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.

The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180-and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180؉p105؉p55 dCAF-1 complex is active for DNA replicationcoupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and the Drosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo in Drosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.

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The Drosophila Polycomb Group (PcG) proteins are required for stable long term transcriptional silencing of the homeotic genes. Among PcG genes, esc is unique in being critically required for establishment of PcG-mediated silencing during early embryogenesis, but not for its subsequent maintenance throughout development. We previously showed that ESC is physically associated in vivo with the PcG protein E(Z). We report here that ESC, together with E(Z), is present in a 600 kDa complex that is distinct from complexes containing other PcG proteins. We have purified this ESC complex and show that it also contains the histone deacetylase RPD3 and the histone-binding protein p55, which is also a component of the chromatin remodeling complex NURF and the chromatin assembly complex CAF-1. The association of ESC and E(Z) with p55 and RPD3 is conserved in mammals. We show that RPD3 is required for silencing mediated by a Polycomb response element (PRE) in vivo and that E(Z) and RPD3 are bound to the Ubx PRE in vivo, suggesting that they act directly at the PRE. We propose that histone deacetylation by this complex is a prerequisite for establishment of stable long-term silencing by other continuously required PcG complexes.

Transcription in 9A-11A aneuploid mosaic female larvae of Drosophila melanogaster has been assessed autoradiographically. Eleven larvae were found to exhibit mosaicism out of sixty-six larvae scanned and the percentage of XO and XX nuclei varied from approximately 9 to 100. Irrespective of the number of XX nuclei present the XO nuclei (duplicated for 9A-11A) invariably showed hyperactivity for both the segments. The XX nucleus exhibited a dosage effect for all the three segments of 9A-11 A. Results support the transcriptional constancy of the entire X chromosome, as proposed by Maroni and Lucchesi. Cellular autonomy of hyperactivity of the single X chromosome even at the level of segments of the X is thus evident from the present results. Autonomy