absent, small, or homeotic discs 1
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 UbxOFF and UbxONcells 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, these 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).
Previous studies have shown that PhoRC contains the DNA-binding PcG protein Pho that targets the complex to PREs, and Scm-related gene containing four mbt domains (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) (Klymenko, 2006). 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 dimethylated H3-K9 and H4-K20 in peptide-binding assays (Klymenko. 2006). 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).
Polycomb group (PcG) and trithorax group (trxG) proteins act in an epigenetic fashion to maintain active and repressive states of expression of the Hox and other target genes by altering their chromatin structure. Genetically, mutations in trxG and PcG genes can antagonize each other's function, whereas mutations of genes within each group have synergistic effects. This study showd in Drosophila that multiple trxG and PcG proteins act through the same or juxtaposed sequences in the maintenance element (ME) of the homeotic gene Ultrabithorax. Surprisingly, trxG or PcG proteins, but not both, associate in vivo in any one cell in a salivary gland with the ME of an activated or repressed Ultrabithorax transgene, respectively. Among several trxG and PcG proteins, only Ash1 and Asx require Trithorax in order to bind to their target genes. Together, these data argue that at the single-cell level, association of repressors and activators correlates with gene silencing and activation, respectively. There is, however, no overall synergism or antagonism between and within the trxG and PcG proteins and, instead, only subsets of trxG proteins act synergistically (Petruk, 2008).
Despite much interest, there is little understanding of how the epigenetic TRE/PRE-containing MEs function. One key unresolved issue pertains to the organization of these complex transcription regulatory elements with regard to the response elements/binding sites of particular trxG and PcG proteins. Response elements for several PcG proteins were mapped in the bxd ME previously, and some PcG proteins were detected at this DNA element in ChIP assays. However, information about the association of trxG proteins in the bxd ME is very limited. Several Trx-dependent TREs have been mapped in the bxd ME. In addition, Trx and Ash1 proteins have been detected at the bxd ME in ChIP assays. Given the apparent functional heterogeneity of the trxG proteins, it is revealing that besides Trx, many other trxG genes are essential for functioning of the bxd ME. Two of the interacting genes, skd and kto, encode components of the Drosophila Mediator complex, so it is possible that their role in the functioning of the bxd ME relates to the transcription of some of the non-coding RNAs that are known to be transcribed through this element. Ash2 is a component of several purified MLL (a human homolog of Trx) protein complexes. The identification of an ash2 response element in the bxd ME suggests that a second putative Trx-containing MLL-like complex might reside at the bxd ME. The genes urd and sls have only been minimally characterized, mainly as suppressors of Pc phenotypes. Therefore, it is premature to speculate about their function at this element, although they clearly interact there in some capacity (Petruk, 2008).
Identification of multiple TREs and PREs within the same ME raises an important question with regard to potential interdependency or competition in the association of proteins from the same and different protein families. To address this, focus was placed on the fine mapping of response elements for several major trxG genes that are essential for functioning of the bxd ME: ash1, the brm component of the BRM chromatin remodeling complex, and the ETP gene Asx. These proteins or components of their protein complexes (i.e. Snr1, a component of BRM) can physically associate with Trx. Thus, finding their response elements either in DNA fragments that are juxtaposed to (brm and ash1) or the same as (Asx) the previously mapped trx response element is consistent with direct interactions of these proteins with Trx. It should be noted, however, that all these proteins are components of protein complexes other than the Trx complex TAC1. Nevertheless, this suggests that there might be interdependency in recruitment and/or association of these protein complexes at the bxd ME. However, the results indicate that this suggestion is only partially true. Binding of the components of the BRM complex and of another trxG protein, Kis, were not affected by elimination of Trx. However, the association of Ash1 and Asx at all their sites on the salivary gland polytene chromosomes is completely dependent on the presence of Trx. Previous results of the reciprocal experiments indicated that binding of Trx is strongly decreased in ash1 mutant animals. This suggests that Trx, Ash1 and Asx represent a special, and at least partially interdependent, set of trxG proteins. This also suggests, in contrast to the previously mentioned genetic studies, that not all trxG proteins are mutually dependent in their functioning (Petruk, 2008).
Close proximity or even overlap between some TREs and PREs in the bxd ME suggests the existence of potential competitive relationships with regard to the binding of these functionally opposing groups of proteins. Furthermore, some ChIP assays indicate that some trxG and PcG proteins can bind to the bxd ME of both the activated and silenced gene, suggesting a potential interaction of these proteins on DNA. This was tested by asking whether binding of the components of two major PcG complexes, PRC1 and PRC2, is affected by elimination of Trx. No significant change was detected in the number or intensity of immunostained bands for all tested PcG proteins on the polytene chromosomes of trx mutant larvae. This suggests that not only is the association of PcG proteins independent of Trx, but also that Trx is not essential for preventing binding of the PcG proteins to their response elements. This is an important conclusion because some genetic studies have proposed that the main function of Trx and Ash1 is to prevent silencing by the PcG proteins (Petruk, 2008).
