Gene name - Polycomb
Cytological map position - 78C9
Function - transcription factor
Keywords - Polycomb group
Symbol - Pc
Genetic map position - 3-47.1
Classification - chromo domain
Cellular location - nuclear
|Recent literature||Du, J., Zhang, J., He, T., Li, Y., Su, Y., Tie, F., Liu, M., Harte, P. J. and Zhu, A. J. (2016). Stuxnet facilitates the degradation of Polycomb protein during development. Dev Cell 37: 507-519. PubMed ID: 27326929
Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. This study reports a Drosophila gene, stuxnet (stx), that controls Pc protein stability. Heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; Stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. These results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity.
|Loubiere, V., Delest, A., Thomas, A., Bonev, B., Schuettengruber, B., Sati, S., Martinez, A. M. and Cavalli, G. (2016). Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development. Nat Genet [Epub ahead of print]. PubMed ID: 27643538
Polycomb group proteins form two main complexes, PRC2 and PRC1, which generally coregulate their target genes. This study shows that PRC1 components act as neoplastic tumor suppressors independently of PRC2 function. By mapping the distribution of PRC1 components and trimethylation of histone H3 at Lys27 (H3K27me3) across the genome, a large set of genes were identified that acquire PRC1 in the absence of H3K27me3 in Drosophila larval tissues. These genes massively outnumber canonical targets and are mainly involved in the regulation of cell proliferation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. In human embryonic stem cells, PRC1 components colocalize with H3K27me3 as in Drosophila embryos, whereas in differentiated cell types they are selectively recruited to a large set of proliferation and signaling-associated genes that lack H3K27me3, suggesting that the redeployment of PRC1 components during development is evolutionarily conserved.
What are the targets of Polycomb-group (Pc-G) proteins and how do they regulate gene silencing? Pc-G proteins are usually considered to be inhibitors of homeotic genes, since Pc-G mutants were originally identified on the basis of their causing expression of homeotic genes in unusual locations, areas in which they normally would not be expressed. This so-called ectopic expression is attributed to the failure of proper gene silencing in Pc-G mutants. In fact bithorax complex contains Pc-G response elements (PREs) that are involved in initiating and maintaining silencing of the whole bithorax complex (Busturia, 1993).
Pc-G genes also regulate gap genes giant and knirps, restructuring their transcription to the posterior half of the embryo. This silencing is inititated by high levels of Hunchback protein in the anterior portion of the embryo, and maintained by Pc-G action (Pelegri, 1994).
The action of Pc-G proteins is thought to be mediated through chromatin. Chromatin is a combination of DNA and protein that derives its name from a staining reaction with dyes. About a third of the DNA in chromosomes is held in heterochromatin, a type of chromatin with which few genes are associated. Placing active genes next to heterochromatin results in their inactivation or variation in expression, an effect termed position effect variation. It is thought that heterochromatin is capable of spreading to active genes, resulting in their silencing (Spofford, 1976).
These classic biological observations have taken on a new and more literal meaning recently with the discovery of common sequence domains in Pc-G genes. Polycomb itself has a domain called the chromodomain, which is shared with HP1, a heterochromatin-associated protein of Drosophila (Paro, 1991). Many of the Pc-G proteins, including Polycomb itself, are unable to bind DNA.
Considering that they lack the ability to bind DNA, how do Pc-G proteins function to establish gene silencing? It is thought that the initial repression of a gene is carried out by transcription factors that possess the ability to recognize DNA. In the cases of giant and knirps, for example, the maternal protein Hunchback represses their expression in the anterior part of the embryo (Pelegri, 1994). Pc-G proteins provide a mechanism whereby initial repression becomes permanent. They carry out this role by assembling at the site of initial repression and forming a multiprotein complex involved in modifying chromatin to promote gene silencing. Polycomb itself has this ability to self associate (Franke, 1992).
Thus many of the roles of Pc-G proteins are played out not in association with DNA but in association with each other and with other chromosomal proteins, especially histones, the principle scaffolding protein of chromosomes. Interest in the silencing of gene expression has shifted from a concern about DNA-protein interaction to an emphasis on protein-protein interaction, and the maintenance and modification of chromatin structure (Reviewed by Orlando, 1995, Pirrotta, 1995 and Simon, 1995).
Trithorax group (trx-G) genes are able to reverse the inactivating effects of chromatin. These proteins are thought to function as transcriptional activators, removing the block on gene expression put in place by arrays of inactivating proteins.
