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.

Fig 1

UTX and BRM are associated with Drosophila CBP in vivo. (A) Specificity of anti-BRM and anti-UTX antibodies. Whole S2 cell extracts (5 × 10⁵ cells in left and 2.5 ×10⁵ cells in right two panels) were separated by SDS-PAGE and visualized by Coomassie staining or Western blotting with anti-BRM and anti-UTX antibodies as indicated. Arrows indicate BRM and UTX bands that disappeared upon S2 cell treatment by Brm and Utx dsRNAs (see Fig. 7B and data not shown). (B) Immunoblots of proteins after IP from embryo nuclear extracts (NE) with guinea pig anti-CBP, anti-UTX, anti-BRM, anti-PHO, and anti-SU(Z)12 antibodies (lanes 3 to 7). Preimmune serum in lane 2 serves as a negative control. NEs were treated with ethidium bromide prior to IP. (C) Fractionation of NE on a Superose 6 size exclusion column. Fraction numbers and sizes are indicated at top. Void volume ends with fraction 6. Note that UTX but not BRM or CBP was also detected in smaller-size fractions (fractions 18 to 20, ∼150 kDa). PRC2, identified by the distribution of its SU(Z)12 subunit, peaks at fraction 15 (670 kDa). (D) IP by anti-CBP antibodies from fraction 11 in panel C. An asterisk indicates IgG heavy chain.

Fig 2

UTX and BRM colocalize with CBP on many genes marked by H3K27ac. (A and B) ChIP-chip plots across the BX-C (Ubx, abd-A, and Abd-B in panel A) marked with H3K27me3 and genes (He189B, srp, GATAe, and pnr in panel B) marked with H3K27ac in DRSC S2 cells. Antibodies used in ChIP are indicated on the left of each plot. Vertical lines below each plot indicate the regions where binding is enriched (ChIP/Input ratio of ≥2). Arrows at TSS of Abd-B and srp indicate direction of transcription. (C) Heat map illustrating the extent of overlap of CBP, UTX, BRM, H3K27ac, H3K27me3, and PC signals across chr3R. The 5 kb on each side of peak midpoints is shown. ChIP/Input signals are scaled to a range of −3 to +3. Note that H3K27ac signals overlap with CBP, UTX, and BRM in clusters 1 and 4, where H3K27me3 is absent. (D) Aggregate plots (average value at peak midpoint and 5 kb on each side) of CBP, UTX, BRM, H3K27ac, H3K27me3, and PC following clustering shown in panel C.

Fig 3

UTX and BRM bind to zinc fingers of CBP. (A and B) Immunoblots of UTX and BRM recovered in GST-CBP pulldown assays from ethidium bromide-treated NE (top) and Coomassie staining of purified GST-CBP fusion proteins, indicated by arrowheads (bottom). Some degraded forms of GST-CBP proteins are present. A nonspecific band is indicated by an asterisk in panel A. (C) Schematic summary of GST-CBP fragments tested and results of UTX and BRM pulldowns from NE. Conserved domains in CBP include three zinc fingers (ZF1, residues 509 to 594; PHD finger or ZF2, 1858 to 1932; and ZF3, 2397 to 2470), KIX domain (940 to 1012), BrD (1699 to 1806), and HAT (1858 to 2378).

Fig 4

The TPR motifs of UTX and the C-terminal BrD-containing region of BRM bind directly to CBP. (A) Immunoblots of proteins pulled down from NE by GST-UTX (top). Purified GST-UTX fragments were stained with Coomassie (bottom). (B) Immunoblots of purified H₆-tagged UTX fragments pulled down by GST-CBP fragments (left). UTX fragments were detected by anti-Penta-His antibodies (Qiagen). Coomassie staining of the two purified H₆-tagged UTX fragments is at the right. (C) Schematic summary of recombinant UTX fragments tested for binding to CBP. UTX (total of 1,136 residues) contains five TPR motifs (115 to 216 and 307 to 402) and a JmjC catalytic domain (870 to 978). Note that UTX(1-262) and UTX(79-363) both contain the first two TPR motifs, strongly suggesting that they are sufficient for CBP binding. (D) GST-CBP fragments e to i (see Fig. 3C) were used to pull down purified BRM as in panel B. Input (left lane) is 10% of the purified BRM proteins used in the assay. (E) Schematic summary of recombinant BRM fragments tested for binding to GST-CBP in vitro. Conserved domains in Drosophila BRM include the ATPase domain (735 to 1364) and a BrD (1421 to 1526).