An important issue in understanding the molecular mechanism of trxG/PcG functioning is to correlate their association at MEs with the state of expression of their target genes. Although most of the existing data were obtained in cultured cells, two studies addressed this issue in Drosophila larval tissues. ChIP analysis in larval imaginal discs suggests that some trxG and PcG proteins are associated with the bxd ME irrespective of the status of gene expression. However, the results of another study suggest alternative association of Trx and Pc at the site of the endogenous BX-C on polytene chromosomes from both fat body and salivary glands, where BX-C is correspondingly activated or repressed. Ideally, to resolve this issue it is essential to investigate the association of PcG and trxG proteins with the ME in the same tissue at the single-cell level and at a gene of defined expression status. Such a test system was established. In this system the bxd-ME-containing transgene is either activated or repressed in cells within the same salivary gland. Direct visualization of the association of different proteins to the site of insertion of this transgene clearly indicates that major trxG and PcG proteins bind to the bxd ME in an alternative fashion. Importantly, using markers for activated and repressed transcription, it was possible to correlate binding of trxG and PcG proteins in a single cell with either the activated or repressed bxd transgene, respectively. The differences between these results and those of Papp (2006) might be explained by technical differences and by the fact that trxG and PcG proteins may behave differently in different tissues and/or in polyploid versus diploid cells. It is important to note that although the current analysis is limited to studies of a transgene, the detected alternative association of Trx and Pc on the bxd ME transgene correlates well with the results obtained at the endogenous BX-C on polytene chromosomes. It is concluded, therefore, that on a cell-by-cell basis, binding of trxG and PcG proteins is strictly dependent on the status of gene expression, in that they bind alternatively to the epigenetic regulatory elements of either activated or repressed target genes, respectively (Petruk, 2008).
In summary, this is the first work on the fine mapping of multiple TREs at any target gene. This is also the first assessment of mutual dependencies within the trxG group of activators and between the trxG and PcG of antagonistic proteins. It provides a glance of the enormously complex regulatory element that binds proteins with opposite transcriptional regulatory activities. The main conclusions of this study are that two major trxG proteins, Trx and Ash1, and the ETP protein Asx, constitute a specific subgroup of interacting proteins that depend on each other in their functioning at the bxd ME and throughout the genome. Although multiple trxG proteins are essential for epigenetic functioning of the bxd ME, their association with this element and other binding sites in the genome might not necessarily require Trx and associated proteins, as exemplified by the components of the BRM complex and Kis. The components of the major PcG complexes, PRC1 and PRC2, also associate with target genes independently of Trx, Ash1 and Asx. Another important conclusion of this work is that trxG and PcG proteins are associated with the bxd ME only at activated and repressed genes, respectively. It will be important to determine whether the choice between the establishment of trxG-mediated activation or PcG-mediated repression occurs only at very specific early stages of development, or whether it can also occur at later developmental stages (Petruk, 2008).
Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (Trx) and Ash1. This study has analyzed the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins Trx and Ash1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, it was shown that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in Ash1, the N-terminal, but not C-terminal moiety of Trx and H3K27ac. Ash1/Trx N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a 'balanced' state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a 'void' state, lacking transcription, H3K27ac, or binding of Trx or Ash1. It is concluded that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, Trx, and activators. N-ter Trx plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to Trx/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the 'bivalent' chromatin state of embryonic stem cells, and gene expression in development (Schwartz, 2010).
Key to the current understanding of PcG mechanisms is the fact that, while PcG proteins are present in most kinds of cells, the decision whether or not to repress a target gene depends crucially on whether that gene had been repressed in the previous cell cycle. This effect is responsible for the epigenetic maintenance of the repressed state and associated chromatin modifications. Similarly, through the action of Trx and Ash1, a PcG target gene that had not been repressed tends not to become repressed in the subsequent cell cycle and remains susceptible to transcriptional activators. By comparing PcG/TrxG and transcriptional landscapes in three lines of Drosophila cultured cells it was found that the full repertoire of chromatin states that PcG target genes can assume is not limited to the repressed state dominated by PcG mechanisms and the transcriptionally active state governed by TrxG proteins but in addition includes transcriptionally active 'balanced' states subjected to simultaneous or at least rapidly alternating control by both PcG and TrxG proteins, and a transcriptionally inactive 'void' state lacking both PcG and TrxG control. Thus, although PcG mechanisms first achieved fame for producing stable long-term silenced states in Drosophila homeotic genes, it is clear that, in the general case, PcG states are not necessarily stable nor long-term (Schwartz, 2010).
