible for H3-K9 and H4-K20 trimethylation, but analysis of E(z) mutants indicate that these modifications may be generated in a sequential manner, following H3-K27 trimethylation by PRC2. The molecular mechanisms that permit locally tethered HMTases such as PRE-bound PRC2 to maintain such extended chromatin stretches in a trimethylated state are only poorly understood. However, a recent study showed that the PhoRC subunit dSfmbt selectively binds to mono- and di-methylated H3-K9 and H4-K20 in peptide-binding assays. One possibility would be that dSfmbt participates in the process that ensures that repressed Ubx chromatin is trimethylated at H3-K27, H3-K9, and H4-K20. For example, dSfmbt, tethered to PREs by Pho, may interact with nucleosomes of lower methylated states (i.e., H3-K9me1/2 or H4-K20me1/2) in the flanking chromatin and thereby bring them into the vicinity of PRE-anchored HMTases that will hypermethylate them to the trimethylated state (Papp, 2006).

These analyses suggest that H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region is critical for Polycomb repression. (1) Although H3-K27, H3-K9, and H4-K20 trimethylation is present at the inactive and active Ubx gene, it is specifically depleted in the promoter and coding region in the active Ubx gene. (2) Misexpression of Ubx in wing discs with impaired E(z) activity correlates well with loss of H3-K27 and H3-K9 trimethylation at the promoter and 5' coding region. It is possible that the persisting H3-K27 and H3-K9 trimethylation in the 3' coding region is responsible for maintenance of repression in those E(z) mutant wing discs cells that do not show misexpression of Ubx. (3) In haltere and third-leg discs of ash1 mutants, the promoter and coding region become extensively trimethylated at H3-K27 and H3-K9, and this correlates with loss of Ubx expression. Previous studies showed that Ubx expression is restored in ash1 mutants cells that also lack E(z) function. Together, these findings therefore provide strong evidence that Ash1 is required to prevent PRC2 and other HMTases from trimethylating the promoter and coding region at H3-K27 and H3-K9. The loss of H3-K4 trimethylation in ash1 mutants is formally consistent with the idea that Ash1 exerts its antirepressor function by trimethylating H3-K4 in nucleosomes in the promoter and 5' coding region, but other explanations are possible (Papp, 2006).

But how might H3-K27, H3-K9, and H4-K20 trimethylation in the promoter and coding region repress transcription? The observation that TBP and Spt5 are also bound to the promoter in the OFF state suggests that these methylation marks do not prevent assembly of the basic transcription apparatus at the promoter. However, the nucleosome remodeling factor Kis is not recruited in the OFF state, and transcription thus appears to be blocked at a late step of transcriptional intiation prior to elongation. It was found that the low-level binding of Pc in the coding region correlates with the presence of H3-K27 trimethylation; i.e., Pc and H3-K27me3 are both present in the OFF state, but are absent in the ON state. One possible scenario would thus be that H3-K27 trimethylation in the promoter and coding region permits direct recruitment of PRC1. According to this view, locally recruited PRC1 would then repress transcription; e.g., by inhibiting nucleosome remodeling in the promoter region. However, several observations are not easily reconciled with such a simple 'recruitment-by-methylation' model. First, peak levels of all PRC1 components are present at PREs and, apart from Pc, very little binding is observed outside of PREs. Second, excision of PRE sequences from a PRE reporter gene during development leads to a rapid loss of silencing, suggesting that transcriptional repression requires the continuous presence of PREs and the proteins that are bound to them. A second, more plausible scenario would therefore be that DNA-binding factors first target PcG protein complexes to PREs, and that these PRE-tethered complexes then interact with trimethylated nucleosomes in the flanking chromatin in order to repress transcription. For example, it is possible that bridging interactions between the Pc chromodomain in PRE-tethered PRC1 and H3-K27me3-marked chromatin in the promoter or coding region permit other PRE-tethered PcG proteins to recognize the chromatin interval across which they should act, e.g., to inhibit nucleosome remodeling in the case of PRC1 or to trimethylate H3-K27 at hypomethylated nucleosomes in the case of PRC2 (Papp, 2006).

The analysis of a HOX gene in developing Drosophila suggests that histone trimethylation at H3-K27, H3-K9, and H4-K20 in the promoter and coding region plays a central role in generating and maintaining of a PcG-repressed state. Contrary to previous reports, the current findings provide no evidence that H3-K27 trimethylation is specifically localized at PREs and could thus recruit PRC1 to PREs; widespread H3-K27 trimethylation is found across the whole transcription unit. The data presented in this study provide evidence that PREs serve as assembly platforms for PcG protein complexes such as PRC2 that act over considerable distances to trimethylate H3-K27 across long stretches of chromatin. The presence of this trimethylation mark in the chromatin that flanks PREs may in turn serve as a signal to define the chromatin interval that is targeted by other PRE-tethered PcG protein complexes such as PRC1. The results reported here also provide a molecular explanation for the previously reported antirepressor function of trxG HMTases; selective binding of Ash1 to the active HOX gene blocks PcG repression by preventing PRC2 from trimethylating the promoter and coding region. It is possible that the extended domain of combined H3-K27, H3-K9, and H4-K20 trimethylation creates not only the necessary stability for transcriptional repression, but that it also provides the molecular marks that permits PcG repression to be heritably maintained through cell division (Papp, 2006).

