Chromatin occupancy of ATF4 at day 1 of cardiomyocytes differentiation [ChIP-seq]

Cardiac development involves large-scale rearrangements of the proteome. How the developing cardiac cells maintain the integrity of the proteome during the rapid lineage transition remains unclear. Here we show that proteotoxic stress visualized by the misfolded protein aggregates appears during early cardiac differentiation of human embryonic stem cells (hESCs) and is resolved by activation of the PERK branch of the unfolded protein response (UPR). PERK depletion increases misfolded protein accumulation, leading to pluripotency exit defect and impaired mesendoderm specification of hESCs. Mechanistically, we found that PERK safeguards cardiogenesis through its conserved downstream effector ATF4, which subsequently activates a novel transcriptional target WARS1, to cope with the differentiation-induced proteotoxic stress. Our results indicate that protein quality control represents a previously unrecognized core component of the cardiogenic regulatory network. Broadly, these findings provide a framework for understanding how UPR is integrated into the developmental program by activating the PERK-ATF4-WARS1 axis. Overall design: Undifferentiated hESCs cultured in E8 medium were dissociated into single cell suspension by Accutase (Stem Cells Technology, 7920) and reseeded onto Matrigel-coated 24-well plate at a density of 105 cells/per well in E8 medium containing 10 µM Y-27632. When reached ~80% confluence 2-3 days after plating, CM differentiation was initiated by switching to the cardiac differentiation medium (DMEM/F-12 (Gibco, 11330032) supplemented with 50 U ml-1 Penicillin-Streptomycin (Gibco, 15070063), Chemically Defined Lipid Concentrate (1:100, Gibco, 11905031), 10.7 µg ml-1 holo-Transferrin human (Sigma, T0665), 71 µg ml-1 L-Ascorbic acid (Sigma, A8960), 14 ng ml-1 Sodium selenite (Sigma, S5261)) and renewed every day. 5 µM CHIR99021 (Selleck, S1263) and 3 µM IWP2 (Selleck, S7085) was added into the cardiac differentiation medium from days 0-1. Then samples were collected following the previously published protocol (Lee, T.I., Johnstone, S.E., and Young, R.A. (2006). Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc 1, 729-748)