Bromodomain Protein BRD4 Is a Transcriptional Repressor of Terminal Erythropoiesis By Interacting with EHMT1/2

Background: Ex vivo production of mature and functional red blood cells from stem cell sources such as induced pluripotent stem cells (iPSCs) holds the promise for alleviating the severe clinical problem of "blood shortage". However, currently, the induced erythroid cells from stem cells, especially iPSCs, do not have the full functions of erythroid cells due to low efficiency of enucleation and immature features. Thus, characterization of the molecular mechanism underlying terminal erythropoiesis is critical. Terminal erythropoiesis is a complex multistep developmental process requiring significant changes in gene expression in the context of dramatic nuclear condensation, which indicates that epigenetic and transcriptional regulators are important players during terminal erythropoiesis.

Methods and results: To find key regulators during terminal erythropoiesis, we performed a screening with more than 300 small molecules targeting epigenetic and transcriptional regulators, including histone acetyltransferases and histone deacetylases; histone methyltransferases and histone demethylases, and bromodomain proteins. Our results showed that inhibition of bromodomain protein BRD4, a well-known epigenetic reader and transcriptional regulator, accelerated terminal erythropoiesis, and thus increased enucleation efficiency. We observed similar phenotype during erythrocyte differentiation from iPSCs, PBMNCs, and cell lines as HUDEP2 and TF-1. Interestingly, the function of BRD4 was independent of CDK9, which was further supported by our finding that both long isoform and short isoform of BRD4 were repressors of terminal erythropoiesis. We further performed RNA-seq, ATAC-seq, and Cut&Tag with BRD4 inhibition, demonstrating that BRD4 regulated erythropoiesis by inhibiting small G proteins. Co-IP and function study illustrated that BRD4 repressed target genes transcription by EHMT1/2.

Conclusion: Taken together, our study demonstrated a non-classical role of BRD4 during terminal erythropoiesis, which provided a new and simple method to improve enucleation efficiency for red blood cells production from stem cells in vitro, and may also supply a therapeutic target to dyserythropoiesis by inducing maturation of erythrocyte progenitor cells.