The results establish clearly that in robust PcG target regions (i.e. Class I PcG target regions) PcG and TrxG regulation are tightly coupled. Considering the role of MLL1 in the regulation of HOX genes and the similarity between PcG complexes in flies and mammals, it is expected that the same holds true for mammalian cells. It is possible that PcG and Trx recruitment to PRE/TREs share some DNA-binding proteins or DNA motifs. It will be important to determine whether PcG and Trx bind simultaneously or alternate over time. The nature of the Trx complex that binds to PRE/TREs remains enigmatic. To date the only Trx complex characterized biochemically is TAC1, purified from Drosophila embryos (Petruk, 2001). It is said to contain uncleaved full length Trx, anti-phosphatase Sbf1 and histone acetyltransferase dCBP. This study could detect no uncleaved Trx in the nuclei of cultured cells indicating that the Trx bound at PRE/TREs of repressed genes does not represent TAC1. Proteolytically cleaved human orthologs of Trx, MLL1 and MLL2 have been purified as part of complexes similar in composition to the yeast COMPASS4. Although the PRE/TRE binds both parts of the cleaved Trx, it lacks some COMPASS components and lacks H3K4 trimethylation, suggesting that it involves a different complex whose composition is yet to be characterized (Schwartz, 2010).
Consistent with genetic evidence, the presence of Ash1 and Trx at PcG target regions is linked to their transcriptional activity. However the two proteins show important differences in their behavior: binding of Ash1 is limited to transcriptionally active (fully derepressed or balanced) PcG targets and is not detected at completely repressed target loci. Trx is more complex. Both N-ter and C-ter parts of the protein associate with PREs regardless of the transcriptional status of their target genes and bind in the vicinity of TSS specifically when a target gene is transcriptionally active. In addition, the N-ter moiety of Trx together with Ash1 forms broad domains that encompass transcriptionally active PcG target genes. The different behavior of N-ter and C-ter parts of Trx may account for the discrepancy between reports of the co-localization of Trx and PcG proteins at many chromosomal sites and reports claiming that Trx binds exclusively to transcriptionally active target genes. The different accounts are due to the use of anti-Trx antibodies specific to different parts of the protein (Schwartz, 2010).
Whether C-ter or N-ter specific antibodies were used, the number of Trx bound regions detected in these experiments is small compared to the number of active genes. This argues against a general role for Trx in transcription and is consistent with the limited number of regions detected on polytene chromosomes by various antibodies directed against C-ter or N-ter Trx. In marked contrast to these observations, Schuettengruber (2009) has recently reported exclusive association of N-ter but not C-ter moiety of Trx with TSS of most active transcription units in the chromatin of embryonic cells. The same report also asserted that in embryonic cells Trx C-ter but not Trx N-ter is bound at PREs. Remarkably the Trx N-ter specific antibody used by Schuettengruber is reportedly the same as one of the two used in experiments from this lab. While the possibility cannot be excluded that the behavior of the N-ter moiety of Trx in embryos is totally different from that in cultured or salivary gland cells, it is suspected that more likely the preparation of the antibody used by Schuettengruber cross-reacted with some general transcription factor particularly abundant in embryonic cells. This emphasizes the importance, even the necessity, of using two or more independent antibodies to verify genome-wide ChIP results (Schwartz, 2010).
The RNAi knockdown experiments show that the broad binding of Ash1 and Trx N-ter within transcriptionally active PcG target regions is interdependent. This is consistent with the reported dissociation of Ash1 from polytene chromosomes of the salivary gland cells subjected to Trx RNAi and the severe reduction of Trx N-ter binding to polytene chromosomes of ash1 mutant larvae. Despite interdependence in binding there is no compelling evidence that Ash1 and Trx N-ter are in the same protein complex. Although an interaction between Trx and Ash1 has been reported, it was said to require the intact SET domain of Trx, which is absent from its N-ter moiety. This study has not found that Trx N-ter and Ash1 co-precipitate from nuclear extracts, strengthening the impression that the two peptides do not interact directly (Schwartz, 2010).
