Abstract
The widely expressed bromodomain and extraterminal motif (BET) proteins bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 are multifunctional transcriptional regulators that bind acetylated chromatin via their conserved tandem bromodomains. Small molecules that target BET bromodomains are being tested for various diseases but typically do not discern between BET family members. Genomic distributions and protein partners of BET proteins have been described, but the basis for differences in BET protein function within a given lineage remains unclear. By establishing a gene knockout-rescue system in a Brd2-null erythroblast cell line, here we compared a series of mutant and chimeric BET proteins for their ability to modulate cell growth, differentiation, and gene expression. We found that the BET N-terminal halves bearing the bromodomains convey marked differences in protein stability but do not account for specificity in BET protein function. Instead, when BET proteins were expressed at comparable levels, their specificity was largely determined by the C-terminal half. Remarkably, a chimeric BET protein comprising the N-terminal half of the structurally similar short BRD4 isoform (BRD4S) and the C-terminal half of BRD2 functioned similarly to intact BRD2. We traced part of the BRD2-specific activity to a previously uncharacterized short segment predicted to harbor a coiled-coil (CC) domain. Deleting the CC segment impaired BRD2's ability to restore growth and differentiation, and the CC region functioned in conjunction with the adjacent ET domain to impart BRD2-like activity onto BRD4S. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities.
Highlights
The widely expressed bromodomain and extraterminal motif (BET) proteins bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 are multifunctional transcriptional regulators that bind acetylated chromatin via their conserved tandem bromodomains
The BET family consists of BRD2, BRD3, and BRD4, which are widely expressed across tissue types, and the testisspecific BRDT [2]
We measured BRD3 and BRD4L protein levels and found them to be unchanged in the BRD2 KO cells (Fig. S1A), indicating that BRD2 is not necessary for BRD3 and BRD4L expression and that any defects in the BRD2 KO cells cannot be attributed to their loss
Summary
BRD2 is essential for GATA1-dependent erythroid differentiation and gene expression. A mechanistic explanation for specific functions of individual BET proteins remains unresolved. To determine whether there are BET-selective functions that are conveyed by domains that do not affect protein levels, we compared the abilities of BRD2 and BRD2-4S and of BRD4S and BRD4-2 proteins to restore BRD2 KO cell growth and differentiation-associated gene expression. To test whether either domain contributes to BRD2 function, we deleted both the ET (BRD2dET) and CC (BRD2dCC) regions individually and expressed these constructs in BRD2 KO cells (Fig. 4C, middle) Both genes were expressed at levels similar to full-length BRD2 based on YFP fluorescence (Fig. 4C, left). To measure effects on gene expression, we isolated BRD2 KO1 cell lines stably expressing HA-tagged versions of these constructs or an empty vector (encoding only GFP) and confirmed by Western blotting that BRD2, BRD2dET, and BRD2dCC protein and RNA levels (based on Brd and GFP in bicistronic transgene) were similar (Fig. S5, A and B). These results suggest that the BRD2 and BRD3 CC domains augment the ability of the ET domains to engage in specific protein contacts
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