The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription

Abstract

Posttranslational histone modifications are crucial for the modulation of chromatin structure and regulation of transcription. Bromodomains present in many chromatin-associated proteins recognize acetylated lysines in the unstructured N-terminal regions of histones. Here, we report that the double bromodomain proteins Brd2 and Brd3 associate preferentially in vivo with hyperacetylated chromatin along the entire lengths of transcribed genes. Brd2- and Brd3-associated chromatin is significantly enriched in H4K5, H4K12, and H3K14 acetylation and contains relatively little dimethylated H3K9. Both Brd2 and Brd3 allowed RNA polymerase II to transcribe through nucleosomes in a defined transcription system. Such activity depended on specific histone H4 modifications known to be recognized by the Brd proteins. We also demonstrate that Brd2 has intrinsic histone chaperone activity and is required for transcription of the cyclin D1 gene in vivo. These data identify proteins that render nucleosomes marked by acetylation permissive to the passage of elongating RNA polymerase II.

Figure 1. Brd2 and Brd3 associate with acetylated chromatin (A)

Alignment of the closely related Brd proteins that contain double bromodomains, shown to scale. Bromodomains in blue, ET domains in red. (B) The association of FLAG-Brd2 and FLAG-Brd3 with chromosomal histones was examined by immunoprecipitation. The proteins present in input chromatin (I), the immunoprecipitation unbound (U) and FLAG-peptide-eluted, bound (B) fractions were examined by SDS-PAGE and Coomassie Blue staining. (C) The proteins recovered from cells synthesizing FLAG-Brd2, FLAG-Brd3, or no tagged protein (Negative) by anti-FLAG immunoprecipitation were resolved by SDS-PAGE and examined by immunoblotting with antibodies specific for the histone modifications indicated.

Figure 2. Brd2 and Brd3 associate with chromatin containing transcribed genes (A)

DNA from the immunoprecipitation reactions shown in Figure 1B was isolated and amplified by PCR for the regions of the genes indicated. All bound reactions contained immunoprecipitated DNA from the same number of cells. Parallel reactions contained input and unbound DNA from the same number of cells (1/10 that of bound reactions). (B) Chromatin-immunoprecipitation was performed with anti-acetyl-H4 (H4TetraAc) or anti-acetyl-H3 (H3K14Ac) antibody and or preblocked A/G beads alone (A/G beads). DNA from immunoprecipitation was then purified and amplified by PCR for the genes indicated. (C) The DNA samples used in the experiment shown in panel A were amplified by PCR for the regions of the cyclin D1 gene shown. The major PCR products observed are those indicated. The fold enrichment of each sequence in the Brd2 and Brd3 associated ChIP relative to the negative control ChIP are listed under each lane. (D) The DNA samples used in the experiment shown in panel B were amplified by PCR for the regions of cyclin D1 gene shown.

Figure 3. RNAi knockdown of Brd2 induces a reduction in cyclin D1 transcription (A)

Immunoblotting of proteins isolated from cells treated with siRNA for Brd2, as indicated. (B) cDNA synthesized from the RNA isolated from cells treated with siRNA for Brd2 was amplified by PCR with primers for the genes indicated.

Figure 4. In vitro chromatin transcription system (A)

Purified RNA polymerase II, the general transcription factor proteins,, the chimeric Gal4 DNA binding domain-c-Myc activation domain (Gal4AD) activator and Rsf. Proteins were resolved by SDS-PAGE and stained with Coomassie Blue. (B) Hypo-acetylated and hyper-acetylated core histones were purified from HeLa cells, resolved by SDS-PAGE (left panel) or TAU-PAGE (middle) and stained with Coomassie Blue. Histones resolved on SDS-PAGE were subjected to immunoblotting with antibodies as indicated (right). (C) In vitro chromatin assembly system. Chromatin assembled with Rsf and either hypo-acetylated or hyper-acetylated histones onto the pG5MLP plasmid was digested with two concentrations of micrococcal nuclease, resolved by electrophoresis in a 1.3% agarose gel and visualized by ethidium bromide staining. (D) Transcription reactions contained a hyper-acetylated chromatin pG5MLP template, TBP, TFIIB, TFIIF, TFIIE, TFIIH and RNA polymerase II. was added as indicated. RNA labeled in vitro was purified and visualized by electrophoresis in 10% polyacrylamide urea gels as described in the Experimental Procedures. (E) Transcription reactions contained either a hypo-acetylated or hyper-acetylated chromatin pG5MLP template and, as indicated.

Figure 5. Brd2 and Brd3 preferentially allow transcription through hyper-acetylated chromatin (A)

Affinity purified FLAG-Brd2 and FLAG-Brd3. Proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining. (B) Hyper-acetylated chromatin templates were transcribed in the presence of and/or FLAG-Brd2 (left panel), or FLAG-Brd3 (right panel), as indicated. (C) The naked pG5MLP template was transcribed in the presence of the GTFs, FLAG-Brd2 and FLAG-Brd3, as indicated. (D) Comparison of transcription from hypo-acetylated (left panel) and hyper-acetylated (right panel) chromatin templates in the presence of, FLAG-Brd2 or FLAG-Brd3, as indicated. (E) Quantification of -, Brd2- and Brd3- dependent transcription from hypo-acetylated and hyper-acetylated chromatin templates. The values represent the mean of four independent experiments.

Figure 6. Brd2 and Brd3 require acetylation to facilitate transcription from chromatin templates (A)

Immunoblotting of histones treated with Hdac2 and Hdac3 with the antibodies indicated. (B) Comparison of transcription from chromatin assembled with hyper-acetylated histones (lanes 1–4) or with hyper-acetylated histones treated with Hdac2 and Hdac3 (lanes 5–8) in the presence of, FLAG-Brd2, or FLAG-Brd3, as indicated. (C) Hyper-acetylated chromatin or naked DNA templates were transcribed in the presence of, FLAG-Brd2 and H4 tail peptides, as indicated. Peptides were at a ~20-fold molar excess over the concentration of histone H4. (D) Hyper-acetylated chromatin templates were transcribed in the presence of equal concentrations of FLAG-Brd2 or FLAG-Bd(1+2)-Y/F, as indicated.

Figure 7. Brd2 functions as a histone chaperone (A)

Schematic diagram of the histone transfer assay. (B) The nucleosome transfer reactions contained sonicated salmon sperm naked DNA (lanes 3–4), hyper-acetylated oligonucleosomes (lanes 5–6) or hyper-acetylated oligonucleosomes treated with Hdac2 and Hdac3 (lanes 7–8) and FLAG-Brd2, as indicated. The first 2 lanes are markers, D: naked DNA, N: mono-nucleosome. (C) The reactions contained hyper-acetylated oligonucleosomes, FLAG-Brd2 and AMP-PNP, as indicated. Markers were as in panel B.