A histone mark associated with Ash1/Trx N-ter domains is H3K27ac. Acetylation of H3K27 can antagonize PcG activity by competing with the placement of the H3K27me3 mark, which in our model is needed for effective contact of the PRE complex with the promoter, as well as for stable PcG binding. In fact, targeting a histone H3 acetylase to a PRE is sufficient to prevent the epigenetic maintenance of repression indicates that in Drosophila the HAT responsible for bulk acetylation of H3K27 is CREB-binding protein (dCBP), encoded by the nejire gene. Direct association of both Trx and Ash1 with dCBP has been previously reported. Consistent with this, the Trx knock-down experiment, which also impairs Ash1 binding, shows that either or both proteins promote acetylation of K27 in the chromatin of active PcG targets. It also shows, however, that the level of H3K27ac is not directly related to the amount of Ash1 or Trx N-ter bound. It is possible that a small amount of Trx and/or Ash1 remaining on the chromosomes after RNAi depletion is sufficient to target enough HAT activity to maintain nearly normal levels of H3K27ac. Alternatively H3K27 acetylation may be produced by a process that is not mechanistically linked to Ash1 or Trx but is promoted by the two. A global reduction of immunostaining of polytene chromosomes with anti-H3K27ac antibodies and a global elevation of immunostaining with anti-H3K27me3 antibodies in trx mutant larvae was recently reported. The current study did not detect any global changes in either H3K27ac or H3K27me3 levels. It is possible that the effects are generally weak and could only be detected on polytene chromosomes that consist of thousands of chromatin fibers bundled together. It is noted, however, that the reported changes of H3K27 acetylation and trimethylation levels involved numerous chromosomal sites, most of which, according to current data, do not stably bind PcG or TrxG, suggesting that in these cases the effect of trx mutation may have been indirect (Schwartz, 2010).
The microarray data show that narrower peaks of H3K27ac are also found near numerous active promoters, at genes not known to be PcG targets. A role of H3K27ac at these promoters may be to antagonize dimethylation of H3K27, which is abundantly distributed throughout the genome and may have a general negative effect on transcription (Schwartz, 2010).
In Drosophila cultured cells most PcG target genes are either completely repressed or fully derepressed or entirely devoid of both PcG and TrxG regulation. However in about 5% of cases, exemplified by the Psc-Su(z)2 or inv-en loci, binding of PcG complexes does not result in complete transcriptional silencing and can coexist with binding of Ash1/Trx N-ter. Whether a PcG target gene is capable of and will assume a 'balanced' state may depend on the nature of the PRE, its binding complexes, and the promoter of the target gene. More likely, however, the major determinants are the nature and concentration of activators and repressors that act in concert with PcG/TrxG. Consistent with this idea, in imaginal disc cells, which are controlled by much more complex regulatory networks than cultured cells, simultaneous presence of both PcG and TrxG proteins at the transcriptionally active PcG target genes appears to be more common. Interestingly a common feature of the 'balanced' chromatin state in both cultured and imaginal disc cells is the confinement of Ash1/Trx N-ter binding to the regions immediately around the promoters. This is taken as a hint that the formation of a broad Ash1/Trx N-ter domain starts in the vicinity of the TSS (Schwartz, 2010).
Much has been said about the 'bivalent' state, i.e. containing both PcG repressive marks and transcriptional activity marks, as characteristic of genes in mammalian embryonic stem cells. In Drosophila, in cases such as those of the Psc-Su(z)2 or inv-en loci, the balanced action of PcG and TrxG results in chromatin states similar in appearance to the 'bivalent' state. It is supposed that, like the 'balanced' chromatin state of Drosophila PcG targets, the 'bivalent' domains of embryonic stem cells would be associated with both PcG proteins and mammalian orthologs of Trx and Ash1 (Schwartz, 2010).
PcG target genes may also assume a state lacking both PcG and TrxG proteins. The fact that in several instances the same locus resides in this 'void' chromatin state in cultured cells of completely different origin argues against it being a product of genomic aberrations. Several experimental observations also indicate that the 'void' state is not a peculiarity of cultured cells. Thus, in salivary glands the hh gene lacks PcG binding and H3K27me3 but remains transcriptionally inactive, as in D23 cells. More recently a comparison of embryonic and imaginal disc cells showed that in many cases lack of PC binding was not accompanied by transcriptional activity. The void chromatin state might be simply interpreted as a derepressed region that is transcriptionally inactive because the needed activator is absent. However, this does not explain why in this state Trx is also absent from the PRE, implying that neither PcG nor Trx binding is the default state of the PRE or that some specific condition prevents the recruitment of both. No other known repressive marks such as H3K9 methylation has been detected at these sites (Schwartz, 2010).
In line with the finding that the lack of PcG repression in the 'void' chromatin state does not automatically lead to the activation of a target gene is the observation that PC knock-down elicits a very specific genomic response. Remarkably Sex combs reduced (Scr), Antennapedia (Antp) and Abd-B, the HOX genes whose derepression in heterozygous +/Pc- flies gives the famous Polycomb phenotype, are also among the genes most sensitive to PC knockdown in BG3 cells. The sensitivity of these genes cannot be explained by intrinsically poor recruitment of PcG proteins as Abd-B and Scr are controlled by multiple strong PREs capable of robust repression when placed next to a reporter gene. It is supposed that the reason for the differential sensitivity to PC levels lies in the availability of the corresponding transcriptional activators. It is propose that in the BG3 cells the transcriptional activators of sensitive PcG target genes are present but at levels insufficient to override repression under normal conditions. The knockdown of PC lowers the threshold required for derepression. It is suggested that a general role of PcG and TrxG mechanisms is to modulate the constraints on the levels of transcriptional activators required to switch the expression of PcG target genes. This concept helps to explain why, despite the implication of the PcG system in the control of all morphogenetic pathways, the reduction of PcG levels during differentiation of mammalian cell lineages or tissue regeneration in flies results in the execution of very specific genomic programs (Schwartz, 2010).
