Mitigating reprogramming stress with dominant negative BET deletion fragments

The iPSC reprogramming is very inefficient and has an early stochastic stage. The cause of the low efficiency and stochastic nature are poorly understood. The bromo- and extra-terminal domain (BET) proteins have impacts on cellular reprogramming, but the molecular mechanisms are elusive. Here we show that the reprogramming factors cause dramatic reprogramming stresses including massive transcriptional turbulence, massive and random dysregulation of stress response genes, cell cycle impairment, and cytotoxicity. BET dominant negative (DN) mutants lacking the characteristic ET and bromo- domain enhanced iPSC reprogramming and downregulated a large set of stress response genes over-activated by the reprogramming factors. These DN BET mutants also restored the cell cycle profile and mitotic genes impaired by the reprogramming factors, and mitigate the reprogramming-associated cytotoxicity. The DN BET fragments additionally reduced illegitimate transcriptional reprogramming imposed by the reprogramming factors. Both the DN BET mutants and the BET chemical inhibitors target a similar set of the stress response genes in the reprogramming cells although their targeting mechanism are different. In human naive fibroblasts, these DN BET fragments also commonly target a large set of stress response genes including inflammatory genes, implying general roles of BET proteins in stress responses. These DN BET peptides constitute a novel approach to targeting BET proteins for disease managements. Overall design: To understand the molecular mechanisms underlying the gained reprogramming activities of the short isoform of human BRD3 (BRD3R), we overexpressed the domain-deletion proteins of BRD2, BRD3 and BRD4S in human normal fibroblasts (BJ cells) in their naive state or in the reprogramming cells (BJ expressing OCT4, SOX2 and KLF4). These deletion mutants lack the ET-containing C terminus and one or both bromodomain (BD). All the domain-deletion mutants enhance reprogramming efficiencies. We then extract total RNA under different conditions (various BET mutants) and conducted RNA-seq.