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Acetylation of lysine residues is a widespread protein post-translational modification (PTM), and extensively relevant to modulation of cellular processes, including protein conformation and interaction. Histone lysine acetylation was historically proposed to be a hallmark of transcriptionally active genes, and so far, deregulation of histone acetylation patterns often drives the aberrant expression of oncogenes leading to proliferation and tumorigenesis. Three types of proteins have been identified to regulate lysine acetylation: histone acetyltransferases (HATs), bromodomain (BRD) proteins, histone deacetylases (HDACs) and sirtuins (SIRTs). Bromodomains, acting as acetyl-lysine binding domains, belong to a family of evolutionarily conserved protein modules that originally are found in proteins associated with chromatin and in almost all nuclear HATs. BRDs could contribute to highly specific histone acetylation through tethering transcriptional HATs to specific chromosomal sites, or to the activity of multi-protein complexes in chromatin remodeling. Therefore, BRDs modulate enzyme activities, protein assembly and protein-protein interactions (PPIs) through lysine acetylation, revealing wide implications for the mechanisms underlying a broad variety of cellular events, such as chromatin remodeling and transcriptional activation.
Structure and function
BRDs are 110 amino acid modules which are highly conserved throughout evolution. The BRD-containing protein family includes a variety of transcriptional coregulators, chromatin modifying enzymes and nuclear scaffold proteins that are able to specifically recognize acetylated lysine residues on histone tails. BRD containing proteins are able to also bind acetylated lysine residues on non-histone proteins. The BRD structure is composed of a left-handed bundle of four antiparallel alpha helices linked by two loop regions (Figure 1). The co-crystal structures of BRDs bound to acetyl-lysine containing peptides suggest that the acetylated lysine is first recognized in a hydrophobic pocket located between the two loops, which are formed by the most highly conserved residues. This includes an asparagine at the core of the binding site, which engages the acetyl-lysine through a hydrogen bond between its NH2 group and the acetyl carbonyl oxygen atom of the acetylated lysine. At the entrance of the binding pocket, residues located in the two loop regions interact with residues adjacent to the acetylated lysine in the target sequence, and further reinforcing the binding through hydrophobic and electrostatic interactions.
Figure 1. BRD4 bromodomain 1 structure showing the binding with the acetylated Lysine 16 on the tail of Histone H4.
The human genome encodes 46 diverse proteins which contain a total of 61 BRDs structurally clustered into eight distinct subfamilies (Figure 2). The first subfamily consists of a functionally diverse group of proteins that includes the HAT P300/CBP-associated factor (PCAF). In fact, it was the solution of the BRD structure of PCAF that proved the ability of BRDs to bind to acetylated lysine’s. The Bromodomain and Extra Terminal (BET) proteins are grouped in the second subfamily and highly specific small molecule inhibitors of this family have recently emerged as promising therapeutic agents in cancer and inflammation. Proteins within the fifth subfamily are structurally characterized by the presence of a methyl-lysine reader domain, the plant homeodomain (PHD) finger in tandem with a BRD. This tandem epigenetic reader module is necessary for the binding to chromatin and highlights a crucial theme in chromatin biology: the multivalent engagement of histone modifications through epigenetic reader proteins that contain more than one reader domain.
Figure 2. Phylogenetic tree based on the structure of the human BRDs.
Roles in gene regulation
BRD-containing proteins have multiple physiological functions, either alone or as part of larger protein complexes and, most notably, are involved in gene regulation through the modulation of transcription. First, these proteins are known to be involved in regulatory chromatin modifications that result in chromatin remodeling and in the introduction of further histone modifications (including methylation and acetylation). BRD-containing proteins can also regulate transcription through the specific recognition of histones and by serving as scaffolds that control the recruitment of other transcriptional regulators to chromatin. Finally, they can modulate the transcriptional machinery itself.
Figure 3. Roles of bromodomain-containing proteins in gene regulation.
Role of BRD-containing proteins in disease
Early evidence for the role of BET proteins in lymphocytes came from the studies of transgenic mice overexpressing Brd2. These mice developed splenic follicular B cell lymphomas, which had a transcriptional profile overlapping that seen in human-derived samples and a propensity for transplantable leukemia. These observations suggested that the BET proteins had a critical role in the specification, maintenance and expansion of lymphocyte lineages known to mediate a number of autoimmune diseases.
Insights into the role of genes in disease can often be gained from human genetic studies; nevertheless, the location of the Brd2 gene within the MHC complex has hampered genetic analysis of its role in immune-mediated diseases. However, variations in the frequency of single nucleotide polymorphisms (SNPs) mapping to the Brd2 gene in rheumatoid arthritis (RA) patients were first observed in high resolution mapping studies by Chissoe and colleagues at GSK and were subsequently independently confirmed in a multivariate study of the interaction of smoking, genotype and citrullinated peptide levels in RA cohorts. These results suggest that variations in Brd2 function or expression may contribute in part to the development of RA disease.
The fundamental role for BET family proteins in cell division has been highlighted by lots of observations. Work from several laboratories has elucidated the role of BET proteins in chromatin binding and macromolecular complex formation and function, pointing to a central role in oncogenesis. This was definitively demonstrated by the finding that transgenic overexpression of Brd2 in lymphocytes produced B cell lymphomas that were transcriptionally identical to activated B cell lymphoma isolates from patients. The expression of Brd4 was found to be deregulated in breast cancer biopsies, and the Brd4 transcriptional signature was found to discriminate rate of disease progression. Recently, the functional effects of Brd4 on metastasis and stem cell transformation have been determined to reside in the C terminus proline-rich domain of the protein.
Brd4 gene translocation with the otherwise testis- and nuclear-restricted protein NUT has been shown to result in a rare but rapidly fatal condition termed NUT midline carcinoma. In cell lines carrying the translocation, administration of siRNA to the Brd4 protein or treatment with JQ1 is sufficient to reduce proliferation and induce differentiation of the cells into keratinocytes. Consistent with these in vitro observations, JQ1 can significantly inhibit tumor growth in mice in two xenograft models of NUT midline carcinoma.
Conclusions and perspectives
The current chemical tool-set to target BRDs provides the possibility to validate BRD-dependent protein-protein interactions, to reveal new functions of BRD-containing proteins and to delineate their roles in normal and diseased tissue, which will demystify their functions. It remains to be seen how the modulation of acylation-dependent signaling and transcriptional circuits that is linked to BRD-driven recognition of these PTMs will lead to novel, improved therapeutics. Generally speaking, current progress in understanding the mechanisms of Kac recognition by BRD modules, together with the elucidation of the functions of BRD-containing proteins in physiology and disease, holds great promise for developing novel therapeutics that can complement current therapies, which are based on the use of histone deacetylase inhibitors to prevent the removal of Kac.