Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4

Abstract

Bromodomain-containing protein 4 (Brd4) contains two tandem bromodomains (BD1 and BD2) that bind preferentially to acetylated lysine residues found in histones and nonhistone proteins. This molecular recognition allows Brd4 to associate with acetylated chromatin throughout the cell cycle and regulates transcription at targeted loci. Recruitment of positive transcription elongation factor b, and possibly the general initiation cofactor Mediator as well, plays an important role in Brd4-regulated transcription. Selective contacts with acetyl-lysines in nucleosomal histones and chromatin-binding factors likely provide a molecular switch modulating the steps from chromatin targeting to transcriptional regulation, thus further expanding the 'acetylation code' for combinatorial regulation in eukaryotes.

Figure 1.. Domain organization of the bromodomain and extraterminal domain (BET) family proteins

The evolutionarily conserved domains found in bromodomain-containing protein 4 (Brd4) and the other BET family proteins include bromodomain 1 (BD1), bromodomain 2 (BD2), extraterminal (ET), motif B and SEED (Ser/Glu/Asp-rich region). Motif A and the carboxyl-terminal motif (CTM) are present only in some family members. Numbers indicate the amino acid boundaries of each protein derived from human (h), Drosophila (d), or yeast (y). The short form of human Brd4 (hBrd4) is also shown for comparison. Alignment of amino acid sequences and the accession number for each protein-coding gene are based on the information described by Wu and Chiang [3]. Bdf, bromodomain factor; Fsh, female sterile homeotic.

Figure 2.. Acetylation of histone H3 and H4 lysine residues modulates Brd4 association with chromatin and the recruitment of Mediator and P-TEFb

Three steps for bromodomain-containing protein 4 (Brd4)-regulated chromatin targeting and transcriptional regulation are highlighted. The first step (left) represents a commitment to target gene transcription illustrated by cooperative binding between Brd4 and a transcriptional activator with acetylated chromatin through Brd4-activator interaction, activator-DNA contact, and Brd4 association, via its tandem bromodomains, with acetylated lysine 5 (K5ac), acetylated lysine 8 (K8ac), acetylated lysine 12 (K12ac), and acetylated lysine 16 (K16ac) of histone H4, and/or acetylated lysine 14 (K14ac) of histone H3. The second step (center) is Brd4-mediated recruitment of the initiation cofactor Mediator to the promoter region, which often leads to phosphorylation of the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) at Ser5 during initiation and post-initiation events. The third step (right) is Brd4-facilitated recruitment of the elongation cofactor P-TEFb (positive transcription elongation factor b) to paused Pol II that results in Ser2 phosphorylation of the CTD, thereby allowing Pol II to resume elongation. The inducible recruitment of Brd4 to an acetylated nucleosome located downstream of the transcription start site (indicated by an arrow) appears to depend on crosstalk between acetylated lysine 9 (K9ac) and phosphorylated serine 10 (S10) of H3 with H4K16ac [38].

Figure 3.. Summary of binding studies performed with various BET family proteins or their derived bromodomains with acetylated histone H3 and H4 peptides in vitro or with chromatin in live cells

Human (h) or mouse (m) bromodomains (BD1 and BD2) or BET (bromodomain and extraterminal domain) proteins purified from bacteria (bac) or present in nuclear extract (NE) or in living cells, such as 293, NIH3T3, HeLa or bone marrow-derived macrophages (BMMΦs), used for binding studies with acetylated histone H3 and H4 are indicated on the left. The assay used for individual analysis and the original reference (Ref.) reporting the results are listed on the right. Only positive interaction (+) is marked. Diacetylated residues in histone H4 required for cooperative binding are bracketed with stronger interaction indicated by thick lines. The two red and black dashed lines indicate the weakest interaction compared to other interactions depicted by bracketed solid lines. The crosstalk between H3S10 phosphorylation and H4K16ac is linked by a dashed blue arrow. ChIP, chromatin immunoprecipitation; IP, immunoprecipitation.