Context-Specific Transcription Factor Functions Regulate Epigenomic and Transcriptional Dynamics During Cardiac Reprogramming [ATAC-seq]. Context-Specific Transcription Factor Functions Regulate Epigenomic and Transcriptional Dynamics During Cardiac Reprogramming [ATAC-seq]

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 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 genome-wide accessibility of induced cardiomyocytes. ATAC-seq analyses were performed with three biological replicates.

为阐明Gata4、Mef2c与Tbx5（GMT）的表达介导心脏成纤维细胞向诱导心肌细胞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学与表观基因组学解析。单细胞RNA测序（single cell RNA sequencing）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，且主要受GMT成功表达的限制。通过ATAC-seq（Assay for Transposase-Accessible Chromatin using sequencing）评估染色质开放状态，验证了基因表达的动态变化，并揭示了重编程早期阶段广泛存在的染色质重塑现象。对各因子单独或联合使用后进行染色质免疫共沉淀测序（ChIP-seq），结果表明GMT可单独或协同结合DNA；在部分情境下，异位表达Mef2c或Tbx5即可提升染色质开放程度。本研究还发现了组合调控下各因子结合产生协同促进与优化作用的证据。整合基因表达动态变化与染色质开放动态区域的随机森林分类器显示，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其他转录因子基序，提示了可能参与重编程过程的新调控因子。整体实验设计：对诱导心肌细胞进行全基因组染色质开放状态检测。ATAC-seq分析设置3次生物学重复。