Unique Transcription Factor Functions Regulate Epigenetic and Transcriptional Dynamics During Cardiac Reprogramming

To determine the molecular mechanisms by which expression of Gata4, Mef2c, and Tbx5 (GMT) induces direct reprogramming from a cardiac fibroblast toward an induced cardiomyocyte, we performed a comprehensive transcriptomic and epigenomic interrogation of the reprogramming process. Single cell RNA sequencing (GEO Series GSE133452) indicated that a reprogramming trajectory was acquired within 48 hours of GMT introduction, did not require cell division, and was limited mainly by successful expression of GMT. Evaluation of chromatin accessibility by ATAC-seq supported the expression dynamics and revealed widespread chromatin remodeling at early stages of the reprogramming process. Chromatin immunoprecipitation followed by sequencing of each factor alone or in combinations revealed that GMT bind DNA individually and in combination, and that ectopic expression of either Mef2c or Tbx5 is sufficient in some contexts to increase accessibility. We also find evidence for cooperative facilitation and refinement of each factor's binding in a combinatorial setting. A random-forest classifier that integrated the observed gene expression dynamics with regions of dynamic chromatin accessibility suggested Tbx5 binding is a primary driver of gene expression changes and revealed additional transcription factor motifs co-segregating with reprogramming factor motifs, suggesting new factors that may be involved in the reprogramming process. Overall design: Examination of methylome of induced cardiomyocytes. Whole genome bisulfite sequencing (WGBS) was performed with three biological replicates on our starting population of neonatal cardiac fibroblasts, and cardiac reporter-positive cells from day 3 and day 7 after reprogramming induction.

为阐明Gata4、Mef2c与Tbx5（合称GMT）的表达介导心脏成纤维细胞向诱导心肌细胞（induced cardiomyocyte）直接重编程的分子机制，本研究对该重编程过程开展了全面的转录组与表观基因组学探查分析。单细胞RNA测序（Single cell RNA sequencing，GEO系列GSE133452）结果显示，GMT导入后48小时内即可形成重编程轨迹，该过程无需细胞分裂，且其进展主要受GMT成功表达的限制。通过ATAC-seq对染色质开放状态进行的分析验证了转录表达的动态特征，并揭示了重编程早期阶段广泛存在的染色质重塑现象。对单个或组合形式的GMT因子进行染色质免疫共沉淀测序（Chromatin immunoprecipitation followed by sequencing, ChIP-seq）后发现，GMT可分别或协同结合DNA；且在部分实验场景下，异位表达Mef2c或Tbx5即可提升染色质开放程度。本研究同时发现，在组合因子体系中存在各因子结合的协同促进与优化现象。将观测到的基因表达动态特征与染色质开放区域动态变化相结合的随机森林分类器分析表明，Tbx5结合是基因表达改变的主要驱动因素；同时还发现了与重编程因子基序共分离的额外转录因子基序，提示可能存在其他参与重编程过程的新因子。实验整体设计：对诱导心肌细胞的甲基化组进行分析。本研究对起始群体新生心脏成纤维细胞，以及重编程诱导后第3天、第7天的心脏报告基因阳性细胞进行了全基因组亚硫酸氢盐测序（Whole genome bisulfite sequencing, WGBS），每组设置3次生物学重复。