Search PubMed for articles about Drosophila Bithoraxoid

Bender, W. and Fitzgerald, D. P. (2002). Transcription activates repressed domains in the Drosophila bithorax complex. Development 129: 4923-4930. Medline abstract: 12397101

Chan, C. S., Rastelli, L. and Pirrotta, V. (1994). A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. EMBO J. 13: 2553-2564. Medline abstract: 7912192

Cumberledge, S., Zaratzian, A. and Sakonju, S. (1990). Characterization of two RNAs transcribed from the cis-regulatory region of the abd-A domain within the Drosophila bithorax complex. Proc. Natl. Acad. Sci. 87: 3259-3263. Medline abstract: 1692133

Faucheux, M., et al. (2003). batman interacts with Polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor. Molec. Cell. Bio. 23: 1181-1195. 12556479

Fritsch C., Brown J. L., Kassis J. A. and Muller, J. (1999). The DNA-binding polycomb group protein pleiohomeotic mediates silencing of a Drosophila homeotic gene. Development 126: 3905-3913. Medline abstract: 10433918

Grimaud, C. et al. (2006), RNAi components are required for nuclear clustering of Polycomb group response elements. Cell 124: 957-971. Medline abstract: 16530043

Hodgson, J. W., Argiropoulos, B. and Brock, H. W. (2001). Site-specific recognition of a 70-base-pair element containing d(GA)n repeats mediates bithoraxoid Polycomb group response element-dependent silencing. Mol. Cell. Biol. 21: 4528-4543. 11416132

Hogga, I. and Karch, F. (2002). Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing. Development 129: 4915-4922. Medline abstract: 12397100

Huang, D.-H., et al. (2002). pipsqueak encodes a factor essential for sequence-specific targeting of a polycomb group protein complex. Mol. Cell. Biol. 22: 6261-6271. 12167718

Lipshitz, H. D., Peattie, D. A. and Hogness, D. S. (1987). Novel transcripts from the Ultrabithorax domain of the bithorax complex. Genes Dev. 1: 307-322. Medline abstract: 3119423

Mishra, K., et al. (2003). Trl-GAGA directly interacts with lola like and both are part of the repressive complex of Polycomb group of genes. Mech. Dev. 120: 681-689. 12834867

Muller, J. and Bienz, M. (1991), Long range repression conferring boundaries of Ultrabithorax expression in the Drosophila embryo. EMBO J. 10: 3147-3155. Medline abstract: 1680676

Muller J., Hart C. M., Francis N. J., Vargas M. L., Sengupta A., Wild B., Miller E. L., OíConnor M. B., Kingston R. E. and Simon, J. A. (2002). Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111: 197-208. Medline abstract: 12408864

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(15): 2041-54. Medline abstract: 16882982

Petruk, S., et al. (2001). Trithorax and dCBP acting in a complex to maintain expression of a homeotic gene. Science 294: 1331-1334. Medline abstract: 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

Rank, G., Prestel, M. and Paro, R. (2002). Transcription through intergenic chromosomal memory elements of the Drosophila bithorax complex correlates with an epigenetic switch. Mol. Cell Biol. 22: 8026-8034. Medline abstract: 12391168

Rinn, J. L., et al. (2007). Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129(7): 1311-23. Medline abstract: 17604720

Ronshaugen, M., Biemar, F., Piel, J., Levine, M. and Lai, E. C. (2005). The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings. Genes Dev. 19: 2947-2952. Medline abstract: 16357215

Sanchez-Elsner, T., Gou, D., Kremmer, E. and Sauer, F. (2006). Noncoding RNAs of trithorax response elements recruit Drosophila Ash1 to Ultrabithorax. Science 311(5764): 1118-23. Medline abstract: 16497925

Sanchez-Herrero, E. and Akam, M. (1989), Spatially ordered transcription of regulatory DNA in the bithorax complex of Drosophila. Development 107: 321-329. Medline abstract: 2632227

Savla, U., Benes, J., Zhang, J. and Jones, R. S. (2008). Recruitment of Drosophila Polycomb-group proteins by Polycomblike, a component of a novel protein complex in larvae. Development 135(5): 813-7. PubMed citation; Online text

Schmitt S, Prestel M, Paro R. (2005). Intergenic transcription through a polycomb group response element counteracts silencing. Genes Dev. 19: 697-708. Medline abstract: 15741315

Sessa, L., et al. (2006). Noncoding RNA synthesis and loss of Polycomb group repression accompanies the colinear activation of the human HOXA cluster. RNA 13(2): 223-39. Medline abstract: 17185360

Simon, J., Chiang, A., Bender, W., Shimell, M. J. and O'Connor, M. (1993). Elements of the Drosophila bithorax complex that mediate repression by Polycomb group products. Dev. Biol. 158: 131-144. Medline abstract: 8101171

Smith, S. T., et al. (2004). Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat. Cell Biol. 6: 162-167. Medline abstract: 14730313

date revised: 25 February 2008

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