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

项目4：多能性和组织特异性基因的标记

基本信息

批准号：
8520352
负责人：
Stephen T Smale
金额：
$ 32.26万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8520352
关键词：
3-Dimensional Albumins Animal Experimentation Architecture Bacterial Artificial Chromosomes Binding Cancer Etiology Cell Line Cell Lineage Cells Chromatin Chromatin Structure Collaborations Comparative Study Competence Complex CpG dinucleotide DNA DNA-Protein Interaction Dendritic Cells Development Developmental Gene Disease Employee Strikes Endoderm Enhancers Evolution Gene Expression Regulation Genes Goals Hepatocyte Human Human Experimentation In Vitro Interleukin-12 Knowledge Learning Liver Malignant Neoplasms Methylation Modeling Molecular Mus Nuclear Plasmids Pluripotent Stem Cells Proliferating Property Proteins Publishing Recruitment Activity Reporter Research Research Personnel Resistance Somatic Cell Stem cells Testing Time Tissues Transcriptional Activation Transfection Work base cell type cytokine embryonic stem cell fascinate histone modification human embryonic stem cell in vivo induced pluripotent stem cell insight interest interleukin-23 macrophage pluripotency promoter research study stem cell differentiation thymocyte tool transcription factor

项目摘要

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.

描述胚胎干细胞（ESC）和诱导的多能干细胞（IPSC）对治疗和研究衰弱的疾病具有巨大的希望。干细胞也对癌症生物学家引起了极大的兴趣，因为证据表明许多人类癌症是由具有干细胞样性质的细胞的异常扩张引起的。尽管已经获得了广泛的知识，但仍有许多关于干细胞和多能状态的基本分子特性的知识，该态被定义为分化为几乎所有细胞谱系的能力。从基因调控的角度来看，多数多能性研究的重点是1。多能性的主要转录调节剂和由这些调节剂控制的基因网络，2。二价组蛋白修饰域，这些二价组蛋白修饰域是特征在于早期发育决策中涉及基因的启动子，以及3。与冰分结构区别的基础差异。最近，我们的实验室和其他人出现了证据表明，先锋转录因子和未甲基化的CPG二核苷酸对典型组织特异性基因的增强子标记也可能对于建立或维持多能状态至关重要。已经假设这些增强子标记为分化细胞类型的转录激活提供了能力。拟议的研究的目标是更好地了解组织特异性增强子如何在ESC中标记并严格检查这些增强子标记的功能意义。在AIM 1中，细菌性人造染色体（BAC）将用于识别和表征DNA图案和转录因子，这些因子和转录因子在良好的特定组织特异性增强子上积极和负面调节在ESC和IPSC中观察到的未甲基化窗户的建立。通过与其他三个项目PI紧密合作，我们将通过检查重编程过程中的建立时间，并评估其在整体三维细胞的3维核结构中评估其组织的时机，从而进一步深入了解增强子标记的相关性。在AIM 2中，将通过有条件地募集能够删除ESC中标记的抑制性染色质复合物来检查增强子标记的功能相关性。在这些实验中，我们将检验以下假设：ESC中的痕迹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因素通过不同抑制性染色质复合物的作用来帮助Zaret博士实现其项目（项目2）的目标（项目2）。拟议的工作将不会进行人类或动物的实验。