Characterization of the Regulation and Function of Epithelial Cell Adhesion Molecule EpCAM in Mouse Embryonic Stem Cells

上皮细胞粘附分子EpCAM是一种膜系蛋白，在胚胎发育和肿瘤进展过程中具有选择性和限制性表达。EpCAM在生理和病理分化过程中的细胞特异性表达是通过在mRNA和蛋白水平上的动态调节实现的。我们和其他人已经研究了在癌症和胚胎发育中这种选择性EpCAM表达的时机和原理。在癌症中，EpCAM通过调节的膜内蛋白水解（RIP）和其细胞内结构域EpICD与细胞周期调节因子的启动子的结合来促进增殖和肿瘤生长。在多能胚胎干细胞（ESC）中，EpCAM强烈表达。EpCAM的选择性调节发生在原肠胚形成开始时的上皮-间充质转化期间、转移级联反应中和治疗抗性肿瘤细胞中。在上一个资助期内，我们证明了在小鼠胚胎发育的早期，中胚层谱系的细胞严格关闭EpCAM，而它在内胚层后代中得以维持。功能获得和功能丧失实验破坏了EpCAM的紧密调节，并抑制了内胚层和中胚层分化细胞（包括心肌细胞）的形成。为了产生我们可以更详细地研究EpCAM调控在分化过程中的作用的系统，我们已经产生了CRISPR/Cas9工程化的EpCAM报告细胞系，其在多能性标志物表达和分化潜力方面保持完全功能。此外，我们还建立了EpCAM基因敲除的ESC细胞系，并利用RNAseq技术对野生型和EpCAM基因敲除的ESC细胞进行了分化过程中转录组的比较。迄今为止，对EpCAM选择性调控的分子机制及其对细胞分化的影响的研究仍不完整。因此，拟议项目将解决以下几个方面：1。 EpCAM在维持ESC的多能基态中起作用吗？2. EpCAM如何支持胚胎干细胞早期分化过程中的多能性存在？3. EpCAM在激活/维持Wnt通路中发挥什么作用？4. EpCAM在分化后期的内胚层和中胚层细胞和组织类型的发育中起什么作用？为此，我们将使用确定的内胚层和中胚层细胞群和EpCAM报告细胞系来解决ESC分化的3D模型中EpCAM的调节和功能。虽然RNA测序方法旨在鉴定EpCAM的下游靶基因，但全基因组CRISPR敲除文库筛选将用于研究在ESC分化过程中发挥作用的上游调控因子。总之，本项目的主要目的是阐明选择性EpCAM调节的分子机制及其在ESC分化调节中的功能。

Epithelial cell adhesion molecule EpCAM is a membrane-tethered protein with a selective and restricted expression during embryonic development and tumor progression. Cell-specific expression of EpCAM during physiological and pathological differentiation is achieved through dynamic regulation at the mRNA and protein level. We and others have investigated timing and rationale for this selective EpCAM expression in cancer and embryonic development. In cancer, EpCAM promotes proliferation and tumor growth through regulated intramembrane proteolysis (RIP) and binding of its intracellular domain EpICD to promoters of cell cycle regulators. In pluripotent embryonic stem cells (ESC) EpCAM is strongly expressed. Selective regulation of EpCAM occurs during epithelial-to-mesenchymal transition at the initiation of gastrulation, in the metastatic cascade, and in therapy-resistant tumor cells. Within the last funding period we demonstrated that early in murine embryonic development, cells of the mesodermal lineage strictly turn off EpCAM, while it is maintained in endodermal progeny. Gain- and loss-of-function experiments disrupted the tight EpCAM regulation and inhibited the formation of endodermal and mesodermal-differentiated cells, including cardiomyocytes. To generate a system with which we can study the role of EpCAM regulation during differentiation in more detail, we have generated CRISPR/Cas9-engineered EpCAM reporter cell lines that remain fully functional in terms of pluripotency marker expression and differentiation potential. Additionally, EpCAM-knockout ESC lines and RNAseq-based transcriptome comparisons of wild-type and EpCAM-knockout ESC during differentiation were generated.Up to now, insights into molecular mechanism of selective EpCAM regulation and its impact on cell differentiation remain fragmentary. Therefore, the proposed project will address the following aspects:1. Is EpCAM functional in maintaining a pluripotent ground state of ESCs?2. How does EpCAM support pluripotency exit during early differentiation of ESCs?3. What role does EpCAM play in activating/sustaining the Wnt pathway?4. What role does EpCAM play in the development of endodermal and mesodermal cell and tissue types in later stages of differentiation?To this end, we will use defined endo- and mesodermal cell populations and EpCAM reporter cell lines to address the regulation and function of EpCAM in 3D models of ESC differentiation. While RNA sequencing approaches aim to identify down-stream target genes of EpCAM, a whole genome CRISPR-knockout library screen will be applied to investigate upstream regulators, which play a role during ESC differentiation. In summary, major aims of this project are the elucidation of molecular mechanisms of selective EpCAM regulation and its function in the regulation of ESC differentiation.