Single cell RNA-seq analysis of Etv2 induced MEFs 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. Single cell RNA sequencing (scRNA seq) was performed on iHA-Etv2 MEF reprogrammed cells with and without Brg1 shRNA knockdown using the previously described protocol9. Briefly, single cells from reprogrammed D1, D2, D7 and D7-Flk1+ sorted iHA-Etv2 MEFs were barcoded using the 10x Chromium Single Cell platform, and cDNA libraries were prepared according to the manufacturer’s protocol (Single Cell 30v3, 10x Genomics, USA) . Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were then sequenced on a NovaSeq S4 Illumina platform (2x150 bp) aiming for 50,000 reads per cell. Lentiviral particles to knockdown Brg1 were obtained using three unique 21-mer shRNA for Brg1 (pLKO.1 -TRC shRNA library). We generated these lentiviral particles using standard protocols and tested each one of them by infecting NIH 3T3 MEFs12. The most efficient lentivirus shRNA construct was used in our studies to knockdown Brg1 in iHA-Etv2 MEFs. Cultured iHA-Etv2 MEFs were infected with lentiviruses using the Lentiblast Premium reagent (OZBiosciences) as per the manufacturer′s instruction. After 72 hrs of infection, doxycycline was added to cells in order to reprogram them. iHA-Etv2 MEFs were harvested for analysis 24 hrs and 7 days following the addition of doxycycline.