To determine if Brm physically interacts with other trithorax group proteins, the Brm complex was purified from Drosophila embryos and its subunit composition analyzed. The Brm complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the Brm complex are not encoded by trithorax group genes. The proteins that consistently copurify with Brm have been designated Brm-associated proteins (BAPs) and are referred to by their molecular mass in kDa (BAP45, BAP47, BAP55, BAP60, BAP74, BAP111 and BAP155). Two different purification schemes identify the same set of seven polypeptides associated with Brm (Papoulas, 1998).
Biochemical evidence is presented for the existence of two additional complexes containing trithorax group proteins: a 2 MDa Ash1 complex and a 500 kDa Ash2 complex. Based on their genetic properties, three of the best candidates for trx-G members that physically interact with Brm are Absent, small or homeotic discs 1 and 2 (Ash1 and Ash2), and Trithorax. In spite of being bona fide members of the trx-G, neither Ash1, Ash2 nor Trithorax are found to be a part of the Brm complex. Affinity-purified polyclonal antibodies against Ash1 detect three prominent bands in embryo extracts, the largest of which is 270 kDa. The predicted size of the Ash1 protein (244 kDa) and the variability in amount of the smaller bands detected in different experiments argues that the 270 kDa band represents full-length Ash1 and that the smaller bands are degradation products. Affinity-purified antibodies against ASH2 detect a single band of 94 kDa. Although the Brm, BAP45/Snr1, Ash1 and Ash2 proteins are readily detected by western blotting in whole embryo extracts, neither the Ash1 nor Ash2 proteins are detected in purified Brm complex. Similar experiments using antibodies against Trx did not yield reproducible results, presumably due to the low abundance and instability of this >350 kDa protein. An examination to see if Ash1 or Ash2 are physically associated with Brm in embryo extracts used a coimmunoprecipitation assay. Neither Ash1 nor Ash2 were found to coimmunoprecipitate with Brn. It is therefore concluded that the Ash1 and Ash2 proteins do not stably interact with the Brm complex. To determine whether Ash1 and Ash2 are components of protein complexes distinct from the Brm complex in the Drosophila embryo, the native molecular mass of both proteins was examined by gel filtration chromatography. The ASH1 protein has a native molecular mass of approximately 2 MDa. By contrast, Ash2 has an apparent native molecular mass of approximately 500 kDa. No monomeric Ash1 or Ash2 is detected in embryo extracts. It is concluded that the Drosophila embryo contains at least three distinct protein complexes containing trx-G proteins: the 2 MDa BRM complex, a 2 MDa Ash1 complex and a 500 kDa Ash2 complex (Papoulas, 1998).
Trithorax (Trx) and Ash1 belong to the trithorax group (trxG) of transcriptional activator proteins -- this group of proteins maintains homeotic gene expression during Drosophila development. Trx and Ash1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and the observation that association of the Trx protein with polytene chromosomes is ash1 dependent, the possibility of a physical linkage between the two proteins was investigated. Endogenous Trx and Ash1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, Trx and Ash1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that Ash1 and Trx act on Ubx in relatively close proximity to each other. Finally, Trx and Ash1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that Trx and Ash1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription (Rozovskaia, 1999).
Since genetic experiments suggest that both trx and ash1 are involved in regulation of homeotic gene expression, it was of particular interest to determine whether binding of the proteins to polytene chromosomes and/or genetic responsiveness is conferred by the same DNA sequences. To test this, an analysis was performed to see whether both proteins bind in vivo to a well-characterized TRE-PRE-containing bxd regulatory module located 25 kb upstream of the Ubx promoter. Indeed, on salivary gland polytene chromosomes, both proteins are found at the site of insertion of a transgene containing this 4-kb bxd subregion. This indicates that Trx and Ash1 DNA binding elements may be close to each other. In addition, it has been shown that Ash1 is required for full function of the same regulatory region in vivo. Since this 4-kb region contains three trx-responsive TREs, this leaves open the possibility that Trx and Ash1 may function through the same DNA elements. Experiments aimed at fine mapping of the ash1 response element(s) within this region of Ubx are currently in progress. Nonetheless, these results suggest that Trx and Ash1 may act in concert on one or more bxd TREs. Two interesting possibilities are that both Trx and Ash1 are components of the same protein complex or that they are interacting components of two separate protein complexes that form on closely situated TREs. The physical association between Trx and Ash1 (probably through interaction of their SET domains) is apparently required for Trx binding to chromosomes, since Trx is only weakly associated with chromosomes in ash1 mutant larvae. These close physical and functional associations on Ubx regulatory DNA provide a biochemical rationale for the genetic interactions between trx and ash1 mutants (Rozovskaia, 1999).
