BIOLOGICAL OVERVIEW

Pleiohomeotic (Pho) is the first Polycomb group (PcG) member to be identified as a DNA binding protein (Brown, 1998). Genes of the Drosophila Polycomb group encode proteins necessary for the maintenance of transcriptional repression of homeotic genes. PcG proteins are thought to act by binding as multiprotein complexes to DNA through Polycomb group response elements (PREs). Although recruitment of PcG proteins to PREs is central to PcG action, it has not been clear whether PcG proteins recognize PREs through interactions with sequence-specific DNA binding proteins or by recognizing a particular chromatin structure. Pleiohomoetic has been identfied as a PRE binding protein in a search for proteins that interact with a PRE from the segment polarity gene engrailed. Sequencing of pho reveals that it codes for a homolog of vertebrate Ying-Yang (YY1), a ubiquitously expressed zinc finger DNA binding protein that is able to act as either a transcriptional repressor or activator in different regulatory contexts (Brown, 1998).

PREs have been shown to have an unusual silencing activity. When a PRE is included in a P-transformation vector with the white gene, it silences white expression in transgenic Drosophila. This silencing is much stronger in flies homozygous for the PRE-white vector; thus, this type of repression has been called pairing-sensitive silencing: it requires two copies of the PRE-white vector. This silencing activity was first reported for a 2.4 kb fragment of DNA from the Drosophila engrailed gene (Kassis, 1991). This fragment of DNA was further dissected and shown to contain two strong and one weak pairing-sensitive silencing fragments (Kassis, 1994). One of the fragments (a 176 bp fragment located at -576 to -400 bp of the engrailed locus) that acts as a strong pairing-sensitive site, has been the focus of an attempt to discover PRE binding proteins (Brown, 1998).

A homologous DNA fragment from the distantly related Drosophila species Drosophila virilis also acts as a pairing-sensitive silencer in D. melanogaster (Kassis, 1994). Sequence conservation between these two fragments reveals five blocks of near sequence identity. The first block is a 17 bp sequence located -547 to -531 bp upstream of the transcription start site of the Drosophila engrailed gene. When this 17 bp is deleted, the 176 bp fragment no longer acts as a pairing-sensitive silencer in transgenic flies. This result shows that the 17 bp sequence is essential for pairing-sensitive silencing. This 17 bp sequence was therefore used to search for DNA binding proteins that might be required for silencing. The 17 bp sequence was radioactively labeled and used as a gel shift probe with nuclear extracts from 0-22 hr Drosophila embryos. Three shifted complexes were detected, all of which competed specifically with the unlabeled probe but not with nonspecific double-stranded probes or the single-stranded 17 base oligonucleotides. A multimer (nine copies) of the 17 bp sequence was prepared and used to screen a cDNA expression library made from 20-24 hr Drosophila embryos. Of three clones coding for a protein that showed specific binding to a labeled fragment, one contained a full-length cDNA for a protein specifically binding the 17 bp oligonucleotide. Sequencing of the cDNA revealed a protein homologous to YY1 (Brown, 1998).

Previous analyses have uncovered GAGA sites as the only obvious sequence similarity between diverse PREs. The sequences of three different PREs were therefore examined for the presence of the YY1 core consensus site CCATNTT or the other reported YY1 consensus binding site 5'(C/g/a)(G/t)(C/t/a)CATN(T/a)(T/g/c)-3'. YY1 consensus binding sites were found within a PRE from Antennapedia (Zink, 1991), a PRE from polyhomeotic (Fauvarque, 1993), and a PRE from Ubx (Chang, 1995 ), suggesting that Pho binds to all of these PREs. Thus, Pho may be important for the function of many different PREs. It is unlikely that Pho acts alone in recruiting PcG proteins to the DNA for three reasons: (1) another fragment of engrailed DNA (from -2407 to -1944) contains potential Pho binding sites but does not exhibit pairing-sensitive repression of white (Kassis, 1994 ); (2) deletion of another block of conserved DNA (from -481 to -470) of the 176 bp silencer also leads to a loss of pairing-sensitive silencing, and (3) a multimerized version of the 17 bp oligonucleotide was not sufficient for pairing-sensitive silencing of mini-white. Thus, it is likely that more than one DNA binding protein is required for pairing-sensitive silencing. It has been suggested that Pho binds to PREs and, in conjunction with other unidentified DNA binding proteins, recruits PcG proteins to the DNA. At different promoters and in different developmental contexts, Pho may interact with different protein complexes. At the engrailed locus, different subsets of PcG complexes may act together to silence engrailed expression (Brown, 1998 and references).

