The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis

Abstract

Previous in vitro studies showed that the bromodomain binds to acetyllysines on histone tails, leading to the proposal that the domain is involved in deciphering the histone code. However, there is little in vivo evidence supporting the binding of bromodomains to acetylated chromatin in the native environment. Brd4 is a member of the BET family that carries two bromodomains. It associates with mitotic chromosomes, a feature characteristic of the family. Here, we studied the interaction of Brd4 with chromatin in living cells by photobleaching. Brd4 was mobile and interacted with chromatin with a rapid ''on and off'' mode of binding. This interaction required both bromodomains. Indicating a preferential interaction with acetylated chromatin, Brd4 became less mobile upon increased chromatin acetylation caused by a histone deacetylase inhibitor. Providing biochemical support, salt solubility of Brd4 was markedly reduced upon increased histone acetylation. This change also required both bromodomains. In peptide binding assays, Brd4 avidly bound to di-and tetraacetylated histone H4 and diacetylated H3, but weakly or not at all to mono-and unacetylated H3 and H4. By contrast, it did not bind to unacetylated H4 or H3. Further, Brd4 colocalized with acetylated H4 and H3 in noncentromeric regions of mitotic chromosomes. This colocalization also required both bromodomains. These observations indicate that Brd4 specifically recognizes acetylated histone codes, and this recognition is passed onto the chromatin of newly divided cells.A cetylation of lysines on histone tails is thought to form distinct histone codes that direct molecular processes important for transcription (1, 2). A bromodomain is a motif present in a number of chromatin-modifying proteins including histone acetylases of the GNAT family, CBP͞p300, general transcription factors including TAFII250, and chromatin remodeling factors of the SWI͞SNF family (3, 4). Structural analyses in vitro have shown that the bromodomain is composed of four ␣-helices and binds to acetylated lysines on histone H3 and H4, although with relatively low affinity (5-9). Based on these studies, the bromodomain has been proposed to act as a chromatin targeting module, deciphering histone acetylation codes (1, 2, 10). However, despite in vitro evidence, it has not been clear whether bromodomain proteins interact with acetylated chromatin in the native nuclear environment in vivo. Besides the question of in vivo interaction, it has not been clear whether differentially acetylated histones are distinguished by bromodomains. The latter question is of interest in view of the fact that bromodomains of different proteins have considerable structural diversity (3, 4). Furthermore, histone acetylation codes are likely to be diverse and translated into distinct processes, as individual lysines on histone H3 and H4 are acetylated in a highly specific and ordered fashion during transcription (11,12). In addition to transcription, histone acetylation codes may play a role in cell growth, as H3 and ...

The bromodomain interaction module

“…There are four proteins in this subfamily, BRD2 [45], BRD3 [45], BRD4 [46] and BRDT [39]. Interestingly BET proteins are recruited to transcriptional start sites during mitosis [37,47,48] and the BET protein BRD4 has been shown to tether the positive transcription elongation factor (P-TEFb) to these sites via its unique C-terminus. As such, their interactions with histones have been studied in detail by several groups (Table 2).…”

The Diasporin Pathway: a tumor progression-related transcriptional network that predicts breast cancer survival

“…Necdin (Ndn) is an imprinted transcription factor on chromosome 7 that has previously been implicated in cellular proliferation, collagen gene expression [25], and regulation of the metastasis-associated gene Hif1a [26,27]. Brd4, on chromosome 17, is a bromodomain-containing protein that associates with chromatin [17]. Intriguingly, Brd4 has been demonstrated to be binding partner of our recently described metastasis efficiency modifier, Sipa1 [16].…”

“…This methodology not only allowed us to identify Rrp1b as a dual ECM and metastasis efficiency modifier, but also to demonstrate that the cell growth regulator Bromodomain 4 (Brd4) [15][16][17], which also binds Sipa1, has similar properties with regards to metastasis and ECM gene expression (Bromodomain 4 activation predicts breast cancer survival; N.Crawford, J.Alsarraj, L.Lukes, R.Walker, H.Yang, M.Lee, K. Ozato, K.Hunter; PNAS, in press). Furthermore, we demonstrated that differential activity of BRD4 in primary breast cancers accurately predicts outcome in five different tumor gene expression datasets.…”

Structural insights into human KAP1 PHD finger–bromodomain and its role in gene silencing

“…Integrated functions in histone interactions have also been shown for tandem modules of the same fold, such as the double bromodomain in human transcriptional proteins BRD2 and BRD4 (ref. 20,21) and Rsc4 of the yeast RSC remodeling complex 22 , the double chromodomain of Drosophila melanogaster CHD1 (chromo-ATPase/helicase-DNA binding) 23 and the double Tudor domain of JMJD2A 24 . However, although tandem modules of different structural folds such as the PHD finger and the bromodomain are found in many transcriptional proteins 25,26 , molecular mechanisms for their possible interactive functions are much less understood.…”