Peter J. Harte

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.

Binding of Trithorax and Polycomb proteins to the bithorax complex: dynamic changes during early Drosophila embryogenesis

In Drosophila, the maintenance of developmentally important transcription patterns is controlled at the level of chromatin structure. The Polycomb group (PcG) and trithorax group (trxG) genes encode proteins involved in chromatin remodelling. PcG genes have been proposed to act by packaging transcriptional repressed chromosomal domains into condensed heterochromatin‐like structures. Some of the trxG proteins characterized so far are members of chromatin opening complexes (e.g. SWI/SNF and GAGA/NURF) which facilitate binding of transcription factors and components of the basal transcriptional machinery. Genetic and biochemical data suggest that these two groups of regulatory factors may act through a common set of DNA elements. In the present study, we have investigated the binding of Trithorax (TRX) and Polycomb (PC) protein in the bithorax complex (BX‐C) during embryogenesis. In addition, we have identified the minimal fragments from the Ultrabithorax (Ubx) regulatory region that are capable of recruiting TRX to chromosomal sites containing them. Comparative analysis of the binding of the two proteins shows that TRX and PC bind target sequences (PcG‐regulated elements, PREs) by cellular blastoderm, when BX‐C transcription begins. At the same stage, TRX but not PC is strongly associated with core promoters. Later, at germ band extension, the time of derepression in Polycomb mutants, PC binding is also detected outside core PREs and additionally binds to the fragments containing promoters.

The Drosophila trithorax proteins contain a novel variant of the nuclear receptor type DNA binding domain and an ancient conserved motif found in other chromosomal proteins

The products of the trithorax gene are required to stably maintain homeotic gene expression patterns established during embryo-genesis by the action of the transiently expressed segmentation genes. We have determined the intron/exon structure of the trx gene and the large alternatively spliced trx RNAs, which are capable of encoding only two protein isoforms. These very large trx proteins differ only in a long Ser- and Gly-rich N-terminal extension, encoded by exon II, which is present only in the larger trx isoform. We have identified a novel variant of the highly conserved nuclear receptor type of DNA binding domain. We have found that the previously identified Cys-rich central region contains multiple novel zinc finger motifs which are also present in the Polycomb-like protein and RBP2, a retinoblastoma binding protein. The trx proteins terminate with another novel conserved domain which we have identified in proteins from three kingdoms, including plants and fungi, indicating that has an ancient origin. Many of these proteins are chromosomally associated, suggesting that this domain may be involved in interactions between trx and other highly conserved components of chromatin involved in transcription regulation. The sequence alterations of trx mutations identify the highly conserved regions of trx as critical for the function of these large proteins. We show that zygotically expressed trx RNAs encoding the larger protein isoform are initially expressed in a spatially restricted pattern which overlaps the expression domains of the BX-C genes Ubx, abd-A and Abd-B. This pattern is transient and evolves into a broader expression domain encompassing the entire germ band during the extended germ band stage.

A 1-megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3.

ABSTRACT Polycomb group (PcG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. Here we identify and characterize a 1-MDa ESC/E(Z) complex that is distinguished from the 600-kDa complex by the presence of the PcG protein Polycomblike (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PcG protein SU(Z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As previously shown for E(Z) and RPD3, PCL and SU(Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo.

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter.

Regulation of intra- and extracellular ion activities (e.g. H+, Cl−, Na+) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na+/HCO3 − cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO3 −transporters are known: a neutral Na+/ HCO3 − cotransporter, a K+/ HCO3 − cotransporter, and a Na+-dependent Cl−-HCO3 − exchanger. We report the cloning and characterization of a Na+-coupled Cl−-HCO3 − exchanger and a physiologically unique bicarbonate transporter superfamily member. ThisDrosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughoutDrosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl−, Na+, H+, and HCO3 − but does not require HCO3 −. Transport is blocked by the stilbene 4,4′-diisothiocyanodihydrostilbene- 2,2′-disulfonates and may not be strictly electroneutral. Our functional data suggest thisNa+ driven anionexchanger (NDAE1) is responsible for the Na+-dependent Cl−-HCO3 − exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.

The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites.

trithorax is required to stably maintain homeotic gene expression patterns established during embryogenesis by the action of the transiently expressed products of the segmentation genes. The large trithorax proteins contain a number of highly conserved novel motifs, some of which have been hypothesized to interact directly with specific DNA sequences in their target genes. Using antibodies directed against trithorax proteins, we show that they are bound to 63 specific sites on the polytene chromosomes of the larval salivary gland. trithorax binding is detected at the sites of its known targets, the Bithorax and Antennapedia complexes, despite the transcriptionally repressed state of these loci in the salivary gland. A temperature‐sensitive trithorax mutation greatly reduces the number of binding sites. Simultaneous localization of trithorax and Polycomb indicates that many of their chromosomal binding sites coincide. We localized one trithorax binding site within a portion of the large 5′ regulatory region of the Ubx gene, to an interval which also contains binding sites for Polycomb group proteins. These results suggest that trithorax exerts its effects by binding directly or indirectly to specific DNA sequences in its target genes. Co‐localization with Polycomb also suggests that interactions between these activators and repressors of the homeotic genes may be a significant feature of their mode of action.