By applying yeast two-hybrid assays as well as other methodologies, it has been found that the SET domains of both Trx and Ash1 proteins can self-associate. The self-associating Trx fragment (aa 3540 to 3759) spans the ~130-aa SET domain and includes an additional ~90 aa of upstream sequence. The self-interacting Ash1 region includes the entire SET domain (residues 1318 to 1448) in addition to upstream sequence (aa 1160 to 1317). An alternative self-associating region of Ash1 (aa 1245 to 1525) also includes the entire SET domain. Mutations within the SET domain of both Trx and Ash1 prevent self-association. Whether those TRX and ASH1 regions can also undergo hetero-oligomerization was examined. Indeed, the two polypeptides interact strongly in yeast, as evidenced by activation of both the HIS and lacZ reporters. To confirm this result, GST pull-down methodology as well as coimmunoprecipitation analysis was performed. A C-terminal Trx polypeptide (Trx SET) was synthesized and radiolabeled in a coupled transcription-translation system and tested for binding to the relevant Ash1 polypeptide (Ash1 SET) linked to GST. The ASH1-linked resin binds 10- to 20-fold more Trx SET than does GST resin alone. For in vitro coimmunoprecipitation analysis, the same Trx polypeptide was radiolabeled and mixed with unlabeled epitope-tagged (T7) Ash1 SET. The labeled Trx SET coimmunoprecipitates with the T7-ASH1 SET but not with two unrelated T7-tagged proteins. Similar results were obtained in a reciprocal experiment. Finally, plasmids encoding the T7-tagged Ash1 SET and HA-tagged Trx SET were transiently cotransfected into COS cells. The epitope-tagged polypeptides produced in vivo were also found to coimmunoprecipitate (Rozovskaia, 1999).
To address the biological significance of this hetero-oligomerization, conserved residues within the SET domain were mutagenized and their effects on interaction in yeast were tested. Thirteen different mutations at either single amino acids or nearby pairs of amino acids were constructed, 10 at highly conserved residues and 3 controls at nonconserved residues within Trx SET. Each of the alterations of conserved amino acids resulted in the loss of most or all of the capacity of TRX SET to interact with Ash1 SET in yeast. In contrast, the three alterations of nonconserved residues, located within the SET domain or immediately upstream of it, did not affect the interaction. A more limited mutagenesis analysis of conserved residues within the Ash1 SET domain shows that conversion of GRG (residues 1310 to 1321) to VRV, PN (1391 and 1392) to AY, I (1414) to A, or DY (1423 and 1424) to AA results in the loss of most or all of the interaction in yeast. These results argue for the functional significance of the TRX SET-ASH1 SET interactions seen in yeast and in vitro and suggest that the association in embryos between full-length Trx and Ash1 is direct and involves binding between their SET domains (Rozovskaia, 1999).
The human ALL-1 gene is involved in acute leukemia through gene fusions, partial tandem duplications or a specific deletion. Several sequence motifs within the ALL-1 protein, such as the SET domain, PHD fingers and the region with homology to DNA methyl transferase are shared with other proteins involved in transcription regulation through chromatin alterations. However, the function of these motifs is still not clear. Studying ALL-1 presents an additional challenge because the gene is the human homologue of Drosophila trithorax. The latter is a member of the trithorax-Polycomb gene family which acts to determine the body pattern of Drosophila by maintaining expression or repression of the Antennapedia-bithorax homeotic gene complex. Yeast two hybrid methodology, in vivo immunoprecipitation and in vitro 'pull down' techniques have been applied to show self association of the SET motifs of ALL-1, Trithorax and Ash1 proteins (Drosophila Ash1 is encoded by a trithorax-group gene). Point mutations in evolutionary conserved residues of Trithorax SET, abolish the interaction. SET-SET interactions might act in integrating the activity of ALL-1 (Trx and Ash1) protein molecules, simultaneously positioned at different maintenance elements and directing expression of the same or different target genes (Rozovskaia, 1999).