One of the other proteins involved in recruiting PcG proteins to the DNA may be GAGA factor, a protein involved in nucleosome remodeling. GAGA factor has been found to colocalize with PC protein on regulatory elements of the bithorax complex. GAGA binding sites are found in some PREs, and mutations in GAGA factor have been found to influence pairing-sensitive silencing mediated by the Fab-7 PRE. Three potential GAGA binding sites are present within the 176 bp silencer. Within the engrailed pairing-sensitive silencer, located from -1944 to -1503, there are two YY1-consensus binding sites and three GAGA binding sites. Interestingly, the engrailed fragment at -2407 to -1944 contains a YY1-consensus site but no GAGA sites and does not act as a pairing-sensitive silencer (Brown, 1998 and references).

What is the relationship between YY1 and the Polycomb group genes? Mammalian YY1 is a ubiquitous multifunctional protein involved in the expression of many different genes (reviewed in Shi, 1997). It can act as an activator, repressor, or initiator of transcription depending on cellular and promoter context. Transcriptional activation by YY1 involves a bipartite transactivation domain (comprising two acidic regions at the N terminus), a spacer region (with modulatory activity), and the DNA binding domain. Transcriptional repression is largely mediated by the zinc finger domain that is required both for protein-protein and protein-DNA interactions. No known transactivation domains were detected in the Pho sequence, and only the zinc finger region and a small region of the spacer of mammalian YY1 were found to be conserved. Thus, the function of Pho may be limited to repression. This would explain why no Drosophila YY1 activity was detected in a previous study (Seto, 1991), since the assays employed required YY1-driven transcriptional initiation of reporter constructs (Brown, 1998 and references).

How does mammalian YY1 repress transcription, and how is this related to the mechanism of repression by the PcG genes? YY1 has been shown to repress transcription by multiple mechanisms, including competition with an activator for overlapping binding sites, direct protein-protein interactions with transcriptional activators, and interference with activator/transcriptional machinery interactions. Mammalian YY1 has also been shown to interact with RPD3 (see Drosophila Rpd3), a histone deacetylase, through a domain not present in Pho. In HIV-1, YY1 seems to interact with another transcription factor, LSF, to repress transcription through an unknown mechanism. Thus, the mechanism of transcriptional repression by YY1 seems to be promoter-specific. It is suggested that Pho may play multiple roles in the regulation of gene expression in Drosophila: that is, Pho may be considered a general transcription factor that plays a role in stable silencing by binding to the DNA and recruiting chromatin-associated silencing proteins. The severe pleiotropic effects of removing pho function from Drosophila eggs may reflect this dual role (Brown, 1998).

The DNA-binding Polycomb-group protein Pleiohomeotic maintains both active and repressed transcriptional states through a single site

Although epigenetic maintenance of either the active or repressed transcriptional state often involves overlapping regulatory elements, the underlying basis of this is not known. Epigenetic and pairing-sensitive silencing are related properties of Polycomb-group proteins, whereas their activities are generally opposed by the trithorax group. Both groups modify chromatin structure, but how their opposing activities are targeted to allow differential maintenance remains a mystery. This study identified a strong pairing-sensitive silencing (PSS) element at the 3' border of the Drosophila even skipped (eve) locus. This element can maintain repression during embryonic as well as adult eye development. Transgenic dissection revealed that silencing activity depends on a binding site for the Polycomb-group protein Pleiohomeotic (Pho) and on pho gene function. Binding sites for the trithorax-group protein GAGA factor also contribute, whereas sites for the known Polycomb response element binding factors Zeste and Dsp1 are dispensible. Normally, eve expression in the nervous system is maintained throughout larval stages. An enhancer that functions fully in embryos does not maintain expression, but the adjacent PSS element confers maintenance. This positive activity also depends on pho gene activity and on Pho binding. Thus, a DNA-binding complex requiring Pho is differentially regulated to facilitate epigenetic transcriptional memory of both the active and the repressed state (Fujioka, 2008).

This study dissected a strong pairing sensitive silencing element from the 3' boundary of the eve locus. It was found that silencing activity depends on a single Pho-binding site, whereas sites for a number of other proteins found in such elements are less important. The element is genetically responsive to PcG-group activity, as it depends on pho gene function. This eve 3' PRE has bona fide PRE activity, which can maintain a silenced state established in embryos (Fujioka, 2008).

Previous studies have suggested that PRE-containing P-element-based transgenes have a tendency to insert near endogenous PREs, and that this can bias reporter gene expression. This study applied both P-element analysis and the {Phi}C31 recombinase-mediated cassette exchange (RMCE) system to compare the effects of mutating binding sites. The data reveal that there is also variation in PRE effects using RMCE into different target sites. Therefore, it would seem important to test several target sites when using RMCE, to ensure that results are not specific to one chromosomal location. Furthermore, where sensitivity to position effects is high, such as with GAGA factor (GAF) site-mutated PRE, it remains valuable to use the standard methodology to probe a variety of insertion sites (Fujioka, 2008).