Polycomb Repressive Complex 2 and Trithorax modulate Drosophila longevity and stress resistance

Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are key epigenetic regulators of global transcription programs. Their antagonistic chromatin-modifying activities modulate the expression of many genes and affect many biological processes. Here we report that heterozygous mutations in two core subunits of Polycomb Repressive Complex 2 (PRC2), the histone H3 lysine 27 (H3K27)-specific methyltransferase E(Z) and its partner, the H3 binding protein ESC, increase longevity and reduce adult levels of trimethylated H3K27 (H3K27me3). Mutations in trithorax (trx), a well known antagonist of Polycomb silencing, elevate the H3K27me3 level of E(z) mutants and suppress their increased longevity. Like many long-lived mutants, E(z) and esc mutants exhibit increased resistance to oxidative stress and starvation, and these phenotypes are also suppressed by trx mutations. This suppression strongly suggests that both the longevity and stress resistance phenotypes of PRC2 mutants are specifically due to their reduced levels of H3K27me3 and the consequent perturbation of Polycomb silencing. Consistent with this, long-lived E(z) mutants exhibit derepression of Abd-B, a well-characterized direct target of Polycomb silencing, and Odc1, a putative direct target implicated in stress resistance. These findings establish a role for PRC2 and TRX in the modulation of organismal longevity and stress resistance and indicate that moderate perturbation of Polycomb silencing can increase longevity.

Histone Demethylase UTX and Chromatin Remodeler BRM Bind Directly to CBP and Modulate Acetylation of Histone H3 Lysine 27

ABSTRACT Trithorax group (TrxG) proteins antagonize Polycomb silencing and are required for maintenance of transcriptionally active states. We previously showed that the Drosophila melanogaster acetyltransferase CREB-binding protein (CBP) acetylates histone H3 lysine 27 (H3K27ac), thereby directly blocking its trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) in Polycomb target genes. Here, we show that H3K27ac levels also depend on other TrxG proteins, including the histone H3K27-specific demethylase UTX and the chromatin-remodeling ATPase Brahma (BRM). We show that UTX and BRM are physically associated with CBP in vivo and that UTX, BRM, and CBP colocalize genome-wide on Polycomb response elements (PREs) and on many active Polycomb target genes marked by H3K27ac. UTX and BRM bind directly to conserved zinc fingers of CBP, suggesting that their individual activities are functionally coupled in vivo. The bromodomain-containing C terminus of BRM binds to the CBP PHD finger, enhances PHD binding to histone H3, and enhances in vitro acetylation of H3K27 by recombinant CBP. brm mutations and knockdown of UTX by RNA interference (RNAi) reduce H3K27ac levels and increase H3K27me3 levels. We propose that direct binding of UTX and BRM to CBP and their modulation of H3K27ac play an important role in antagonizing Polycomb silencing.

SIR2 is required for Polycomb silencing and is associated with an E(Z) histone methyltransferase complex

BACKGROUND SIR2 was originally identified in S. cerevisiae for its role in epigenetic silencing through the creation of specialized chromatin domains. It is the most evolutionarily conserved protein deacetylase, with homologs in all kingdoms. SIR2 orthologs in multicellular eukaryotes have been implicated in lifespan determination and regulation of the activities of transcription factors and other proteins. Although SIR2 has not been widely implicated in epigenetic silencing outside yeast, Drosophila SIR2 mutations were recently shown to perturb position effect variegation, suggesting that the role of SIR2 in epigenetic silencing may not be restricted to yeast. RESULTS Evidence is presented that Drosophila SIR2 is also involved in epigenetic silencing by the Polycomb group proteins. Sir2 mutations enhance the phenotypes of Polycomb group mutants and disrupt silencing of a mini-white reporter transgene mediated by a Polycomb response element. Consistent with this, SIR2 is physically associated with components of an E(Z) histone methyltransferase complex. SIR2 binds to many euchromatic sites on polytene chromosomes and colocalizes with E(Z) at most sites. CONCLUSIONS SIR2 is involved in the epigenetic inheritance of silent chromatin states mediated by the Drosophila Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase.

The N Terminus of Drosophila ESC Binds Directly to Histone H3 and Is Required for E(Z)-Dependent Trimethylation of H3 Lysine 27

ABSTRACT Polycomb group proteins mediate heritable transcriptional silencing and function through multiprotein complexes that methylate and ubiquitinate histones. The 600-kDa E(Z)/ESC complex, also known as Polycomb repressive complex 2 (PRC2), specifically methylates histone H3 lysine 27 (H3 K27) through the intrinsic histone methyltransferase (HMTase) activity of the E(Z) SET domain. By itself, E(Z) exhibits no detectable HMTase activity and requires ESC for methylation of H3 K27. The molecular basis for this requirement is unknown. ESC binds directly, via its C-terminal WD repeats (β-propeller domain), to E(Z). Here, we show that the N-terminal region of ESC that precedes its β-propeller domain interacts directly with histone H3, thereby physically linking E(Z) to its substrate. We show that when expressed in stable S2 cell lines, an N-terminally truncated ESC (FLAG-ESC61-425), like full-length ESC, is incorporated into complexes with E(Z) and binds to a Ubx Polycomb response element in a chromatin immunoprecipitation assay. However, incorporation of this N-terminally truncated ESC into E(Z) complexes prevents trimethylation of histone H3 by E(Z). We also show that a closely related Drosophila melanogaster paralog of ESC, ESC-like (ESCL), and the mammalian homolog of ESC, EED, also interact with histone H3 via their N termini, indicating that the interaction of ESC with histone H3 is evolutionarily conserved, reflecting its functional importance. Our data suggest that one of the roles of ESC (and ESCL and EED) in PRC2 complexes is to enable E(Z) to utilize histone H3 as a substrate by physically linking enzyme and substrate.