Metazoan genomes are partitioned into modular chromosomal domains containing active or repressive chromatin. In flies, Polycomb group (PcG) response elements (PREs) recruit Pho and other DNA-binding factors and act as nucleation sites for the formation of Polycomb repressive domains. The sequence specificity of PREs is not well understood. This study used comparative epigenomics and transgenic assays to show that Drosophila domain organization and PRE specification are evolutionarily conserved despite significant cis-element divergence within Polycomb domains, whereas cis-element evolution is strongly correlated with transcription factor binding divergence outside of Polycomb domains. Cooperative interactions of PcG complexes and their recruiting factor Pho stabilize Pho recruitment to low-specificity sequences. Consistently, Pho recruitment to sites within Polycomb domains is stabilized by PRC1. These data suggest that cooperative rather than hierarchical interactions among low-affinity sequences, DNA-binding factors, and the Polycomb machinery are giving rise to specific and strongly conserved 3D structures in Drosophila (Schuettengruber, 2014).
Comparative epigenomics was used to demonstrate that Polycomb domains are an extremely well conserved feature of the genome during fly evolution. In fact, the evolutionary profile of epigenomic domain organization in embryos of five Drosophila species indicates a complete lack of divergence of H3K27me3- marked Polycomb domains in syntenic regions. A similar high conservation of the H3K27me3 pattern across Drosophila species was recently described. Polycomb domains typically harbor several PH-marked PREs, and a comparative analysis showed that these are also highly conserved and the few loci that show a divergence of PRC1 occupancy patterns are not correlated with overall domain divergence. Likewise, the binding of PHO and DSP1 is highly conserved (to a degree at least as strongly, and possibly more strongly, than binding of individual factors), but even cases of diverged factor occupancies are usually not correlated with overall PRE divergence. In marked contrast, the sequences underlying PREs and Polycomb domains are diverging extensively, and sequence-based prediction of PREs across Drosophila species suggested that divergence of PREs could occur frequently. However, neither ChIP-seq experiments nortransgenic reporter assays support this dynamic behavior. Instead, such sequence divergence is buffered by the epigenetic targeting mechanisms to maintain Polycomb domains. It is suggested that the multilayered organization uses redundancy and cooperativity to facilitate the remarkable Polycomb domain conservation. This is occurring both in cis, where several TFs collaborate to define a regulatory element even when the underlying sequence is imperfect, and at the domain level, where several PREs participate to define the PcG domain structure and possibly stabilize each other (Schuettengruber, 2014).
Although PREs are associated with several known sequence features (such as GAGA- and PHO-binding motifs) in a statistically significant way, these features are not sufficient to distinguish many PREs from the genomic background and from other PHO- or DSP1-bound active chromatin elements. There are many possible explanations for this lack of specificity, including the existence of additional, yet-to-be-characterized sequence-specific recruiting factors; the involvement of nucleosome positioning; transcription of non-coding RNAs; or imperfect modeling of the sequence specificity of the known factors. The data presented in this study, however, introduce a new perspective that can help resolve this conundrum. In contrast to previous hypotheses, the data show that even when strong binding sites are lacking, PHO and DSP1 may bind PREs directly through weak (but highly nonrandom) motifs. Remarkably, sequence affinities that are completely nonspecific on a genomic scale (possibly defining millions of spurious sites) are still highly informative for predicting the binding intensity within the context of a PRE. The strong correlation of PHO binding with weak but nonrandom motifs makes it unlikely that binding to these sites represents indirect binding via interaction/looping with strong binding sites. The data show that in order to understand PRE sequence specificity, multiple potential binding sites with variable affinities and fidelities must be taken into account, and their cooperative interaction must be considered in the context of the PRE chromosomal landscape. This idea is compatible with the evolutionary constraints on PRE sequences, which has been demonstrated in this study to affect a spectrum of binding affinities rather than to conserve classical binding sites alone (Schuettengruber, 2014).
What might be the molecular mechanism that allows the specific binding of weak sites in the context of Polycomb domains? One possibility is that cooperative binding of TFs at PREs supports their occupancy of weak motifs. Indeed, this study found that PHO and DSP1 are bound jointly at PREs (with weak underlying sequence motifs), whereas at other regions of the genome where the factors bind alone, they are usually associated with strong sequence motifs. This observation is in agreement with the recently proposed 'TF collective model,' according to which combinatorial TF binding occurs with little or no apparent sequence motifs for at least a subset of the bound factors (Schuettengruber, 2014).
