Pioneer factor interactions in embryonic stem cells

胚胎干细胞中先锋因子的相互作用

• 批准号: 7570360
• 负责人: Stephen T Smale
• 金额: $ 19.29万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570360
• 关键词: Bacterial Artificial Chromosomes; Binding; Binding Sites; Cancer Etiology; Cells; Chromatin; Chromatin Structure; Collaborations; Complex; CpG dinucleotide; DNA; DNA-Binding Proteins; DNA-Protein Interaction; Dendritic Cells; Development; Developmental Gene; Elements; Embryo; Employee Strikes; Endoderm; Enhancers; Environment; Epigenetic Process; Event; Fibroblasts; Foxes; Gene Expression Regulation; Generations; Genes; Goals; Hematopoietic; Hematopoietic stem cells; Human; Hypersensitivity; In Vitro; Interleukin-12; Interleukin-9; Lead; Liver; Maintenance; Malignant Neoplasms; Methylation; Modeling; Molecular; Mus; NuRD complex; Nucleosomes; Pilot Projects; Plasmids; Property; Proteins; Reporter; Resistance; Staging; Stem cells; Study models; Therapeutic; Tissues; Trans-Activators; Transcriptional Activation; Transfection; embryonic stem cell; gastrulation; histone modification; in vivo; induced pluripotent stem cell; interest; macrophage; novel; nuclease; pluripotency; promoter; public health relevance; research study; thymocyte; tool; transcription factor

DESCRIPTION (provided by applicant): Epigenetic properties responsible for the broad developmental potential of embryonic stem cells (ES cells), hematopoietic stem cells (HSCs), and other types of stem cells are of considerable interest because these cells are advantageous for a number of therapeutic strategies. Stem cells are of equal interest to cancer biologists because of evidence that many human cancers are caused by the aberrant expansion of cells with stem cell-like properties. Recent studies suggest that genes involved in early developmental decisions are poised for activation through their association with bivalent histone modification domains, consisting of both active and repressive epigenetic marks. However, these bivalent domains are not generally associated with typical tissue-specific genes expressed in differentiated cells. Instead, previous studies of the liver-specific Alb1 gene suggested that typical tissue-specific genes are assembled into inaccessible chromatin structures in pluripotent cells and that, during or after gastrulation, pioneer transcription factors initiate a cascade of events that promotes chromatin decondensation and ultimately leads to transcriptional activation. In contrast to this hypothesis, we recently demonstrated that well-characterized enhancers for three tissue-specific genes, Ptcra, Il12b, and Alb1, are selectively marked by unmethylated CpG dinucleotides in pluripotent ES cells. The unmethylated CpGs appear to result from the binding of transcription factors to the enhancers, even though the binding of these factors in ES cells does not lead to nuclease hypersensitivity and does not always promote histone modifications typically associated with active or silent genes. Preliminary functional studies suggest that the enhancer marks we have observed in ES cells may be critical for transcriptional activation of the tissue-specific genes in differentiated cells. In the absence of the enhancer marks, pre-methylated enhancer- promoter-reporter plasmids were resistant to transcriptional activation in differentiated cells, and were unable to establish unmethylated windows at their enhancers. These results lead to the hypothesis that enhancer marks must be established in early development because the more repressive chromatin environment found in differentiated cells is incompatible with transcriptional activation of unmarked genes. To further explore this hypothesis and better understand the establishment and maintenance of the enhancer marks in ES cells, we will continue our studies of the same three model genes used for our preliminary experiments, the thymocyte- specific Ptcra gene, macrophage/dendritic cell-specific Il12b gene, and liver-specific Alb1 gene. A major goal will be to identify the specific DNA elements required for establishment of the enhancer marks, as a first step toward rigorously analyzing the functional significance of the marks. The enhancer marks will also be evaluated in induced pluripotent stem cells (iPS), as a strategy for better evaluating the establishment and significance of the marks during epigenetic reprogramming. PUBLIC HEALTH RELEVANCE: The molecular features of embryonic stem cells, hematopoietic stem cells, and many other types of stem cells are of considerable interest because of the therapeutic potential of stem cell-derived tissues. This study will explore a recently discovered feature of mouse embryonic stem cells that may be critical for their unique properties.

描述（由申请人提供）：胚胎干细胞（ES细胞）、造血干细胞（hsc）和其他类型干细胞的广泛发育潜力的表观遗传特性引起了相当大的兴趣，因为这些细胞在许多治疗策略中都是有利的。癌症生物学家对干细胞同样感兴趣，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异常扩增引起的。最近的研究表明，参与早期发育决策的基因通过与二价组蛋白修饰域的关联而被激活，二价组蛋白修饰域包括活性和抑制性表观遗传标记。然而，这些二价结构域通常与分化细胞中表达的典型组织特异性基因无关。相反，先前对肝脏特异性Alb1基因的研究表明，典型的组织特异性基因在多能细胞中组装成不可接近的染色质结构，并且在原肠胚形成期间或之后，先驱转录因子启动一系列事件，促进染色质去浓缩并最终导致转录激活。与这一假设相反，我们最近证明，在多能胚胎干细胞中，三种组织特异性基因Ptcra、Il12b和Alb1的增强子被未甲基化的CpG二核苷酸选择性地标记。未甲基化的CpGs似乎是转录因子与增强子结合的结果，尽管这些因子在胚胎干细胞中的结合不会导致核酸酶过敏，也并不总是促进与活性或沉默基因相关的组蛋白修饰。初步的功能研究表明，我们在胚胎干细胞中观察到的增强子标记可能对分化细胞中组织特异性基因的转录激活至关重要。在缺乏增强子标记的情况下，预甲基化的增强子-启动子报告质粒在分化细胞中对转录激活具有抗性，并且无法在其增强子上建立非甲基化窗口。这些结果导致增强子标记必须在早期发育中建立的假设，因为在分化细胞中发现的更具抑制性的染色质环境与未标记基因的转录激活不相容。为了进一步探索这一假设，更好地理解ES细胞中增强子标记的建立和维持，我们将继续研究与我们初步实验相同的三个模型基因，胸腺细胞特异性Ptcra基因，巨噬细胞/树突状细胞特异性Il12b基因和肝脏特异性Alb1基因。一个主要目标将是确定建立增强子标记所需的特定DNA元素，作为严格分析标记功能意义的第一步。增强子标记也将在诱导多能干细胞（iPS）中进行评估，作为更好地评估标记在表观遗传重编程过程中的建立和意义的策略。公共卫生相关性：由于干细胞衍生组织的治疗潜力，胚胎干细胞、造血干细胞和许多其他类型干细胞的分子特征引起了相当大的兴趣。这项研究将探索最近发现的小鼠胚胎干细胞的一个特征，这个特征可能对它们的独特特性至关重要。