CREB-binding protein (CBP) is a coactivator for multiple transcription factors that transduce a variety of signaling pathways. Current models propose that CBP enhances gene expression by bridging the signal-responsive transcription factors with components of the basal transcriptional machinery and by augmenting the access of transcription factors to DNA through the acetylation of histones. To define the pathways and proteins that require CBP function in a living organism, a genetic analysis of CBP has been initiated in flies. Drosophila CBP (dCBP) has been overexpressed in a variety of cell types and distinct adult phenotypes were obtained. An uninflated-wing phenotype, caused by the overexpression of dCBP in specific central nervous system cells, was used to screen for suppressors of dCBP overactivity. Two genes with mutant versions that act as dominant suppressors of the wing phenotype were identified: the PKA-C1/DCO gene, encoding the catalytic subunit of cyclic AMP protein kinase, and ash1, a member of the trithorax group (trxG) of chromatin modifiers. Using immunocolocalization, it has been shown that the ASH1 protein is specifically expressed in the majority of the dCBP-overexpressing cells, suggesting that these proteins have the potential to interact biochemically. This model was confirmed by the findings that the proteins interact strongly in vitro and colocalize at specific sites on polytene chromosomes. The trxG proteins are thought to maintain gene expression during development by creating domains of open chromatin structure. These results thus implicate a second class of chromatin-associated proteins in mediating dCBP function and imply that dCBP might be involved in the regulation of higher-order chromatin structure (Bantignies, 2000).
Screens for enhancers and suppressors of overexpression phenotypes have been useful in identifying components of regulatory pathways. Nevertheless, overexpression systems have drawbacks and can potentially identify secondary effectors of a nonspecific phenotype. However, it is thought that this screen has identified genes that affect dCBP function for several reasons. (1) The number of deficiencies that suppress the uninflated-wing phenotype is small. A large number of suppressors might suggest that the overexpression of dCBP is not eliciting a specific cell phenotype. (2) Two of the deletions suppress both the wing and the eye overexpression phenotypes, suggesting that the overexpression of dCBP in the two tissues has some common effects. One of the deletions demonstrates that the dosage of PKA can affect the dCBP overexpression phenotype. CBP and dCBP are known to play a role in PKA signaling, so the fact that PKA was identified in this screen is consistent with the idea that dCBP overexpression reflects an overactivation of the PKA pathway. Trivial explanations for the suppression of dCBP overexpression by ASH1 have been ruled out; dCBP overexpression does not cause the death of ASH1-expressing cells, nor do ash1 mutations affect the overexpression of dCBP. A characterization of dCBP loss of function in these cells both in wild-type and ash1 mutant backgrounds is necessary to complete this analysis. A clonal analysis of dCBP mutant cells is not feasible because dCBP is required for cell viability and only small clones can be generated. This analysis will have to await reagents that allow dCBP function to be knocked out in the GAL4-386 cells in the ash1 mutant background. In addition, it will be important to identify the targets of dCBP and ASH1 in these cells as well as the pathways that activate them. Although the genetic analysis is not complete, it is likely that the genetic suppression of dCBP overexpression by ash1 mutations reflects a functional association between ASH1 and dCBP because these two proteins have specific interactions in vitro (Bantignies, 2000).
Overexpression of dCBP in specific CNS cells causes wing inflation defects. In many tissues, overexpression of dCBP causes lethality, suggesting that the dose of this effector is important for its function. The overproduction of dCBP in specific cells of the CNS with two different GAL4 lines produces defects in wing inflation with various degrees of penetrance. However, overexpression of dCBP in wing tissues throughout development does not interfere with wing inflation (Bantignies, 2000).
Previous studies have implicated specific CNS cells in the regulation of wing inflation. In Drosophila, the death of specific cells is triggered after eclosion and is strongly correlated with wing inflation behavior. In addition, two specific neurons in the fly brain are responsible for the production of the neuropeptide eclosion hormone (EH). The specific knockout of EH-producing cells (EH cells) during early development results in eclosion delays and a disruption of eclosion behaviors, such as wing inflation. In the moth Manduca sexta, EH triggers a neuroendocrine cascade that regulates both ecdysis and postecdysis processes such as wing inflation. It was suggested that the frequent failure of EH cell knockout flies to inflate their wings successfully is due to a lack of excitability of neuroendocrine-responsive EH cells that release important signals for proper eclosion behaviors. In Manduca, different neuropeptides, such as bursicon and the cardioacceleratory peptides, are usually released after eclosion to aid in wing expansion. It may be that the neurons that overexpress dCBP are the neurosecretory cells that are targeted by the EH cascade and that produce the peptides that signal the wing inflation process. In this case, the overexpression of dCBP interferes with normal cell function. Of course the wing inflation defect could be due to the death of the neurons caused by the overexpression of dCBP. However, the pattern of cells that overexpress LacZ and dCBP in the GAL4-386 background remains the same throughout development, and cells that overexpress dCBP and express ASH1 are viable at least 24 h posteclosion, so the overexpression of dCBP does not appear to affect the viability of these cells. Two additional GAL4 lines, GAL4-c929 and GAL4-c191, also drive specific expression in the CNS, specifically in most of the peptidergic neurons of the brain and ventral ganglion. At 25°C, escapers were obtained only with the GAL4-c191 line. Approximately 30% of these flies have uninflated or partially inflated wings (Bantignies, 2000).