Surprisingly, it ws found that the eve PRE is also required for positive maintenance of expression in the larval CNS, and that this activity requires both the Pho-binding site and pho gene function. Together, these data strongly suggest that Pho is directly involved in positive maintenance of gene activity. This is surprising because Pho has heretofore been associated only with direct repression of target genes, by recruiting the PRC2 complex and other PcG proteins. However, recent studies have blurred the distinction between PcG genes and trxG genes, as some members of each class appear to have dual functions. Furthermore, PREs usually reside in close proximity to TREs, and an element from the promoter region of engrailed that mediates PSS and can act as a PRE was recently shown to have an activating role in its natural context (Devido, 2008). Recent studies of the Ubx locus have indicated that PcG proteins are present at PREs in both the off and the on state, and that binding of Ash1 prevents silencing by the PRC complex in cells where Ubx is expressed, suggesting that silencing is actively prevented. A similar situation may pertain to Pho function in the eve locus (Fujioka, 2008).

Because trxG proteins are known to be involved in positive regulation by other maintenance elements, it was of interest to see in whether they are involved in pho-dependent positive maintenance by the eve PRE. It was also of interest to see in whether other PcG proteins are involved. Because the positive maintenance assay requires survival to the third larval instar, so far it has not been possible to test only weak alleles of trx, Trl and E(z), none of which showed discernable effects in the assays that were used. At this point, it cannot definitively be said whether other trxG or PcG proteins are involved in the positive maintenance function of the eve PRE. However, the observation that a consensus Grh binding site is present in the more active half of PRE300 suggests the involvement of Grh. Indeed, Grh has been shown to interact genetically with Pho, and to facilitate cooperative interaction with Pho in vitro (Fujioka, 2008).

Consistent with the broad overexpression of eve seen in the CNS of ph mutants, the eve PRE may silence expression in many cells by forming a silencing complex. In wild-type embryos, in the subset of CNS cells where eve is expressed, the same Pho-dependent DNA binding platform may recruit a distinct complex that maintains the active state. Consistent with this model, it was found that expression driven by the eve RP2+a/pCC enhancer fades prematurely in late stage embryos in ph mutants, at the same time that endogenous eve is broadly overexpressed. It will be interesting to determine the composition of Pho-dependent complexes in cells where eve is on, and in those where eve is off (Fujioka, 2008).

How can a region 9 kb away from the basal promoter affect the state of gene expression? There are accumulating data suggesting that locus-wide regulation occurs through direct interactions of the promoter with enhancers and locus control regions. For example, a recent study showed that silencing by the bxd PRE directly affects the activity of the transcriptional machinery at the promoter. In the eve locus, there are PSEs both at the 3' end of the locus and at the promoter. Both contain clusters of binding sites typical of a PRE/TRE. It has been suggested that PRE-containing transgenes have a tendency to insert near endogenous PREs, which might be expected if they mediate long-range interactions. Putting these ideas together, the eve 3' PRE may physically interact with the promoter region in a Pho-dependent manner. This may serve to keep eve on in some cells and to keep it off in others, depending on whether activating or repressive complexes mediate the association (Fujioka, 2008).

Dual functionality of cis-regulatory elements as developmental enhancers and Polycomb response elements

Developmental gene expression is tightly regulated through enhancer elements, which initiate dynamic spatio-temporal expression, and Polycomb response elements (PREs), which maintain stable gene silencing. These two cis-regulatory functions are thought to operate through distinct dedicated elements. By examining the occupancy of the Drosophila pleiohomeotic repressive complex (PhoRC) during embryogenesis, extensive co-occupancy was revealed at developmental enhancers. Using an established in vivo assay for PRE activity, it was demonstrated that a subset of characterized developmental enhancers can function as PREs, silencing transcription in a Polycomb-dependent manner. Conversely, some classic Drosophila PREs can function as developmental enhancers in vivo, activating spatio-temporal expression. This study therefore uncovers elements with dual function: activating transcription in some cells (enhancers) while stably maintaining transcriptional silencing in others (PREs). Given that enhancers initiate spatio-temporal gene expression, reuse of the same elements by the Polycomb group (PcG) system may help fine-tune gene expression and ensure the timely maintenance of cell identities (Erceg, 2017).

While enhancers initiate spatio-temporal transcriptional activity, PREs maintain a previously determined transcriptional state of their target genes, thus leading to transcriptional memory. PREs are generally thought to be dedicated solely to gene silencing and not to contain enhancer-like features to activate gene expression. This study presents evidence to the contrary, that both functions can be encoded in the same cis-regulatory element, depending on the cellular context. This is not a rare event -- almost 25% of PhoRC occupancy is at developmental enhancers. Of the 16 elements that this study tested experimentally (either enhancers for PRE activity or PREs for enhancer activity), nine have dual function, being sufficient to activate transcription in a specific spatio-temporal pattern and mediate PcG-dependent silencing in vivo (Erceg, 2017).

