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. 2009 Sep;136(18):3131-41.
doi: 10.1242/dev.037127.

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

Feng Tie  1 , Rakhee Banerjee, Carl A Stratton, Jayashree Prasad-Sinha, Vincent Stepanik, Andrei Zlobin, Manuel O Diaz, Peter C Scacheri, Peter J Harte
Affiliations

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

Feng Tie et al. Development. 2009 Sep.

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
H3K27 is acetylated by Drosophila CBP. (A) Western blots (top two panels) and Coomassie Blue staining (bottom) of calf thymus histones (lane 1), recombinant Drosophila H3 and H4 from E. coli (lane 2) and Drosophila embryo histones (0-22 hour, lanes 3 and 4) extracted with acid (a) or high salt (s). The asterisk marks N-terminally degraded H3. (B) Western blots of whole S2 cell extracts after a 5-day treatment with CBP or GCN5 dsRNAs (lanes 3 and 4). Recombinant (unmodified) H3 from E. coli in lane 5 serves as a general control for the modification-specific antibodies used. β-tubulin and histone H3 (c and h) serve as loading controls. (C,D) Western blots of whole S2 cell extracts after transient transfection to co-express (C) full-length CBP and GST-H3 (wild-type or K27Q mutant) or (D) truncated FLAG-CBP [CBPΔN and CBPΔ(N+HAT)] and GST-H3. Endogenous H3K27ac was strongly detected in lanes 1-4 (not shown). (E) Immunoblots of in vitro acetylation assays using purified FLAG-CBPΔN (lane 3) and FLAG-p300 (lanes 6 and 7). Lane 1 is input substrate (unmodified H3 from E. coli) and lane 2 is a parallel control using an anti-FLAG-purified fraction from non-transfected S2 cells. Lane 4 (embryo histones) serves as a positive control. The two asterisks mark a C-terminally degraded H3.
Fig. 2.
Fig. 2.
RPD3 is involved in deacetylation of histone H3K27ac. Western analysis of S2 cell extracts, carried out as described in Fig. 1B, after knockdown of CBP, SIR2, RPD3 and CREB2 (as indicated above each lane 2-6), demonstrating the effects of each knockdown on (A) levels of proteins listed on left and (B) levels of histone H3 modifications listed on right. The top two panels in B show different exposures of the same anti-H3K27ac western. β-tubulin and H3 serve as loading controls.
Fig. 3.
Fig. 3.
Complementary changes in H3K27ac and H3K27me3 during embryogenesis. Western analysis of histone H3 extracted from Drosophila embryos in high salt as described in Fig. 1B. Embryo ages (hours after egg lay) are indicated above each lane. The band below K27me2 and K27me1 in lane 3 is partially degraded H3. In 0-4 hour embryos, H3K27me3 could be detected only in 4× concentrated extracts (data not shown).
Fig. 4.
Fig. 4.
Knockdown of E(Z) leads to reciprocal changes in H3K27me3 and H3K27ac. (A) Western analysis of different numbers of S2 cells (0.15, 0.3 and 0.45 million cells), either untreated (lanes 1-3) or after E(Z) knockdown (lanes 4-6). Note the decrease in H3K27me3 and H3K27me2 and the increase in H3K27ac after knockdown. The two lanes to the right show efficient depletion of E(Z). (B) Western analysis of whole S2 cell extracts (lanes 1-3) and adult fly extracts from controls (w1118, lane 4) and flies constitutively overexpressing E(Z) from a hs-E(z) transgene raised at 29°C (lane 5). Lane 1, untreated cells; lane 2, control knockdown (GFP); lane 3, partial knockdown of E(Z). Note the residual E(Z) and that H3K27me2 was present at the control level. (C) Reduction of H3K27me3 and appearance of H3K27ac on the abd-A promoter after knockdown of E(Z). ChIP efficiency, in terms of the percentage of input DNA recovered by immunoprecipitation, was determined by real-time PCR and is shown for E(Z), CBP, H3K27me3 and H3K27ac. Rabbit pre-immune serum was used as a background control. The ratio of signal to background is indicated at the bottom of each column (left panel). Means and s.d. from two separate ChIP real-time PCRs are shown. Note that E(Z) and H3K27me3 signals at the abd-A promoter (left) were above background (2.6 and 7.6 times, respectively) in untreated S2 cells (gray), but were decreased to 1.3 and 2.7 times background after E(Z) knockdown (black). In the transcribed region of abd-A in untreated and E(Z) knockdown S2 cells, signals for E(Z), CBP and H3K27ac were not significantly above background (right-hand panel), indicating their absence.
Fig. 5.
Fig. 5.
Knockdown or overexpression of CBP leads to reciprocal changes in H3K27ac and H3K27me3. (A,B) Western analysis of histones and proteins from adult flies in which CBP has either been (A) moderately knocked down by RNAi (hsp70-GAL4/+; UAS-CBP RNAi/+) or (B) moderately overexpressed (hsp70-GAL4/+; UAS-CBP/+) (lane 2). Flies expressing GAL4 alone (hsp70-GAL4) serve as a control (lane 1). The H3K27me3 level (relative to total H3) was decreased by ∼30% by CBP overexpression, as determined by a Li-Cor imager. Consistent with the CBP knockdown results, moderate CBP overexpression also increased the level of H3K18ac (lane 2 in B), but not of K23ac, K14ac or K9ac. (C) The small rough eye phenotype caused by stronger eye-specific overexpression of CBP is suppressed by simultaneous overexpression of E(Z). Eyes are shown from (a) a wild-type control (w1118), (b,d) a CBP overexpresser and (c,e) a CBP+E(Z) overexpresser at 29°C and at 18°C as indicated. Homozygous hs-E(z) flies, raised at 29°C, have wild-type eyes (not shown).
Fig. 6.
Fig. 6.
TRX is required for H3K27 acetylation. (A) Polytene chromosomes from the temperature-sensitive trx1 mutant grown at permissive (18°C) or restrictive (29°C) temperature were stained under identical conditions with the antibodies indicated at the top of each panel. DAPI co-staining (left column) of the chromosomes stained with anti-TRX antibody (second column), indicates that the general chromosome structure appears unperturbed by the trx1 mutation. The two right-hand panels show simultaneous co-staining with guinea pig anti-CBP and rabbit anti-H3K27ac antibodies; the two signals appear similar to those in preparations stained with either anti-CBP or anti-H3K27ac alone (data not shown). (B) Western analysis of histones extracted from adult trx1 flies with antibodies specific for total H3 and post-translationally modified H3 as indicated. The CBP level appeared unchanged in the trx1 mutant at 29°C (data not shown). (C) Polytene chromosomes from control (top) and TRX-overexpressing (da-GAL4 driver, bottom) larvae stained as in A. (D) Western analysis of histones extracted from control flies (hs-GAL4, lane 1) and flies overexpressing TRX under control of the hs-GAL4 driver at 29°C (lane 2).
Fig. 7.
Fig. 7.
Model of Polycomb silencing and its dynamic regulation. (A) Summary of enzymatic reactions on H3K27. (B) PRC2 and RPD3 promote Polycomb silencing by deacetylating H3K27ac and trimethylating H3K27 (blue oval) at promoter nucleosomes, which are bound by PC-containing PRC1. TRX, CBP and UTX collaborate to prevent Polycomb silencing by demethylating and acetylating H3K27 (red square) at promoter nucleosomes and stimulating transcription by trimethylation of H3K4 (red oval). The five nucleosomes displayed represent either inactive (left) or active (right) genes.
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