Acetylation-Dependent Interaction of GATA-1 with the Potential Mitotic “Bookmarking” Protein Brd3.

Abstract 2598

Acetylation of the transcription factor GATA-1 facilitates its ability to drive erythroid differentiation by enhancing its association with in vivo target sites. However, the underlying mechanism through which GATA-1 acetylation functions has remained elusive. To test whether GATA-1 acetylation serves to recruit essential cofactors, we performed a peptide affinity screen and identified Brd3 as an acetylated GATA-1 interacting partner. Brd3 belongs to the BET protein family that also includes Brd2, Brd4, and Brdt, and is characterized by tandem bromodomains (BD1 and BD2) and an extraterminal (ET) domain. We show that Brd3 and GATA-1 physically interact in an acetylation-dependent manner in vitro and in vivo. Mapping studies revealed that the interaction depends on BD1 of Brd3 and one of the two major acetylation sites that resides near the C terminal zinc finger of GATA-1. By ChIP-seq and ChIP-qPCR, endogenous Brd3 is recruited to virtually all GATA-1-occupied regulatory elements in erythroid cells, including both GATA-1activated and repressed genes. Although Brd3 has been reported to associate with acetylated histones along the entire length of transcribed genes, we found that Brd3 recruitment correlates poorly with histone acetylation along gene bodies. In agreement with our biochemical data, an intact BD1 is essential for the in vivo recruitment of Brd3 to GATA-1-occupied elements, further demonstrating that acetylation of GATA-1 is essential for Brd3 association in vivo. Notably, a pharmacological compound that targets acetyl lysine binding sites in BD1 and BD2 disrupts the Brd3/GATA-1 interaction in vitro, diminishes Brd3 and GATA-1 association at key erythroid genes in vivo, and impairs GATA-1 target gene expression and erythroid maturation. In concert, these findings suggest a mechanism by which the first bromodomain of Brd3 recognizes acetyl-lysines on GATA-1 to facilitate GATA-1 chromatin occupancy. These studies raise an interesting question: In contrast to the great majority of transcription factors, BET family proteins bind to chromatin during mitosis and might serve an epigenetic memory function to properly reactivate gene transcription upon exit of mitosis. We are currently investigating whether Brd3 functions by bookmarking GATA-1-bound sites throughout mitosis to aid in transcriptional memory and stability of lineage specific gene expression.