Vincenzo Pirrotta

Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites.

Enhancer of Zeste is a Polycomb Group protein essential for the establishment and maintenance of repression of homeotic and other genes. In the early embryo it is found in a complex that includes ESC and is recruited to Polycomb Response Elements. We show that this complex contains a methyltransferase activity that methylates lysine 9 and lysine 27 of histone H3, but the activity is lost when the E(Z) SET domain is mutated. The lysine 9 position is trimethylated and this mark is closely associated with Polycomb binding sites on polytene chromosomes but is also found in centric heterochromatin, chromosome 4, and telomeric sites. Histone H3 methylated in vitro by the E(Z)/ESC complex binds specifically to Polycomb protein.

Identification of functional elements and regulatory circuits by Drosophila modENCODE

From Genome to Regulatory Networks For biologists, having a genome in hand is only the beginning—much more investigation is still needed to characterize how the genome is used to help to produce a functional organism (see the Perspective by Blaxter). In this vein, Gerstein et al. (p. 1775) summarize for the Caenorhabditis elegans genome, and The modENCODE Consortium (p. 1787) summarize for the Drosophila melanogaster genome, full transcriptome analyses over developmental stages, genome-wide identification of transcription factor binding sites, and high-resolution maps of chromatin organization. Both studies identified regions of the nematode and fly genomes that show highly occupied targets (or HOT) regions where DNA was bound by more than 15 of the transcription factors analyzed and the expression of related genes were characterized. Overall, the studies provide insights into the organization, structure, and function of the two genomes and provide basic information needed to guide and correlate both focused and genome-wide studies. The Drosophila modENCODE project demonstrates the functional regulatory network of flies. To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

Polycomb silencing mechanisms and the management of genomic programmes

Polycomb group complexes, which are known to regulate homeotic genes, have now been found to control hundreds of other genes in mammals and insects. First believed to progressively assemble and package chromatin, they are now thought to be localized, but induce a methylation mark on histone H3 over a broad chromatin domain. Recent progress has changed our view of how these complexes are recruited, and how they affect chromatin and repress gene activity. Polycomb complexes function as global enforcers of epigenetically repressed states, balanced by an antagonistic state that is mediated by Trithorax. These epigenetic states must be reprogrammed when cells become committed to differentiation.

Role of the polycomb protein EED in the propagation of repressive histone marks.

Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. Here we show that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells.

Comprehensive analysis of the chromatin landscape in Drosophila melanogaster

Chromatin is composed of DNA and a variety of modified histones and non-histone proteins, which have an impact on cell differentiation, gene regulation and other key cellular processes. Here we present a genome-wide chromatin landscape for Drosophila melanogaster based on eighteen histone modifications, summarized by nine prevalent combinatorial patterns. Integrative analysis with other data (non-histone chromatin proteins, DNase I hypersensitivity, GRO-Seq reads produced by engaged polymerase, short/long RNA products) reveals discrete characteristics of chromosomes, genes, regulatory elements and other functional domains. We find that active genes display distinct chromatin signatures that are correlated with disparate gene lengths, exon patterns, regulatory functions and genomic contexts. We also demonstrate a diversity of signatures among Polycomb targets that include a subset with paused polymerase. This systematic profiling and integrative analysis of chromatin signatures provides insights into how genomic elements are regulated, and will serve as a resource for future experimental investigations of genome structure and function.

Genome-wide analysis of Polycomb targets in Drosophila melanogaster

Polycomb group (PcG) complexes are multiprotein assemblages that bind to chromatin and establish chromatin states leading to epigenetic silencing. PcG proteins regulate homeotic genes in flies and vertebrates, but little is known about other PcG targets and the role of the PcG in development, differentiation and disease. Here, we determined the distribution of the PcG proteins PC, E(Z) and PSC and of trimethylation of histone H3 Lys27 (me3K27) in the D. melanogaster genome. At more than 200 PcG target genes, binding sites for the three PcG proteins colocalize to presumptive Polycomb response elements (PREs). In contrast, H3 me3K27 forms broad domains including the entire transcription unit and regulatory regions. PcG targets are highly enriched in genes encoding transcription factors, but they also include genes coding for receptors, signaling proteins, morphogens and regulators representing all major developmental pathways.

