Transcription factor EBF1 non-cell autonomously regulates cardiac growth and differentiation

Reciprocal interactions between non-myocytes and cardiomyocytes are implicated in the control of cardiac growth and differentiation. Here, we report the identification of early B-cell factor 1 (Ebf1) as a transcription factor highly enriched in non-myocytes that potently regulates heart development. Postnatal Ebf1 deficient hearts are characterized by marked myocardial hypercellularity and reduced cardiomyocyte size, as well as ventricular conduction system hypoplasia and conduction system disease. Growth abnormalities in Ebf1 knockout hearts are observed as early as embryonic day 13.5, with dysregulated cell cycling, myocardial hyperplasia, and immature trabecular morphology. Transcriptional profiling of Ebf1-deficient non-myocytes isolated from embryonic hearts demonstrates dysregulation of Polycomb repressive complex 2 (PRC2) targets, while ATAC-Seq reveals differences in chromatin accessibility near many of these same genes. Gene set enrichment analysis of differentially expressed genes in cardiomyocytes isolated from E13.5 hearts of wildtype and Ebf1 deficientmice reveals significant enrichment of MYC targets, and consistent with this finding, we observe increased abundance of MYC in mutant hearts. Mechanistically, we show that EBF1-deficient non-myocytes, but not wildtype non-myocytes, are sufficient to induce excessive accumulation of nuclear-localized MYC protein in co-cultured wildtype cardiomyocytes. Finally, we demonstrate that BMP signaling induces Ebf1 expression in embryonic heart cultures and controls a gene program enriched in EBF1 targets. Taken together, these results reveal a novel non-cell autonomous pathway controlling cardiac growth and development. Overall design: Gene transcirptional profiling was performed on embryonic day 13.5 mouse heart cells after dissociation and FACS sorting using PECAM1 antibody and TMRM dye to identify endocardial cells and cardiomyocytes respectively. ATAC-Seq was performed on non-myocyte cells from E13.5 mouse hearts isolated by preplating.

非肌细胞与心肌细胞之间的双向互作参与心脏生长与分化的调控。本研究报道了早期B细胞因子1（early B-cell factor 1, Ebf1）的鉴定结果：该转录因子在非肌细胞中高度富集，可强效调控心脏发育。产后Ebf1缺陷型心脏表现为显著的心肌细胞增多症、心肌细胞体积缩小，同时伴随心室传导系统发育不全与传导系统疾病。Ebf1基因敲除小鼠的心脏生长异常早在胚胎期13.5天（E13.5）即可观测到，具体表现为细胞周期紊乱、心肌细胞增生及心肌小梁形态未成熟。对从胚胎心脏分离的Ebf1缺陷型非肌细胞开展转录谱分析（transcriptional profiling）发现，多梳抑制复合体2（Polycomb repressive complex 2, PRC2）的靶基因表达失调；而ATAC-Seq结果显示，上述多数靶基因附近的染色质开放状态存在差异。对野生型与Ebf1缺陷型小鼠E13.5心脏分离的心肌细胞中差异表达基因进行基因集富集分析（Gene Set Enrichment Analysis, GSEA），结果显示MYC靶基因集显著富集；与该发现一致，我们在突变体心脏中观测到MYC蛋白丰度升高。机制研究表明，仅EBF1缺陷型非肌细胞（而非野生型非肌细胞）即可在共培养的野生型心肌细胞中诱导核定位MYC蛋白的过度积累。最后，我们证实骨形态发生蛋白（Bone Morphogenetic Protein, BMP）信号可在胚胎心脏培养体系中诱导Ebf1表达，并调控一组富含EBF1靶标的基因程序。综上，本研究揭示了一条全新的非细胞自主性通路，可调控心脏生长与发育。实验整体设计：本研究对胚胎期13.5天小鼠心脏细胞进行解离后，采用血小板内皮细胞黏附分子1（Platelet Endothelial Cell Adhesion Molecule 1, PECAM1）抗体与四甲基罗丹明甲酯（Tetramethylrhodamine Methyl Ester, TMRM）染料分别标记心内膜细胞与心肌细胞，随后通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离两类细胞并进行转录谱分析。此外，我们通过预铺板法分离E13.5小鼠心脏的非肌细胞，并对其开展ATAC-Seq检测。