Chromatin Open/Close Logic in Cellular Reprogramming [ChIP-Seq]. Mus musculus

The reprogramming of fibroblasts into pluripotent stem cells by Oct4, Sox2, Myc and Klf4 represents a unique system to understand the logic of cell fate decisions. The reprogramming process can be divided into initial, middle and maturation phases that are coupled to the ordered execution of multiple biological processes, of which one of the earliest is a mesenchymal to epithelial transition (MET). Yet, despite these critical insights, less is known about how the reprogramming factors alter the chromatin state to convert somatic cells back to pluripotent cells. Here we report the global chromatin accessibility dynamics as cells reprogram from a somatic to a pluripotent state as assessed by assay for transposase-accessible chromatin with sequencing (ATAC-seq). We show that chromatin changes during reprogramming are defined by a fast initial wave of chromatin changes from accessible/open to inaccessible/closed (OC), which is followed by a slower opening up of chromatin from closed to open (CO), and ends with a climactic wave of CO. The initial OC wave includes loci enriched with motifs for AP-1, TEAD, RUNX and ETS-family transcription factors. Consistently, over-expression of one of the AP-1-family members c-Jun, c-Fos and Fra1blocked reprogramming. c-Jun inhibits reprogramming by preventing OSK-driven OC and skewing the overall OC-CO dynamics. Paradoxically, we show that Sox2 and to a less extent Oct4 and Klf4 are CO-, not OC-, mediators , suggesting that they must first activate potent OC factors to remodel chromatin. Indeed, we show that Sap30, a component of the Sin3 complex8, functions as such an OC factor. Together, our results reveal the chromatin accessibility logic during reprogramming and identify Sap30 as one of the first responders that help silence somatic genes. Overall design: chromatin accessible profile of reprogramming cell were generated by ATAC-seq, in two replicates, using illumina nextseq 500

通过Oct4、Sox2、Myc和Klf4将成纤维细胞重编程为多能干细胞（pluripotent stem cells），是解析细胞命运决定逻辑的独特研究体系。重编程过程可分为初始、中间和成熟三个阶段，与多项生物过程的有序执行相耦合，其中最早发生的事件之一即为间质上皮转化（mesenchymal to epithelial transition, MET）。尽管已有这些重要发现，但我们对重编程因子如何改变染色质状态，从而将体细胞逆转至多能干细胞的机制仍知之甚少。
本研究通过转座酶可及性测序（assay for transposase-accessible chromatin with sequencing, ATAC-seq），解析了体细胞重编程为多能干细胞过程中全基因组染色质可及性的动态变化。研究发现，重编程过程中的染色质变化可分为三个阶段：首先是快速的初始染色质开放向闭合（accessible/open to inaccessible/closed, OC）变化浪潮，随后是较慢的染色质闭合向开放（closed to open, CO）重塑，最终以一轮高潮性的CO变化浪潮收尾。初始OC浪潮所涉及的染色质位点，富集有AP-1、TEAD、RUNX及ETS家族转录因子的结合基序。与之一致的是，过表达AP-1家族成员c-Jun、c-Fos及Fra1均会阻断重编程过程。c-Jun通过阻断OSK介导的OC过程，并扰乱整体OC-CO动态平衡，从而抑制重编程。矛盾的是，本研究发现Sox2以及程度较弱的Oct4和Klf4，是CO过程的介导因子而非OC过程的介导因子，这提示它们需首先激活强效的OC因子以重塑染色质。实际上，本研究证实Sin3复合体（Sin3 complex）的组分Sap30正是此类OC因子。综上，本研究揭示了重编程过程中染色质可及性的调控逻辑，并证实Sap30是参与沉默体细胞基因的早期响应因子之一。
实验设计：本研究通过ATAC-seq构建重编程细胞的染色质可及性图谱，设置两个生物学重复，测序平台为Illumina NextSeq 500。