Pioneer factor interactions in embryonic stem cells

描述(由申请人提供)：胚胎干细胞(ES细胞)、造血干细胞(HSCs)和其他类型的干细胞具有广泛的发展潜力的表观遗传学特性引起了人们的极大兴趣，因为这些细胞对一些治疗策略是有利的。干细胞同样引起癌症生物学家的兴趣，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张引起的。最近的研究表明，参与早期发育决定的基因可能通过与二价组蛋白修饰结构域相关联而被激活，组蛋白修饰结构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并不相关。相反，之前对肝脏特异的Alb1基因的研究表明，在多能细胞中，典型的组织特异性基因组装成难以接近的染色质结构，在原肠形成过程中或之后，先锋转录因子启动一系列事件，促进染色质解缩，最终导致转录激活。与这一假设相反，我们最近证明了在多能ES细胞中，三个组织特异性基因Ptcra、Il12b和Alb1的特征良好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，即使这些因子在ES细胞中的结合不会导致核酸酶过敏，也不总是促进通常与活跃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ES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。在没有增强子标记的情况下，预甲基化的增强子-启动子-报告载体在分化的细胞中对转录激活具有抵抗力，并且不能在其增强子上建立未甲基化的窗口。这些结果导致了这样的假设，即必须在发育早期建立增强子标记，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说并更好地了解ES细胞中增强子标记的建立和维持，我们将继续研究用于初步实验的三个相同的模型基因，即胸腺细胞特异性的PtcrA基因、巨噬细胞/树突状细胞特异性的IL12b基因和肝脏特异性的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元件，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，作为一种更好地评估标记在表观遗传重新编程期间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的干细胞的分子特征引起了人们的极大兴趣。这项研究将探索最近发现的小鼠胚胎干细胞的一个特征，这可能是它们独特特性的关键。

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.