Characterization of epigenomic and transcriptomic changes upon TGF-beta treatment in NMuMG cells [ChIP-seq]

TGFß cytokines have crucial roles in development, proliferation, tissue homeostasis, differentiation, and immune regulation. Consequently, alterations in TGFß signaling underlie numerous diseases, including cancer. Moreover TGFß is one of the most potent inductors of EMT (epithelial to mesenchymal transition) in normal and oncogenic epithelial cells from different origins. During EMT, cells undergo an extensive reorganization of cell adhesion complexes, cytoskeletal architecture, and extracellular matrix interactions and acquire increased motility and invasion properties. However, little is known about the genomic repertoire of enhancers activated by TGFß, the chromatin dynamics during this process, or the SMAD (main effectors of TGFß pathway) partners in epithelial cells. To address these outstanding questions about the genomic regulation mediated by TGFß in epithelial cells we determine and characterize the enhancer atlas of the TGFß response in normal murine mammary gland (NMuMG) epithelial cells, a well-established model for TGFß-dependent EMT. To achieve this we performed ATAC-seq, ChIP-seq against typical histone modifications for enhancers and promoters (H3K27ac, H3K4me1 and H3K4me3) and ChromRNA-seq (to identify enhancer RNAs) analyses at two different time points after TGFß treatment (2h and 12h) to study the enhancers dynamics during this process. We also performed a transcriptomic analyses (RNA-seq) at same time points to correlate genomic regulation to transcriptional changes upon TGFß treatment. We show that TGFß promotes a fast and widespread increase of chromatin accessibility in most enhancers of the cell line, irrespectively of whether the enhancer will become activated or repressed. We also observed that activated enhancers are strongly enriched for SMAD2/3/4 and AP-1 footprints. In contrast, decommissioned enhancers present footprints for TEAD, HNF1A, or HNF1B TF (and not for SMAD2/3/4). Strikingly, analyses of the regulated genes around TGFß-regulated enhancers revealed TGFß regulatory domains that can encompass several genes and that are constrained by 3D chromatin conformation. In these domains enhancer targeting is more promiscuous than previously anticipated. Overall design: Binding profiles of H3K27ac, H3K4me1 and H3K4me3 from NMuMG mouse glandular epithelial cells treated with TGFß1 at 2h and 12h or with vehicle as control were generated by deep sequencing, in duplicates, using Illumina HiSeq2000.

转化生长因子β（TGF-β）细胞因子在发育、细胞增殖、组织稳态、细胞分化及免疫调控中发挥关键作用。因此，TGF-β信号通路的异常是包括癌症在内的多种疾病的发病基础。此外，TGF-β是不同来源正常上皮细胞与致癌上皮细胞中，上皮间质转化（EMT，epithelial to mesenchymal transition）最有效的诱导因子之一。
在上皮间质转化过程中，细胞会经历细胞黏附复合物、细胞骨架结构及细胞外基质相互作用的广泛重排，并获得增强的迁移与侵袭能力。然而，目前对于TGF-β激活的增强子基因组图谱、该过程中的染色质动态变化，以及上皮细胞中TGF-β通路核心效应物SMAD的互作蛋白，我们仍知之甚少。
为解答上述关于TGF-β介导的上皮细胞基因组调控的关键科学问题，我们对正常小鼠乳腺上皮（NMuMG）细胞——一种被广泛应用于TGF-β依赖型上皮间质转化研究的经典模型——的TGF-β应答增强子图谱进行了系统鉴定与表征。
为达成这一研究目标，我们在TGF-β处理后的两个不同时间点（2小时与12小时）开展了多组学分析：针对增强子与启动子典型组蛋白修饰（H3K27ac、H3K4me1及H3K4me3）的染色质免疫共沉淀测序（ChIP-seq）、转座酶可及性测序（ATAC-seq），以及用于识别增强子RNA的染色质RNA测序（ChromRNA-seq），以探究该过程中增强子的动态调控特征。同时，我们在相同时间点开展了转录组测序（RNA-seq）分析，以将基因组层面的调控变化与TGF-β处理后引发的转录改变相关联。
研究结果显示，无论最终增强子是被激活还是被抑制，TGF-β均可快速且广泛地提升该细胞系多数增强子区域的染色质可及性。我们还观察到，激活的增强子显著富集SMAD2/3/4与AP-1的转录因子结合足迹。与之相反，失活的增强子则存在TEAD、HNF1A或HNF1B转录因子的结合足迹（而非SMAD2/3/4）。值得注意的是，对TGF-β调控增强子附近的靶基因进行分析后发现，TGF-β的调控结构域可涵盖多个基因，且受三维染色质构象约束；在此类结构域中，增强子的靶向性比此前预期更为宽泛。
整体实验设计：我们采用Illumina HiSeq2000测序平台，对经TGF-β1处理2小时、12小时的NMuMG小鼠腺上皮细胞，以及经溶剂处理的对照组细胞，进行了H3K27ac、H3K4me1及H3K4me3的结合谱分析，所有实验均设置生物学重复。