In addition, it was shown that transient interactions of DNA-binding proteins with weak affinity sites are stabilized by the presence of the PcG proteins themselves. A similar observation of a positive feedback of PRC1 on PHO binding was recently reported (Kahn, 2014) and is further supported by the fact that cooperative binding of PHO and Polycomb to PREs can occur even in vitro. In vivo, long-range contacts involving remote PREs within the same (or even a different) Polycomb domain may contribute to this process. Clustering of multiple flanking PREs in the 3D space of the nucleus might generate Polycomb compartments characterized by high concentrations of PcG proteins as well as their recruiting DNA-binding proteins. In this scenario, loss of occupancy following the dissociation of any of these factors from DNA may be more easily replenished by the concentrated stock of factor within a Polycomb compartment compared with individual binding sites present elsewhere in the genome. This may push the equilibrium toward increased PHO and DSP1 binding to low-affinity sites and partially reduce the evolutionary pressure to maintain the nucleotidic sequence of recruiter motifs at PREs. Structural long-range effects may also inhibit PcG recruitment in cases where active enhancers and TSSs are in proximity to a candidate PRE sequence. This analysis suggests that H3K4me3-marked loci are also highly conserved, but the low-affinity PHO- or DSP1-binding sites in them are completely uncorrelated with occupancy of these factors, further supporting a model of highly organized and cooperative epigenomic organization (Schuettengruber, 2014).
In conclusion, the data indicate that sequence conservation collaborates with 3D chromatin architecture to maintain an exceptional evolutionary stability of Polycomb-regulated loci in fly genomes. This phenomenon highlights the contribution of chromosome domains and their particular looping structures to epigenomic specificity and genome evolution. Hi-C analysis in mammals has revealed that topological domains are a strikingly conserved feature between the mouse and human genomes. The current data raise the possibility that, beyond combinatorial contributions by TF-binding sites in close proximity, the confinement of regulatory elements within TADs and their frequent DNA contacts constitute significant driving forces that also affect DNA sequence evolution in these and possibly many other specie (Schuettengruber, 2014).
Bases in 5' UTR - 107
Exons - two - The intron is centered in the chromo domain (Paro, 1993).
Bases in 3' UTR - 1170
The PC protein exhibits homology to the heterochromatin associated protein HP1 (Platero, 1995). The homology is confined to a 37 amino acid domain in the N-terminal part of the two proteins. This region is termed the chromo domain, standing for chromatin organization modifier (Paro, 1991 and Messmer, 1992). Carboxy-terminal truncations of the PC protein do not affect chromosomal binding of PC. However, mutations affecting only the chromo domain (including in vitro generated deletions, as well as point mutations) abolish chromosomal binding. Thus the chromo domain is important for the function of PC and it is absolutely required for binding of PC protein to chromatin. Some of the nuclear patterns generated by the mutated forms of the fusion proteins suggest that the chromo domain could be involved in a packaging mechanism, essential for compacting chromosomal proteins within heterochromatin or heterochromatin-like complexes (Messmer, 1992).
Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. The nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH) was examined in this study. Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, it was possible to analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing (Cheutin, 2012).
This study showed that PC bodies co-localize with H3K27me3 and form small nuclear domains of heterogeneous intensity. Surprisingly, PC bodies are found in DAPI poor regions, often adjacent to DAPI and histone-dense euchromatic regions. This result thus indicates that PC bodies are not among the most condensed chromatin portions of the euchromatic part of the genome. This localization of PC bodies is consistent with a previous study with electron microscopy, which has shown that PC is concentrated in the perichromatin compartment of the mammalian nucleus. In contrast, these data are in apparent contrast with a series of papers reporting PcG protein-dependent chromatin condensation. PcG complexes have been shown to compact chromatin in vitro and reduce DNA accessibility in vivo. Moreover, recent works show that PcG proteins are required to maintain compaction of Hox loci in mammalian embryonic stem cells and of the mouse Kcnq1 imprinted cluster. In those studies, condensation has been addressed by measuring either the compaction of nucleosomal fibers in electron microscopy, or the distance between close genomic loci by FISH. It is difficult to relate in vitro data to the current in vivo analysis. In particular, FISH analyses do not directly distinguish between a truly dense 3D organization and other types of conformations, such as a multi-looped architecture that would not necessarily induce an increase in chromatin density. Therefore, PcG target chromatin is probably organized in higher-order 3D structures that involve nucleosome-nucleosome and protein-protein interactions, but the net density of DNA (as seen by DAPI) or histones (as seen by tagged-histone microscopy) is not particularly high in these structures (Cheutin, 2012).
