KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency [ATAC-seq]

Summary: Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors (TFs), as is strikingly exemplified in somatic cell reprogramming to pluripotent stem cells (PSCs) by OCT4, KLF4, SOX2 and cMYC. How TFs orchestrate the complex molecular changes around their targets in a temporal manner remains largely elusive. Here, using KLF4 as a paradigm, we provide the first TF-centric view of chromatin reorganization and the association to 3D enhancer rewiring and the transcriptional changes of linked genes during reprogramming of mouse embryonic fibroblasts to (MEFs) to PSCs. Inducible depletion of KLF factors in PSC caused a genome-wide decrease in connectivity of enhancers and disruption of KLF4 binding site from PSC-specific enhancers was sufficient to reduce expression of genes within the enhancer hub partly by impairing long-range contacts. Our study provides an integrative view of the intricate activities of a master regulator during a controlled cell fate transition and offers novel insights into the order and nature of molecular events that follow TF binding. Purpose: We captured on a genome-wide scale the binding of KLF4 during iPSC formation and its effects on chromatin state, transcriptional activity and chromatin topology around its targets. Methods: We used a well-characterized reprogramming system to apply genome-wide assays that map KLF4 binding (ChIP-seq), chromatin accessibility (ATAC-seq), enhancer and gene activity (H3K27ac ChIP-seq and RNA-seq), enhancer connectivity (H3K27ac Hi-ChIP) as well as KLF4-centric chromatin looping (KLF4 Hi-ChIP) at different stages during acquisition of pluripotency. Results: Integrative analysis of our results generated a reference map of stage-specific chromatin changes around KLF4 bound loci and established strong links with enhancer. rewiring and concordant transcriptional changes. Genetic manipulation of KLF4 binding from a PSC enhancer further supported the ability of KLF4 to function both as a transcriptional regulator and a chromatin organizer. Conclusions: Our study offers novel insights into the intricate roles of a master regulator during cell fate transition. Mouse embryonic fibroblasts (MEFs) (Rosa26-M2rtTA/Col1a1-OKSM) induced with doxycycline (dox) in the presence of ascorbic acid. We collected bulk populations on day 3 after dox treatment, whereas at later stages, on day 6 and day 9, we sorted SSEA1 positive cells to enrich for cells on the trajectory towards induced pluripotency. Finally, we used pluripotent stem cells (PSCs) inclusive of embryonic stem cells (ESCs) as a reference point for established pluripotency. Biological replicates were collected to assay for chromatin accessibility (paired-end ATAC-seq), gene activity (single-end RNA-seq), H3K27ac ChIP-seq (single-end) in MEFs, day3, 6, 9 and PSCs. KLF4 binding (single-end ChIP-seq) was assessed in day3, 6, 9 and PSCs. KLF4/H3K27ac centric looping (paired-end Hi-ChIP) was studied in either day3, day6 and PSCs for KLF4 or MEFs and PSCs for H3K27ac.

研究摘要：
细胞命运转变伴随由转录因子（Transcription Factors, TFs）驱动的全局转录、表观遗传及染色质拓扑结构变化，这一点在OCT4、KLF4、SOX2与cMYC将体细胞重编程为多能干细胞（Pluripotent Stem Cells, PSCs）的过程中得到了极具代表性的体现。目前，转录因子如何以时序性方式调控其靶位点周围的复杂分子变化，在很大程度上仍未明确。本研究以KLF4为模型，首次从转录因子中心视角，揭示了小鼠胚胎成纤维细胞（Mouse Embryonic Fibroblasts, MEFs）重编程为多能干细胞过程中的染色质重塑现象，及其与3D增强子重编程和靶基因转录变化的关联。在多能干细胞中诱导性敲除KLF家族因子会导致全基因组范围内增强子连接性下降；而破坏KLF4在多能干细胞特异性增强子上的结合位点，可通过削弱长距离染色质相互作用，部分降低该增强子枢纽内基因的表达水平。本研究整合揭示了核心调控因子在可控细胞命运转变过程中的复杂调控活性，并为转录因子结合后分子事件的时序与本质提供了全新见解。

研究目的：
本研究在全基因组范围内解析了诱导多能干细胞（Induced Pluripotent Stem Cells, iPSCs）形成过程中KLF4的结合情况，及其对靶位点周围染色质状态、转录活性与染色质拓扑结构的调控作用。

研究方法：
本研究采用经过充分验证的重编程系统，在多能性获得的不同阶段，开展了一系列全基因组检测实验：包括定位KLF4结合位点的染色质免疫共沉淀测序（ChIP-seq）、检测染色质开放性的转座酶可及性测序（ATAC-seq）、分析增强子与基因活性的H3K27ac ChIP-seq和RNA测序（RNA-seq）、检测增强子连接性的H3K27ac Hi-ChIP，以及以KLF4为中心的染色质环分析（KLF4 Hi-ChIP）。

研究结果：
整合分析本研究数据后，我们构建了KLF4结合位点周围阶段特异性染色质变化的参考图谱，并明确了其与增强子重编程及协同转录变化之间的紧密关联。对多能干细胞增强子上KLF4结合位点进行遗传操纵的实验结果，进一步证实KLF4兼具转录调控因子与染色质组织者的双重功能。

研究结论：
本研究为核心调控因子在细胞命运转变过程中的复杂功能提供了全新见解。
本研究使用的实验材料为：在抗坏血酸存在下经强力霉素（Doxycycline, dox）诱导的小鼠胚胎成纤维细胞（MEFs，基因型为Rosa26-M2rtTA/Col1a1-OKSM）。我们在强力霉素处理后第3天收集整体细胞群；在后续阶段（第6天和第9天），则通过分选SSEA1阳性细胞以富集处于诱导多能性获得轨迹中的细胞。最终，我们以包含胚胎干细胞（Embryonic Stem Cells, ESCs）在内的多能干细胞（PSCs）作为已建立多能性状态的参照样本。
我们收集了生物学重复样本，以检测以下样本的相关指标：MEFs、第3、6、9天样本及PSCs的染色质开放性（双端ATAC-seq）、基因活性（单端RNA-seq）及H3K27ac ChIP-seq（单端）；在第3、6天样本及PSCs中检测KLF4结合情况（单端ChIP-seq）；针对KLF4的染色质环分析（双端Hi-ChIP）在第3、6天样本及PSCs中开展，而针对H3K27ac的染色质环分析则在MEFs及PSCs中开展。