TGFβ-SMAD信号通路对干细胞分化的调控机制

The TGFβ family of cytokines and their SMAD signal transduction pathway are central to metazoan development and tissue regeneration. Disruption of TGFβ signaling underlies many diseases, both inherited and somatic. The basic mechanism of TGFβ-SMAD signal transduction from the membrane to the nucleus is understood to a degree, but the cardinal role of TGFβ-SMAD in embryonic stem cell (ESC) differentiation remains poorly understood. .. In order to acquire the necessary data and conceptual framework for the present work, we launched a genome-wide ChIP-seq and RNA-seq analysis of Smad and Trim33 proteins in mouse ESCs. We defined that the differentiation gene responses require nodal-driven mediators Smad2/3, Trim33 and Smad4. According to their genome occupancy maps in ESCs and d2.5 embryonic bodies, interestingly, the highest density of Smad and Trim33 sequence tags in most target genes occurs not at proximal promoters but at distal sites located up to 50kb from the transcriptional start site (TSS). These sites near mesendoderm specification genes are frequently co-occupied by Smad2/3, Smad4 and Trim33. Taking Goosecoid (Gsc) as a template, a model Smad-regulated master differentiation gene， We found proximal GC-rich motif and the distal CAGAC motifs are essential for Gsc transcription and act as potential binding sites for Smad recognition. These findings laid basic foundation to the question of how the SMAD transcriptional complexes target master differentiation genes for regulation. We hypothesized that the Smad2-Smad3-Smad4 trimer would link the promoter and distal enhancer region of mesendoderm differentiation genes. We will address the structural determinants of Smad recognition of these master differentiation genes, test a model for the trimeric nature of Smad complexes and probe the model functionally in ESCs and in mice. We additionally challenge the long-held dogma that Smad2 ¬–the most essential of all Smad family members in vertebrates– does not bind DNA. This knowledge will help explain many extant puzzles in the TGFβ field and beyond.

TGFβ-SMAD信号通路是掌控胚胎干细胞命运的核心信号之一，但是其调控干细胞分化的机制尚不清楚。我们通过全基因组的ChIP-seq 和RNA-seq分析,发现Smad2/3, Smad4和Trim33共同占据并直接转录调控绝大部分中内胚层基因的表达。我们发现在启动子和增强子上均有经典的Smad3/4结合位点，并在启动子上找到了潜在的Smad2结合序列。这一发现与目前认为Smad2蛋白由于3号外显子（Exon 3，E3）的插入而无法与DNA结合的观点相悖。通过CRISPR技术，我们证实Smad2的E3是决定鼠胚胎干细胞中内胚层分化的关键。基于以上重要发现，我们提出Smad2-Smad3-Smad4三聚体可以环状连接分化基因的启动子和增强子，从而促进中内胚层调控基因转录和干细胞分化。因此，本课题将分析Smad复合体与分化基因的启动子和增强子结合的结构基础，以及调控干细胞分化的分子机制。

TGFβ家族的细胞因子及其下游的SMAD信号转导通路是多细胞动物发育和组织再生的控制核心。许多遗传性和细胞性疾病都是由TGFβ信号传导的紊乱所造成。近年来，TGFβ在干细胞中的调控作用受到较大的关注。但是，对于TGFβ-SMAD信号通路如何掌控和决定胚胎干细胞的多能性与分化，我们仍然知之甚少。. TGFβ受体磷酸化SMAD2和SMAD3转录因子，然后与SMAD4形成异源三聚体复合物并与环境特异性的转录因子合作从而激活靶基因。SMAD2和SMAD3的蛋白结构高度相似，但全长SMAD2的MH1结构域多了一个3号外显子（Exon3，E3）。当这个外显子被剪接后，SMAD2将产生在结构和功能上类似于SMAD3的短蛋白质产物SMAD2β。但只有SMAD2-/-小鼠因为原肠胚发育障碍而死亡，而SMAD3-/-小鼠的胚胎却基本正常。虽然SMAD2在原肠胚发生以及其它发育阶段中起着至关重要的作用，但是一直以来，E3被普遍认为阻挡了SMAD2和DNA的结合。我们通过生化研究和结构解析发现，SMAD2与DNA的结合其实取决于其独有的E3的构象转换。我们进一步发现，SMAD2和SMAD3在先驱因子FOXH1介导的TGFβ信号转导中扮演不同的角色。在小鼠的中内胚层调控基因上，FOXH1预先绑定到其中的目标位点，并在不依赖TGFβ信号的情况下招募SMAD3；而SMAD2在基础状态下主要保留在细胞质中。当分化启动时，TGF-β信号激活的SMAD2与 SMAD4 结合并在目标基因的启动子处与SMAD3:FOXH1汇合。因此我们的研究提出一个全新的分化调控模型：SMAD3和FOXH1在多能性状态下，预先与中内胚层分化基因的启动子结合，而受TGFβ信号驱动的SMAD2:SMAD4复合物只有结合到先驱复合物 SMAD3:FOXH1之后，该分化基因才得以转录激活。. 这项工作解决了信号驱动的转录因子如何与先驱因子合作激活分化基因的普遍问题，并更新了对这一经典信号通路的在发育和疾病发生中的作用机制的理解。 在ESC分化调控中，SMAD2充当经典的受体激活信号转导物，而先驱因子FOXH1和SMAD3在分化基因上的预结合，可以帮助信号驱动的SMAD2-SMAD4复合物结合和迅速激活基因的表达。这项研究对TGFβ的信号传导，先驱转录因子和干细胞生物学等领域将具有广泛的影响。

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