POLYCOMBING THE GENOME : PCG, TRXG, AND CHROMATIN SILENCING

A characteristic feature of PcG complexes is their self-maintaining property or cellular memory. If the PcG complex is an extended structure, we might suppose that parts of the silenced region could undergo DNA replication while other parts with their complexed proteins constitute a sufficient nucleus to reassemble the silenced state as the replication wave passes through. However, if the PRE is excised from a reporter construct during development, using the FLP recombinase, silencing cannot be maintained (Busturia et al. 1997xBusturia, A, Wightman, C.D, and Sakonju, S. Development. 1997; 124: 4343–4350PubMedSee all ReferencesBusturia et al. 1997). The PcG chromatin complex does not organize flanking chromatin in a self-renewing structure. It is probably dissociated at each mitotic cycle, requiring the PRE not only for initiating but also for maintaining the silenced state. To account for the cellular memory, that is, the reconstitution of the complex only at PREs that were previously silenced but not those that had no previous complexes, we might suppose that some residual proteins remain associated with the PRE to “mark” it for rapid reassembly, or that the PRE chromatin has been modified by the silencing, for example by deacetylating the nucleosomes. Similarly, the PREs of “open” genes might be marked by some proteins that prevent the de novo assembly of PcG complexes (Michelotti et al. 1997xMichelotti, E.F, Sanford, S, and Levens, D. Nature. 1997; 388: 895–899Crossref | PubMed | Scopus (97)See all ReferencesMichelotti et al. 1997) or by a modification of the chromatin such as acetylation. The state of acetylation would constitute a marker with the required properties. During DNA replication, the semiconservative partitioning of the old nucleosomes on the daughter DNA molecules could provide the link with the previous chromatin state, provided that the presence of acetylated nucleosomes activates a function to acetylate the newly deposed nucleosomes and maintain the fully acetylated state. This might be a role for Trx and related proteins (Figure 3Figure 3).Figure 3A Scenario for the Transmission of an Open Chromatin StateThe PRE is a target for both PcG complexes and Trx. Successful repression by the PRE (possibly mediated by a histone deacetylase HDAC) can be overcome by massive expression of the activator GAL4 that binds to its target site and either directly or indirectly recruits a histone acetylase (HAT) and causes acetylation of the nucleosomes (red dot) and displacement of the PcG complex. Upon DNA replication, the old acetylated nucleosomes are partitioned semiconservatively to the daughter DNA molecules. The TRX protein, activated by the acetylated state recruits a maintenance HAT to acetylate the newly deposed nucleosomes.View Large Image | View Hi-Res Image | Download PowerPoint SlideCavalli and Paro 1998xCavalli, G and Paro, R. Cell, in press. 1998; See all ReferencesCavalli and Paro 1998 now add a surprising new dimension to the question of cellular memory. They used a lacZ reporter gene construct activated by a GAL4 UAS and containing the Fab-7 PRE from the bithorax complex. The transposon construct also contains the white gene as a marker to identify the transgenic flies. In these flies, the PRE represses the basal expression of lacZ and strongly silences the white gene, resulting in weak and variegated eye color. Massive production of GAL4 from another construct driven by a heat shock promoter activates the lacZ transgene and displaces the PcG complex from the PRE. Remarkably, when the activation is induced during embryonic development, the derepressed state of both lacZ and white persists through larval and pupal development. The PRE is evidently so completely stripped of PcG proteins that it cannot reestablish silencing. A simple interpretation of these results might be that silencing complexes can only be established de novo in the early embryo. However, if GAL4 activation takes place during larval development, the derepression is only transient and silencing returns. This could be explained by increased stability of silencing complexes as trans-interactions become possible with longer interphases. In larval cells, PcG proteins might not be completely displaced by GAL4 or might reassemble more easily at the PRE. Hypersilenced states, as well as derepressed states, can be inherited by cell progeny. For PREs such hypersilenced states are induced by growing the embryos at higher temperature (in contrast, for unknown reasons, heterochromatic silencing is alleviated by higher temperatures). When they are returned to lower temperature for the remainder of development, the resulting adults still display higher levels of silencing. These results suggest that some components of the PcG complex remain associated with the chromatin or modify it in a way that persists through DNA replication and mitosis.Yet, none of these explanations can easily account for the further finding of Cavalli and Paro 1998xCavalli, G and Paro, R. Cell, in press. 1998; See all ReferencesCavalli and Paro 1998 that the unsilenced configuration is not only maintained through many cell divisions but to some extent also through meiosis, fertilization, and development of the next generation. The derepressed state resulting from GAL4 induction in germ line cells apparently survives the extensive chromatin reconfiguration that takes place in gametogenesis, and in some way repression fails to be reestablished in up to one-fourth of the G1 embryos. One explanation for this failure would require a maintenance mechanism for the active state. That is, activation would result in a chromatin modification by a mechanism that is self-renewing every cell cycle and that persists through meiosis. Cavalli and Paro propose that trxG proteins might maintain the “open” state, preventing the reconstitution of the silencing complex. In fact, GAGA factor and Trx bind to Fab-7 and other PREs both in the silent and in the active state. Perhaps they lie in wait for the opportunity to institute the open state when the silencing complex is displaced. However, the fact that derepression in larvae is only transient argues against a simple version of this scenario.A more adventurous speculation is that the silent state is the normal state of chromatin in the germ line, where most somatic genes, including homeotic genes, would be inactive. The massive expression of the GAL4 activator in this experiment is obtained by induction of a heat shock promoter, hence GAL4-dependent activation occurs also in the germ line. As a consequence, the zygote would begin life with the reporter construct in a derepressed state. In fact, a global silencing system exists in the germ line cells of C. elegans (Seydoux et al. 1996xSeydoux, G, Mello, C.C, Pettit, J, Wood, W.B, Priess, J.R, and Fire, A. Nature. 1996; 382: 713–716Crossref | PubMed | Scopus (208)See all ReferencesSeydoux et al. 1996). Clearly more surprises are in store. What is needed now is a better understanding of the structural and molecular changes associated with silenced chromatin.

