NOMeseq analysis of Etv2 induced MEF reprogramming

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and thus relevant to the treatment of ischemic diseases, injury-induced regeneration, and solid tumor growth. Therefore, the definition of pioneer factors and regulatory pathways that govern the specification and differentiation of endothelial progenitors will serve as a platform for targeted therapies to promote cardiovascular regeneration and treatment of solid organ tumors. Previously, we demonstrated that ETV2 was an essential transcription factor for the development of cardiac, endothelial and hematopoietic lineages. Here, we report that ETV2 functions as a novel pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with single cell RNA-seq, ATAC-seq and ChIP-seq techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and function as a pioneer factor independent of the cellular context. We determined that ETV2 executes a pioneering role by recruiting BRG1 to remodel chromatin around endothelial genes and help maintain an open configuration. ETV2 chromatin binding also resulted in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular and oncological diseases. NOMe-seq was performed in duplicates using 250,000 cells from control and day 1 reprogrammed MEFs per sample following the protocol described in Lay et al.29 The reagents used in the studies are GpC Methyltransferase (M.CviPI) (NEB # M0227), Klenow Fragment (3'→5' exo-) (NEB # M0210S), EZ DNA Methylation™ Kit (VWR # 77001-534), End-It™ DNA End-Repair Kit (VWR # 75927-936), Rapid DNA Ligation Kit (ThermoFisher # K1422), xGen™ Methyl UDI-UMI Adapters, 1-16 (IDT # 10006644), xGen Library Amplification Primer Mix (IDT # 1077677), KAPA HiFi HotStart Uracil+ Kit (Roche # 07959052001). 200-500 bp size gel purified libraries were sequenced with the The NOVASeq S4 flow cell with 150 bp paired end reads.