A reference map of cardiac transcription factor chromatin occupancy identifies dynamic and conserved transcriptional enhancers

Mapping the chromatin occupancy of transcription factors (TFs) is a key step in deciphering the transcriptional programs that orchestrate organ development and homeostasis. Here we used biotinylated knockin alleles of seven key TFs (GATA4, NKX2-5, MEF2A, MEF2C, SRF, TBX5, TEAD1) that regulate heart development and homeostasis to sensitively and reproducibly map their genome-wide occupancy in the fetal and adult heart. Our results show that these TFs have dynamic chromatin occupancy between developmental stages. We observed that multiple TFs often co-occupy the same chromatin region, exhibiting collaborative binding that overall was most consistent with indirect cooperativity. Regions with multiple TF binding were more likely to exhibit features of functional regulatory elements, including evolutionary conservation, chromatin accessibility, and activity in transcriptional enhancer assays. Although histone H3 acetylation on lysine 27 (H3K27ac) has been used to identify active enhancers, many regions occupied by multiple TFs lacked H3K27ac, and TF-bound regions with or without H3K27ac co-occupancy had similar region conservation and enhancer activity. Specific studies of TEAD1 demonstrated that it is a core component of the cardiac transcriptional network: physically interacted with NKX2-5 and MEF2C and co-occupied regulatory loci with other cardiac TFs to regulate cardiomyocyte-specific gene functions. Our study shows that co-occupancy by multiple TFs is a hallmark of a subset of functional transcriptional regulatory elements and provides a rich resource for deciphering the cardiac transcriptional regulatory network. Examination of cardiac TF binding in fetal (E12.5) and adult mouse heart ventricles using BioChIP-Seq. Examination of cardiac TF binding in fetal (E12.5) and adult mouse heart ventricles.绘制转录因子（transcription factors, TFs）的染色质占据图谱，是解析调控器官发育与内稳态的转录程序的关键环节。本研究利用7种调控心脏发育与内稳态的关键转录因子的生物素标记敲入等位基因（GATA4、NKX2-5、MEF2A、MEF2C、SRF、TBX5、TEAD1），实现了其在胎鼠与成年小鼠心脏中全基因组占据图谱的高灵敏度、可重复绘制。研究结果显示，这些转录因子在不同发育阶段的染色质占据模式呈现动态变化特征。我们观察到，多个转录因子常共同占据同一染色质区域，其协同结合模式整体上与间接协同作用最为契合。带有多个转录因子结合位点的区域，更易展现出功能性调控元件的特征，包括进化保守性、染色质可及性，以及在转录增强子检测实验中的活性。尽管组蛋白H3赖氨酸27乙酰化（H3K27ac）常被用于识别活性增强子，但诸多被多个转录因子占据的区域并不带有H3K27ac标记；且带有或不带有H3K27ac共占据的转录因子结合区域，其区域保守性与增强子活性均较为相近。针对TEAD1的专项研究证实，其是心脏转录调控网络的核心组分：可与NKX2-5和MEF2C发生物理互作，并与其他心脏转录因子共同占据调控位点，从而调控心肌细胞特异性基因的功能。本研究表明，多个转录因子的共占据现象是一类功能性转录调控元件的标志性特征，同时为解析心脏转录调控网络提供了丰富的研究资源。本研究采用BioChIP-Seq技术，对胎鼠（E12.5）与成年小鼠心室的心脏转录因子结合情况进行了检测。本研究采用BioChIP-Seq技术，对胎鼠（E12.5）与成年小鼠心室的心脏转录因子结合情况进行了检测。