It is proposed that the overexpression of dCBP in specific CNS cells affects the regulation of signaling pathways that involve dCBP and that are important for proper eclosion behaviors. Preliminary data suggest that at least some of the cells that overexpress dCBP are neuropeptidergic neurons and colocalize with the neuropeptides FMRFamide and PHM. However, antibody incompatibility does not allow for a determination of whether these cells also express ASH1. Clearly, more characterization will be required to determine the exact pathways affected by dCBP. The dominant wing phenotype obtained by overexpressing dCBP with GAL4-386 is a good model to elucidate some of the cells and signaling pathways involved in wing inflation (Bantignies, 2000).
Biochemical experiments show that coactivator dCBP binds strongly to trxG protein ASH1. This observation supports the idea that ASH1 and dCBP interact in vivo and implicates a novel class of chromatin binding proteins in mediating dCBP function. The ASH1 protein contains three motifs that are characteristic of some proteins that regulate transcription and/or are bound to chromosomes: there are two AT hook motifs in the N-terminal region, a SET domain, and a PHD finger in the C-terminal domain. The AT hook motif is important for the binding of some proteins to DNA. PHD fingers are Cys-rich Zn finger-like motifs implicated in protein-protein interactions and are found in other trxG proteins. The SET domain is an approximately 130-aa region found in a number of other chromatin-associated proteins, including the TRX factor, PcG protein Enhancer of Zeste [E(Z)], and the modifier of position effect variegation Suppressor of variegation 3-9. The TRX SET domains have been proposed to mediate association with components of chromatin-remodeling complexes, and ASH1 and TRX interact directly through their SET domains. Binding assays indicate that two N-terminal regions and the SET domain of ASH1 interact strongly with dCBP. However, no interaction with the PHD domain was observed. Thus, the SET and the PHD domains of ASH1 might function for the recruitment of other chromatin-associated proteins, such as TRX, and the N-terminal region could serve to interact with the DNA, possibly through the AT motifs, to direct the targeting of HATs to the promoter. Further biochemical characterization will be necessary to confirm this model, but the interaction between dCBP and ASH1 provides new insights on the possible function of ASH1 in gene regulation (Bantignies, 2000).
The binding of ASH1 to dCBP requires the C-terminal C/H3 domain. In mammalian CBP and p300, this region mediates interactions with numerous sequence-specific transcription factors, the adenovirus E1A protein, TFIIB, RNA helicase A, and P/CAF, a GCN5-like histone acetylase. In dCBP, the C/H3 domain mediates the interaction with transcription factor dTCF and Mad, demonstrating an important role for this domain in dCBP function. This domain contributes to the interaction with chromatin-associated protein ASH1, suggesting that dCBP may function in epigenetic regulatory complexes. The C/H3 domain is adjacent to HAT and might contribute to the regulation of the histone acetylation activity of CBP and p300 or might recruit targets of acetylation close to the enzymatic domain. Thus, it will be interesting to determine whether ASH1 has any effect on dCBP HAT functions or if it is a target of dCBP acetyltransferase activity (Bantignies, 2000).
The bromodomain of P/CAF has been shown to bind histone peptides in an acetylation-dependent manner. The bromodomain of GCN5, a member of the SAGA complex, is required for SWI/SNF remodeling of the nucleosome and stabilizing the SWI/SNF complex on the promoter. Thus, it appears that the bromodomain interacts with acetylated proteins and may form a link between different regulatory complexes. Although the full-length ASH1 does not interact with the bromodomain of dCBP, both the ASH1-458-853 polypeptide and the SET domain do interact with this domain. It may be that full-length ASH1 undergoes a modification, upon binding with the dCBP C/H3 domain, that allows other regions of ASH1 to interact with the dCBP bromodomain. In this case, it would appear that the interaction is not dependent on acetylation (Bantignies, 2000).
These results also show that dCBP and ASH1 colocalized to a number of specific sites on polytene chromosomes, suggesting that they might serve as coregulators of a specific set of genes including the homeotic selector genes. The mapping of the specific sites where dCBP and ASH1 colocalize will help identify target genes that are regulated by ASH1 and dCBP. An analysis of these genes, their promoters, and their regulation by dCBP and ASH1 will further define the functional role of the dCBP-ASH1 interaction (Bantignies, 2000).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.