Transcription factor binding dynamics during human ES cell differentiation

Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage specific behavior of selected factors. In addition to the orchestrated remodeling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signaling effectors, and the epigenome during human embryonic stem cell differentiation. 200 ChIP-seq experiments profiling 38 transcription factors (TFs) and several chromatin marks in 5 cell types--male human ES cell line HUES64 and directed differentiation of HUES64 towards mesendoderm (dMS, 12 hours), endoderm (dEN, 120 hours), mesoderm (dME, 120 hours), and ectoderm (dEC, 120 hours). In addition, three ES cell lines were derived with shRNA mediated knockdown of GATA4 and differention toward endoderm (dEN_shGATA4) and mesoderm (dME_shGATA4). These cell lines were used for MNChIP-seq of GATA4, SMAD1, and H3K27Ac and for 4 RRBS experiments in GATA4 knockdown and control cell lines.

多能干细胞（pluripotent stem cells）为解析哺乳动物发育进程中调控细胞命运转变的内在分子动态机制提供了强有力的研究系统。本研究结合大量表观基因组与转录组数据，对人类胚胎干细胞向三胚层分化全过程中38种转录因子的全基因组结合数据开展了整合分析。我们阐明了核心调控动态，并展示了筛选得到的转录因子的谱系特异性行为。除染色质图谱的协同重塑之外，我们还发现多种转录因子的结合与单一生殖层中特定的DNA甲基化丢失存在显著关联，且在多数情况下与另外两个生殖层中DNA甲基化的特异性增加呈反向对应关系。综上，本研究揭示了人类胚胎干细胞分化过程中，转录因子结合、下游信号效应因子与表观基因组的环境依赖性重布线调控。

本研究共开展200次染色质免疫共沉淀测序（ChIP-seq）实验，对5种细胞类型中的38种转录因子（transcription factors, TFs）与多种染色质修饰标记进行了表征：包括雄性人类胚胎干细胞系HUES64，以及HUES64定向分化为中内胚层（dMS，12小时）、内胚层（dEN，120小时）、中胚层（dME，120小时）与外胚层（dEC，120小时）的细胞。此外，本研究构建了3株经短发夹RNA（short hairpin RNA, shRNA）介导敲低GATA4的胚胎干细胞系，并将其定向分化为内胚层（dEN_shGATA4）与中胚层（dME_shGATA4）。上述细胞系被用于GATA4、SMAD1与H3K27Ac的微球菌核酸酶染色质免疫共沉淀测序（MNChIP-seq），同时在GATA4敲低与对照细胞系中开展了4次简化代表性重亚硫酸盐测序（RRBS）实验。