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

心脏发育涉及蛋白质组的大规模重排。目前尚不明确发育中的心肌细胞在快速谱系转换过程中如何维持蛋白质组的完整性。本研究显示，在人类胚胎干细胞（human embryonic stem cells, hESCs）的早期心脏分化阶段，可观察到以错误折叠蛋白质聚集为可视化标志的蛋白毒性应激，该应激可通过激活未折叠蛋白反应（unfolded protein response, UPR）的PERK分支得以缓解。敲低PERK会增加错误折叠蛋白质的积累，进而导致hESCs出现多能性退出缺陷及中内胚层特化受损。机制层面，本研究发现PERK通过其保守的下游效应因子ATF4保障心脏发生过程，ATF4随后激活新型转录靶基因WARS1，以应对分化诱导的蛋白毒性应激。本研究结果表明，蛋白质质量控制是此前未被发现的心脏发生调控网络的核心组成部分。广义而言，本研究结果为理解未折叠蛋白反应如何通过激活PERK-ATF4-WARS1轴整合至发育程序提供了研究框架。

整体实验设计：将培养于E8培养基中的未分化hESCs用Accutase（Stem Cells Technology，货号7920）消化为单细胞悬液，以1×10^5个细胞/每孔的密度重新接种于包被Matrigel的24孔板中，所用培养基为添加10 μM Y-27632的E8培养基。接种后2-3天，当细胞汇合度达到约80%时，更换为心脏分化培养基以启动心肌细胞分化：该培养基为DMEM/F-12（Gibco，货号11330032），添加有50 U·ml^-1青霉素-链霉素（Gibco，货号15070063）、化学限定脂质浓缩液（体积比1:100，Gibco，货号11905031）、10.7 μg·ml^-1人全转铁蛋白（Sigma，货号T0665）、71 μg·ml^-1 L-抗坏血酸（Sigma，货号A8960）、14 ng·ml^-1亚硒酸钠（Sigma，货号S5261），且培养基每日更换。在分化第0至1天，向心脏分化培养基中添加5 μM CHIR99021（Selleck，货号S1263）与3 μM IWP2（Selleck，货号S7085）。后续样本收集参照已发表的实验方案（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）进行。