Related chromosome binding sites for zeste, suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zeste function.

Polycomb group genes are necessary for maintaining homeotic genes repressed in appropriate parts of the body plan. Some of these genes, e.g. Psc, Su(z)2 and E(z), are also modifiers of the zeste‐white interaction. The products of Psc and Su(z)2 were immunohistochemically detected at 80–90 sites on polytene chromosomes. The chromosomal binding sites of these two proteins were compared with those of zeste protein and two other Polycomb group proteins, Polycomb and polyhomeotic. The five proteins co‐localize at a large number of sites, suggesting that they frequently act together on target genes. In larvae carrying a temperature sensitive mutation in another Polycomb group gene, E(z), the Su(z)2 and Psc products become dissociated from chromatin at non‐permissive temperatures from most but not all sites, while the binding of the zeste protein is unaffected. The polytene chromosomes in these mutant larvae acquire a decondensed appearance, frequently losing characteristic constrictions. These results suggest that the binding of at least some Polycomb group proteins requires interactions with other members of the group and, although zeste can bind independently, its repressive effect on white involves the presence of at least some of the Polycomb group proteins.

A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression.

Segmentation genes provide the signals for the activation and regulation of homeotic genes in Drosophila but cannot maintain the resulting pattern of expression because their activity ceases halfway through embryogenesis. Maintenance of the pattern is due to the Polycomb group of genes (Pc‐G) and the trithorax group of genes (trx‐G), responsible for the persistence of the active or repressed state of homeotic genes. We have identified a regulatory element in the Ubx gene that responds to Pc‐G and trx‐G genes. Transposons carrying this element create new binding sites for Pc‐G products in the polytene chromosomes. This Pc‐G maintenance element (PRE), establishes a repressive complex that keeps enhancers repressed in cells in which they were originally repressed and maintains this state through many cell divisions. The trx‐G products stimulate the expression of enhancers in cells in which they were originally active. This mechanism is responsible for the correct regulation of imaginal disc enhancers, which lack themselves antero‐posterior positional information. The PRE also causes severe variegation of the mini‐white gene present in the transposon, a phenomenon very similar to heterochromatic position‐effect variegation. The significance of this mechanism for homeotic gene regulation is discussed.

Polycomb complexes and epigenetic states

Important advances in the study of Polycomb Group (PcG) complexes in the past two years have focused on the role of this repressive system in programing the genome. Genome-wide analyses have shown that PcG mechanisms control a large number of genes regulating many cellular functions and all developmental pathways. Current evidence shows that, contrary to the classical picture of their role, PcG complexes do not set a repressed chromatin state that is maintained throughout development but have a much more dynamic role. PcG target genes can become repressed or be reactivated or exist in intermediate states. What controls the balance between repression and derepression is a crucial question in understanding development and differentiation in higher organisms.