Project 4: Pluripotency and the Marking of Tissue-Specific Genes

描述 胚胎干细胞（ESC）和诱导多能干细胞（IPSC）在衰弱性疾病的治疗和研究中具有巨大的前景。干细胞也引起了癌症生物学家的极大兴趣，因为有证据表明许多人类癌症是由具有干细胞样特性的细胞的异常扩增引起的。虽然已经获得了相当多的知识，但关于干细胞和多能状态的基本分子特性仍有许多需要了解，多能状态被定义为分化成几乎所有细胞谱系的能力。从基因调控的角度来看，大多数多能性的研究集中在，1。掌握多能性的转录调节因子和由这些调节因子控制的基因网络，2.表征参与早期发育决定的基因的启动子的二价组蛋白修饰结构域，和3.区分多能细胞和分化细胞的染色质结构的根本差异。最近，我们实验室和其他实验室的证据表明，通过先驱转录因子和未甲基化的CpG二核苷酸标记典型组织特异性基因的增强子也可能对建立或维持多能性状态至关重要。已经假设这些增强子标记提供了在分化的细胞类型中转录激活的能力。这项研究的目的是更好地了解ESC中组织特异性增强子的标记方式，并严格检查这些增强子标记的功能意义。在目标1中，细菌人工染色体（BAC）将被用来识别和表征DNA基序和转录因子，积极和消极地调节在ESC和IPSC中观察到的非甲基化窗口的建立在良好表征的组织特异性增强子。通过与其他三个项目Pis密切合作，我们将进一步深入了解增强子标记的相关性，通过表征它们在人类和小鼠ESC和IPSC之间的保守性，通过检查它们在重编程期间建立的时间，以及通过评估它们在多能细胞的整体三维核结构中的组织。在目标2中，增强子标记的功能相关性将通过能够在ESC中擦除标记的抑制性染色质复合物的条件性募集来检查。在这些实验中，我们将测试这一假设，即在ESC中擦除标记会损害分化后组织特异性基因的转录激活。最后，使用为这些实验生成的相同工具，我们将通过评估多能性因子通过不同抑制性染色质复合物的作用获得体内组装的沉默染色质的能力来帮助Zaret博士实现其项目（项目2）的目标。 不会有人类或动物实验与拟议的工作。

Description Embryonic stem cells (ESC) and induced pluripotent stem cells (IPSC) hold great promise for the treatment and study of debilitating diseases. Stem cells are also of considerable interest to cancer biologists because of evidence that many human cancers are caused by the aberrant expansion of cells with stem cell-like properties. Although considerable knowledge has been acquired, much remains to be learned about the fundamental molecular properties of stem cells and of the pluripotent state, which is defined as the capacity to differentiate into nearly all cell lineages. From a gene regulation perspective, most studies of pluripotency have focused on, 1. master transcriptional regulators of pluripotency and the gene networks controlled by these regulators, 2. bivalent histone modification domains that characterize the promoters of genes involved in early developmental decisions, and 3. fundamental differences in chromatin structure that distinguish pluripotent cells from differentiated cells. Recently, evidence has emerged from our lab and others that the marking of enhancers for typical tissue-specific genes by pioneer transcription factors and unmethylated CpG dinucleotides may also be critical for establishing or maintaining the pluripotency state. It has been hypothesized that these enhancer marks provide competence for transcriptional activation in differentiated cell types. The goals of the proposed research are to better understand how tissue-specific enhancers are marked in ESC and to rigorously examine the functional significance of these enhancer marks. In Aim 1, bacterial artificial chromosomes (BACs) will be used to identify and characterize DNA motifs and transcription factors that positively and negatively regulate the establishment of unmethylated windows observed in ESC and IPSC at well-characterized tissue-specific enhancers. By working closely with the other three project Pis, we will gain further insight into the relevance of the enhancer marks by characterizing their conservation between human and mouse ESC and IPSC, by examining the timing of their establishment during reprogramming, and by evaluating their organization within the overall 3-dimensional nuclear architecture of pluripotent cells. In Aim 2, the functional relevance of the enhancer marks will be examined through the conditional recruitment of repressive chromatin complexes capable of erasing the marks in ESC. In these experiments, we will test the hypothesis that erasure of the marks in ESC compromises transcriptional activation of the tissue-specific genes following differentiation. Finally, using the same tools that are generated for these experiments, we will assist Dr. Zaret with the goals of his project (Project 2) by evaluating the ability of pluripotency factors to gain access to silent chromatin assembled in vivo through the action of different repressive chromatin complexes. There will be no human or animal experimentation with the proposed work.