Bulk RNA seq of P0 aCMs and P28 aCMs

Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa, and conversely, increased the expression of ventricular identity genes including Myl2. Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 74.8% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.阐明心脏心房与心室腔室如何维持其独特的腔室身份，是治疗腔室特异性疾病的必要前提。本研究通过在新生小鼠心脏的心房工作心肌中特异性敲除转录因子Tbx5，证实其对维持心房身份具有不可或缺的作用。心房Tbx5的失活会下调Myl7、Nppa等高度腔室特异性基因的表达，反之则会上调Myl2等心室身份相关基因的转录水平。我们联合运用单细胞核转录组与开放染色质分析（single nucleus transcriptome and open chromatin profiling），对驱动心房身份表达程序改变的基因组可及性变化进行了系统评估，鉴定出1846个相较于敲除组心房心肌细胞（atrial cardiomyocytes, aCMs）在对照心房心肌细胞中可及性更高的基因组位点。其中69%的对照富集型ATAC（Assay for Transposase-Accessible Chromatin）区域可与TBX5结合，证实TBX5在维持心房基因组可及性中发挥关键调控作用。这些区域与在对照心房心肌细胞中相较于敲除组表达水平更高的基因密切相关，提示它们属于TBX5依赖性增强子。我们利用HiChIP技术分析增强子染色质环，鉴定出510个对TBX5剂量敏感的染色质环。在对照心房心肌细胞中富集的染色质环中，74.8%的锚点位于对照富集型ATAC区域内。综上，本研究证实TBX5通过结合心房增强子并维持心房增强子的组织特异性染色质架构，在基因组层面发挥维持心房基因表达程序的调控作用。