Fig 5

BRM binds to histone H3 and enhances H3 binding to CBP PHD finger. (A) BRM binds to unmodified full-length H3. Three BRM fragments (see Coomassie staining in Fig. 6D) were tested for binding to GST-H3 proteins (lanes 3 and 4). Lane 1 shows 15% of BRM proteins used in GST pulldown. Coomassie staining of purified GST, GST-H3 N-terminal tail, and GST-H3(full length) from E. coli is at the bottom. (B) Cooperative interactions among GST-CBP-PHD, BRM, and histone H3. Western blotting of BRM(1417-1634) and histone H3 in GST-CBP PHD pulldown in the presence/absence of BRM (total 1 μg or ∼0.16 mM) and core histones (1 to 3 μg indicated on the top of each lane) were performed. Lane 1 shows 15% of total proteins used in the assay. The ratios of pulldown BRM signals to lane 1 (set as 100%) are listed below each lane.

Fig 6

CBP HAT activity on histone H3 is enhanced by BRM(1417-1634) in vitro. (A) HAT activity of recombinant GST-CBP proteins on free histone H3. Two purified GST-CBP proteins (Coomassie stained at the bottom) were incubated with recombinant histones H3 and H4 in an in vitro HAT assay (see Materials and Methods). Note that only GST-CBP(1603-2678) in lane 2 possesses HAT activity. Lane 1 is a negative control (no GST-CBP) for the HAT assay. (B and C) Enhancement of CBP and p300 HAT activities by BRM polypeptides added to HAT assays. A total of 0.1, 0.2, and 0.4 μg of BRM(1417-1634) (top in panel B), BRM(1417-1578), BRM(1461-1634) (bottom two panels in panel B), or BSA (middle in panel B) were added to a mixture of purified GST-CBP(1603-2678) and free histones H3 and H4. For p300 HAT assay, 0.1, 0.4, and 0.8 μg of BRM(1421-1638) was added to the HAT reaction (lanes 2 to 4). Western analyses of histone H3, H3K27ac, and H3K18ac were performed with the ECL system (Pierce). Signals in panel C were quantified relative to lane 1. (D) Coomassie staining of the three purified BRM fragments used in panel B.

Fig 7

BRM and UTX are required for normal H3K27ac and H3K27me3 levels. (A) Control and brm² mutant embryos coimmunostained with anti-CBP and anti-H3K27ac (columns a and b, c and d) or anti-H3K27me3 (columns e and f) or stained with anti-BRM alone. Note that H3K27me3 is detected in wild-type embryos at stage 13 (column f) but not at stage 4 (embryo indicated by the arrow in top panel of f), when H3K27me3 is barely detectable by Western blotting (49). The bottom two panels show the same embryos as the top two panels at a 4× higher magnification. (B and C) Knockdown of UTX in vivo increases the H3K27me3 level and reduces the H3K27ac level. Immunoblots of proteins (top panels) and histone H3 (bottom panels) from S2 cells after RNAi knockdown of UTX (lanes 2 and 3 in panel A) and E(Z) (lane 4 in panel A) for 6 days and from adult hsp70-Gal4 flies (control) and UTX RNAi line (UTX RNAi/tub-Gal4) (lane 2 in panel B). Quantified H3K27ac signals relative to lane 1 are listed at the bottom. (D) Overexpression of UTX in vivo reduces H3K27me3 and increases H3K27ac. Quantitative Western analysis (49) of histone H3 extracted from adult male flies overexpressing UTX (UAS-UTX 4 M/tub-Gal4 and UAS-UTX 6F/tub-Gal4 in columns 2 and 3) and from wild-type (w¹¹¹⁸) and tub-Gal4 control males (columns 1 and 4). Western blots of protein extracts are shown at the top of the left panel (lanes 1 and 4, control; lanes 2 and 3, UTX overexpression). (E) Wild type control and Df(3L)BSC831 embryos (stage 14) coimmunostained as in panel A with anti-CBP and anti-H3K27me3 (top panel) or anti-CBP and anti-H3K27ac (bottom panel).