Earlier studies indicated that PcG proteins rapidly exchange between the nucleoplasm and PC bodies, suggesting that PC bodies consist of a local transient accumulation of PcG proteins in the cell nucleus. Earlier studies have detected the same number of PC bodies inside the nucleus as the number of bands observed on polytene chromosomes, suggesting that PC bodies are formed by PcG proteins binding to their target chromatin. The observed colocalization of PcG target genes with PC bodies in diploid cells confirms this view. An alternative scenario posits that PC bodies could form nucleation sites onto which PcG-target genes move to become silenced. Two lines of evidence from this work suggest the first scenario to be closer to reality. Firstly, it was found that the amount of PC within a PC body depends on the linear size of the genomic region coated by PC and H3K27me3. Secondly, the higher enrichment of PC in PC bodies after homologous chromosome pairing strongly suggests that PC bodies are the nuclear counterparts of linear genomic domains identified in genome-wide studies rather than nuclear structures to which Polycomb target genes have to be localized for their silencing (Cheutin, 2012).
In the head of embryos, where the Antp and Abd-B genes are silenced, they localize in large PC bodies in all cell nuclei. In contrast, loci where PC coating is restricted to smaller genomic regions do not always localize within PC bodies in interphase cell nuclei. Interestingly, time-lapse imaging shows that large PC bodies are stable structures that can be visualized in all frames of time series, whereas small PC bodies are apparently less stable because they are not visible in all of the frames. One possible explanation for the lack of colocalization between PC target genes and PC bodies is that small genomic regions may not be coated by PC in every cell. Alternatively, the amount of PC within the PC body in which small genomic regions localize might be too small to be directly observed, and only become visible when several small PC bodies interact together. For instance a previous study showed that a transgene containing only two copies of a PRE could be detected in about 50% of cell nuclei (Cheutin, 2012).
Intense PC bodies can be visualized during entire time-lapse experiments, allowing the study of their motion. The interpretation of these time-lapse experiments is not straightforward because the MSD of PC bodies only weakly correlates with the MSC. Interestingly, tracks of PC bodies are mainly composed of narrow angles. The analysis of the motion of chromatin domains containing H2Av-GFP gave similar results, but gave unambiguous evidence for the coordinated motion of several chromatin domains. By using the Lac repressor/lac operator system, two components of chromatin motion in early G2 Drosophila spermatocyte nuclei have been reported: a short range motion which occurs in approximately 0.5 µm radius domains, and long-range motion confined to a large, chromosome-sized domain. Another study has also identified a two-regime motion of a chromatin locus inside mammalian nucleus by using a two-photon microscope, which provides high spatial and temporal resolution. This work indicated that chromatin loci undergo apparent constrained diffusion during long periods, interrupted by jumps of 150 nm lasting less than 2 s. However, none of these previous works reported any coordinated motion of adjacent chromatin domains, and therefore they both described the motion of chromatin as being consistent with a random walk (Cheutin, 2012).
In tracking experiments, it was realized that the fast regime of motion is tightly constrained within volumes much smaller than chromosome territories. This suggests that any given locus will normally explore a restricted three-dimensional environment in the cell nucleus. Since this applies generally to chromatin at all developmental stages, one can deduce that each genomic locus is likely to locate in the vicinity of neighboring loci in the three-dimensional nuclear space. The prediction is thus that each locus should most frequently contact other loci that are in its linear neighborhood along the chromosome. This behavior matches the results observed in chromosome conformation capture on chip (4C) experiments, where each 4C bait had most contacts within few hundred kb to a few Mb of surrounding chromatin. Thus, the current results provide a possible scenario for the explanation of these results obtained from large cell populations. Recent studies showed that homeotic gene clusters form an extensive network of contacts with other PcG target loci. This is consistent with the observation of multiple PC body collisions that can be stable for prolonged times in the nucleus. In contrast, the fact that PC intensity correlates with the linear extension of genomic PC and H3K27me3 domains suggests that PC-mediated associations are relatively rare, at least during embryogenesis (Cheutin, 2012).
The slower regime of long-range motion depends on coordinated large-scale chromatin movements that were not documented before. This may depend on the tools used in previous studies. Time-lapse experiments performed by using the Lac repressor/lac operator system only follow one or a few points inside the cell nucleus, limiting the probability to observe coordinated motions, especially in species containing many chromosomes. In contrast, this study followed many chromatin domains inside Drosophila nuclei and long-range coordinated motions were easily identified when at least two distinct nuclear structures moved simultaneously with a similar trajectory. This motion is directional and chromatin domains and PC bodies can cover up to 1 µm in 10 sec. Different objects having coordinated motion probably belong to the same structure, which suggests that the ensemble of chromatin domains and PC bodies displaying a similar coordinated motion forms a single higher-order nuclear structure. This kind of motion is perfectly consistent with the observation of a chromosome territory, which implies that chromosomes form distinct nuclear structures in interphase cells. A displacement of an entire chromosome, or of a chromosome arm, or a large part thereof, would induce the coordinated motion of all chromatin domains and PC bodies associated to the corresponding chromosome portion (Cheutin, 2012).