These dual elements have interesting implications for transcriptional regulation during embryonic development. First, at the level of PcG protein recruitment, this subset of enhancers is highly enriched in the Pho motif, which distinguishes them from other developmental enhancers. This suggests that the recruitment of Pho to PhoRC enhancers is direct via sequence-specific DNA binding, consistent with an instructive model of recruitment, although other factors are likely involved. PcG proteins and developmental TFs bind in close proximity to each other within the same element (a single DNase hypersensitive site), raising the possibility of direct interplay between the two. The results indicate that the activity of PhoRC-bound enhancers is dominated by tissue-specific TFs that activate transcription in some cells while being dominated by a functional PcG complex in other cells. Is this due to mutually exclusive occupancy of developmental TFs and PcG proteins in different tissues, or do they compete functionally at these elements? The dramatic derepression of enhancer activity in different cell types upon PcG protein removal suggests that other tissue-specific TFs must occupy these enhancers in the PcG silenced cell. This has interesting implications for enhancer activity, as it is well known that TFs bind to thousands of sites (tens of thousands in mammalian cells), but only a subset of associated target genes changes expression when the TF is removed. This has led to the general assumption that the majority of binding events is nonfunctional or neutral. These data suggest that at least a subset of this embryonic occupancy can be functional if not actively antagonized by the presence of PcGs (Erceg, 2017).

Second, enhancer-mediated polycomb recruitment has interesting implications for the mechanism of PcG-mediated silencing. The current models suggest that PcG proteins silence transcription mainly by silencing a gene's promoter, in keeping with PcG recruitment to CpG islands in vertebrates, or by coordinating a three-dimensional repressive topology, where the entire gene's locus is silenced. In either mode, a gene's promoter would not be permissive to enhancer activation. The data suggest that there may be a third mode of very local silencing at an individual enhancer, leaving the promoter and the rest of the gene's regulatory landscape open for activation by other enhancers, as was observed at the prat2 locus. This would allow for much more fine-tuning of silencing in individual tissues and stages. It also suggests that PcG proteins could play a more dynamic role, similar to a 'standard' transcriptional repressor at enhancers (Erceg, 2017).

Third, this may have broader implications for cell fate decisions during rapid developmental transitions. When multipotent cells become specified into different lineages, a specific transcriptional program often needs to be activated in one cell while being repressed in other cells from the same progenitor population. Having active enhancers in the precursor cells remain accessible to directly recruit the PcG complexes would ensure that these enhancers become silenced in a timely manner. Conversely, having maternally deposited PcG proteins already bound to enhancers early in development may serve as placeholders to ensure that these dual elements remain open and available for TFs to activate at the appropriate development stage. Interestingly, in the majority of the tested cases, PcG proteins and developmental TFs use these dual elements to regulate the same target gene, the vast majority of which is key developmental regulators of cell identity (Erceg, 2017).

The identification of PREs in other species has remained a key challenge, with only a handful of PREs identified in mammals and plants to date. In mammals, the PcG system is recruited to inactive CpG islands, with few specific sequence features. Although there are mammalian homologs of the Drosophila Pho and dSfmbt proteins, Yin Yang 1 (YY1) and SFMBT, respectively, the conservation of PhoRC as a complex and its involvement in mammalian PcG silencing remain unclear. It is proposed that such dual enhancers/PREs will also exist in mammals, although, given this apparent lack of conservation of YY1 function, their mechanism of PcG recruitment may have diverged (Erceg, 2017).

PROTEIN STRUCTURE

Amino Acids - 520

Structural Domains

Comparison of the Pleiohomeotic sequence with GenBank shows that it encodes a Drosophila homolog of Yin Yang-1 (YY1). YY1 is a DNA binding zinc finger transcription factor known to be conserved from Xenopus laevis to mammals. Pho shows a remarkable 112/118 amino acid identity with mammalian YY1 over the region encoding the four zinc fingers and is 100% identical over zinc fingers 2 and 3. Pho contains all of the amino acids identified by X-ray crystallography as being involved in contacting the DNA (Houbaviy, 1996 ). In addition to the zinc finger domain, mammalian YY1 contains three other types of domains: two acidic domains, a glycine-alanine-rich domain, and a region called the spacer (Austen, 1997). A small region of the spacer is present in the Drosophila protein, but other regions of similarity between Pho and mammalian YY1 were found (Brown, 1998).

date revised: 22 October 2023

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