WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming

Abstract

Summary: Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality. : To assess the role of chromatin-associated factors in reprogramming of fibroblasts to induced pluripotency, high-content imaging screening with siRNAs targeting chromatin-associated factors was performed to identify colony level phenotypes. RNA sequencing was used as a secondary screen. It was found that WDR5 functionally interacts with BRCA1 and BARD1, and that these factors are important for DNA repair and the mesenchymal-to-epithelial transition. Keywords: reprogramming, iPSCs, mesenchymal-to-epithelial transition, DNA damage repair, functional interactions, chromatin factors, WDR5, BRCA1, BARD1