Bromodomain protein Brd3 associates with acetylated GATA1 to promote its chromatin occupancy at erythroid target genes

Abstract

Acetylation of histones triggers association with bromodomaincontaining proteins that regulate diverse chromatin-related processes. Although acetylation of transcription factors has been appreciated for some time, the mechanistic consequences are less well understood. The hematopoietic transcription factor GATA1 is acetylated at conserved lysines that are required for its stable association with chromatin. We show that the BET family protein Brd3 binds via its first bromodomain (BD1) to GATA1 in an acetylation-dependent manner in vitro and in vivo. Mutation of a single residue in BD1 that is involved in acetyl-lysine binding abrogated recruitment of Brd3 by GATA1, demonstrating that acetylation of GATA1 is essential for Brd3 association with chromatin. Notably, Brd3 is recruited by GATA1 to both active and repressed target genes in a fashion seemingly independent of histone acetylation. Anti-Brd3 ChIP followed by massively parallel sequencing in GATA1-deficient erythroid precursor cells and those that are GATA1 replete revealed that GATA1 is a major determinant of Brd3 recruitment to genomic targets within chromatin. A pharmacologic compound that occupies the acetyl-lysine binding pockets of Brd3 bromodomains disrupts the Brd3-GATA1 interaction, diminishes the chromatin occupancy of both proteins, and inhibits erythroid maturation. Together these findings provide a mechanism for GATA1 acetylation and suggest that Brd3 "reads" acetyl marks on nuclear factors to promote their stable association with chromatin.hematopoiesis | posttranslational modifications | gene regulation P osttranslational acetylation of lysine residues is a widely used cellular signaling mechanism involving histone and nonhistone proteins. Typically, acetylated lysines (acK) serve as docking sites for bromodomains that are found in numerous chromatinrelated proteins (1, 2). Thus, bromodomains serve as "readers" of acK marks in a cascade of signaling events that influence diverse processes within chromatin. The hematopoietic zinc finger protein GATA1 was one of the earliest reported acetylated transcription factors (3, 4). GATA1 is a pivotal regulator of the erythroid, megakaryocyte, and mast cell lineages (5-9). GATA1 activates all known erythroid-specific genes, and mice lacking GATA1 succumb to anemia due to failed maturation and apoptosis of erythroid precursor cells (5, 10, 11). GATA1 also functions as a repressor, down-regulating genes associated with the proliferative, immature state. Mutations in the zinc finger region of GATA1 underlie congenital anemias and thrombocytopenias (12), whereas alterations at the N terminus of GATA1 are associated with megakaryoblastic leukemias in patients with Down syndrome (13). Previously, we found that GATA1 interacts with the acetyltransferase CREB binding protein (CBP) (14) to stimulate histone acetylation at GATA1 target genes (15, 16). CBP and its paralog p300 also acetylate GATA1 at two lysine-rich clusters C-terminal to each of its zinc fingers (Fig. 1A) (3, 4). CBPmediated acetylation of GATA1 c...

RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain

“…It is therefore likely that each BET bromodomain has a distinct function although the combined effect seems to be conferring a regulatory output in gene transcription. We observed in this study that targeting the BD2 had only a modest effect on transcription, an observation supported by previous studies of BRD3 that showed that its recruitment to acetylated sites on GATA1 is mediated by BD1 (40). Notably, deletion of the first bromodomain in BRDT in mice is sufficient to confer sterility by blocking BRDT-dependent sperm maturation (39).…”

“…The cell type-specific roles have been highlighted by recent reports on BRDT-dependent transcription programs that regulate spermatogenesis (37-39) and BRD3-dependent recruitment of GATA1 in hematopoietic cells regulating maturation of erythroid, megakaryocyte, and mast cell lineages (23,40), as well as BRD2-dependent roles regulating differentiation of adipose tissue (25) and neurons (41). Application of BET inhibitors outside the area of oncology, given the pleiotropic nature of BET transcriptional regulation, strongly suggests a requirement for more selective targeting with respect to isoform and/or domains, to avoid adverse effects.…”

The Bromodomain and Extra-Terminal Domain (BET) Family: Functional Anatomy of BET Paralogous Proteins

“…BRD3 has been reported to directly interact with acetylated lysine residues of the transcription factor GATA1, which regulates the expression of all erythroid and megakaryocyte-specific genes [60]. Pull-down of extracts from GATA1-null erythroblasts (G1E cells) expressing normal or mutant forms of HA-tagged BRD3 using acetylated GATA1 peptides shows that lack of BDI interferes with the binding of BRD3 to acetylated peptides, suggesting that recruitment of BRD3 to GATA1-associated Interactions between their bromodomains (BD) and the acetylated lysine (Ac) in histones facilitate the passage of RNA Pol II to elongate nascent transcripts through hyperacetylated nucleosomes.…”

Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation

“…For example, BRD4, a member of the bromo and extra terminal (BET) subfamily of bromodomain-containing proteins, interacts with the acetylated RelA subunit of NF-κB to promote transcriptional activation of inflammatory genes (7). BRD3 has been shown to interact with the acetylated transcription factor GATA1, recruiting this transcription factor to both active and repressed target genes in a histone acetylation-independent fashion (8). Accordingly, there much interest in generating inhibitors to block the interaction between bromodomains and their acetylated binding partners and thus to reverse changes in gene expression in human disease states.…”