The few association and dissociation events of PC bodies observed during this work are related to long-range coordinated motion events that affect both chromatin domains and PC bodies. Therefore, gene kissing depending on PcG proteins could rely on large scale chromatin movements which lead to transient fusion of PC bodies, and may be in turn specifically stabilized by interactions among PcG proteins. Moreover, the association and dissociation of PC bodies seems to be developmentally regulated, because dynamic associations and dissociations were observed during early embryogenesis, but are strongly reduced later in development (Cheutin, 2012).
Condensed chromatin domains and PC bodies move in confined volumes much smaller than chromosome territories. This highly constrained motion prevents chromatin domains from dispersing inside the cell nucleus and can explain why chromosomes form chromosome territories in interphase cells. This movement within highly confined volumes implies that some forces prevent chromatin from diffusing within entire chromosome territories. Interestingly, it was shown before that chromatin loci localized in peri-nucleolar areas or within heterochromatin move less than the ones included in euchromatin, and it was concluded that association of chromatin loci with different nuclear compartments induces specific constraints on their motion. Another time-lapse experiment performed on one Drosophila locus flanking a large block of heterochromatin showed that random association of this locus with pericentric heterochromatin is quite stable and decreases its motion. The motion of larger chromatin structures such as heterochromatin or euchromatin domains cannot be addressed by tracking single loci. By analyzing structures larger than individual chromatin loci, the motion of both bulk chromatin domains and of PC bodies seems to be influenced by their respective local enrichment of histone and PC proteins. Therefore, one key determinant of the motion constraint is an inner property of these structures, which is coherent with the concept of self-organization (Cheutin, 2012).
The most dramatic change of PC body motion occurs during embryogenesis when nuclear volumes strongly decrease, concomitant with a decrease in bulk chromatin motion. Comparison of chromatin motion between early and late G2 Drosophila spermatocytes or between undifferentiated and differentiated cells of eye imaginal discs indicated that the volume in which chromatin loci move decreases during differentiation. However, because of the particularly rapid motion of chromatin domains and PC bodies during early embryogenesis, the slowdown of chromatin motion occurring during embryogenesis is higher than the ones previously described during differentiation. Interestingly, the reduction of the volume of constraint during developmental progression suggests a correlation between the flexibility of chromatin structures and the potential for cell differentiation (Cheutin, 2012).
It is interesting to note that the motion of PC bodies appears less sensitive to temperature than chromatin domains in late embryos, suggesting that Polycomb proteins may specifically buffer environmental effects such as temperature change. This buffering may be an important determinant of the stability of Polycomb-dependent gene silencing during development. During this work, no other fundamental difference was observed between the motion of condensed chromatin domains and of PC bodies. This apparent absence in specificity is coherent with data implying that PC bodies form molecularly specialized chromatin regions, but suggests that the molecular identity of these structures is not the main determinant of their motion. Interestingly, a previous study has shown that the artificial Mx1-YFP nuclear body exhibits a very similar mobility compared with Promyelocytic leukemia and Cajal bodies. Although being molecularly different, no specific motion of these nuclear bodies was observed, indicating that the motion of nuclear bodies mainly depends on structural issues such as their size and the nuclear volume. During fly embryogenesis, PC bodies and condensed chromatin domains move similarly, but PC bodies move in a larger volume than chromatin domains. To explain this difference, one might argue that condensed chromatin domains would form much larger structures than PC bodies. This is difficult to ascertain until the identity of these DAPI- and histone-dense regions is better understood. Genome-wide analysis of chromatin components has recently identified five different types of chromatin in Drosophila cells, among which three contained silent genes (Filion, 2010). In addition to heterochromatin and Polycomb-repressed chromatin, a third type of silent chromatin was uncovered, which is composed of very large genomic domains encompassing half of the genomic euchromatin. It is proposed that this silent chromatin portion of the genome is physically manifested as the DAPI- and histone-dense chromatin that this study has identified to be distinct from PC bodies (Cheutin, 2012).
date revised: 5 August 2016
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