TRANSCRIPTIONAL REGULATION BY GEMININ

描述(申请人提供)：了解维持多能胚胎细胞和控制分化的调节机制与人类健康和疾病的核心科学领域基本相关，包括干细胞、发育和癌症生物学。新的Gem蛋白与SWI/SNF和Polycomb(PcG)染色质调节复合体一起作为胚胎和胚胎干细胞(ES)细胞谱系指定和分化的关键转录调节因子。Gem、SWI/SNF和PcG活性的失调也是多种人类恶性肿瘤的一个关键方面。我的实验室的一个主要重点是了解这个核心转录调控开关是如何运作的，并定义在正常的ES和祖细胞环境中以及在疾病中操纵它的方法。 我们的初步结果支持这样一种模型，即Gem通过直接转录抑制细胞谱系承诺和分化调节基因来维持多能和/或多能祖细胞。我们假设Gem的作用是：1.直接与PcG复合体协同抑制共同靶基因的表达。为了支持这一点，我们最近定义了Gem抑制的靶基因，并发现这些基因与PcG抑制的直接靶基因显著重叠，以阻止细胞谱系承诺。2.GEM还作用于神经前体细胞，调节分化时间。对于这一活性，我们假设Gem通过SWI/SNF和Neurogenin2以及神经元命运承诺和分化所需的NeuroD和NeuroD转录因子的协同活性来拮抗靶基因的反式激活。 在这里，在目标1中，我们将使用一种创新的高通量方法来识别在其直接靶基因中受Neurogenin2和NeuroD结合和调节，并受Gem负调控的增强子。这将通过定义转录调控机制和网络来填补我们现有知识的空白，通过这些机制和网络，Neurogenin2和NeuroD在神经发生中发挥重要作用，并将为分析Gem在这一调控中的作用提供必要的序列背景。在目标2和3中，我们将分析Gem、SWI/SNF和PcG如何被招募并机械地控制Gem靶基因的表达，以及这些活动之间的功能相互作用如何扰乱神经元分化过程中染色质水平的靶基因反式激活。总之，这些研究将阐明维持胚胎干细胞和祖细胞以及调节分化所需的核心转录调控开关的机制和逻辑。我们在这里开发的信息和工具将为在正常干细胞和祖细胞以及在恶性肿瘤等疾病背景下对这种调控开关的诊断和治疗操作提供基础。 与公共卫生相关：这项拟议的工作将确定中央调控开关的作用机制，该开关控制胚胎干细胞中的基因表达，并在许多侵袭性的、治疗耐药的癌症中失控。我们在这里开发的信息和工具将为在正常干细胞环境下以及在治疗人类恶性肿瘤的情况下为诊断和治疗目的操纵这种调控开关提供关键的基础。

DESCRIPTION (provided by applicant): Understanding regulatory mechanisms that maintain pluripotent embryonic cells and control differentiation has fundamental relevance to scientific areas central to human health and disease, including stem cell, developmental, and cancer biology. The novel Geminin (Gem) protein acts together with the SWI/SNF and Polycomb (PcG) chromatin regulatory complexes as key transcriptional regulators of cell lineage specification and differentiation in embryonic and embryonic stem (ES) cells. Dysregulation of Gem, SWI/SNF and PcG activities is also a pivotal aspect of multiple human malignancies. A major focus of my laboratory is to understand how this core transcriptional regulatory switch operates and to define approaches for manipulating it in both normal ES and progenitor cell contexts and in disease. Our preliminary results support a model where Gem maintains pluripotent and/or multipotent progenitor cells by direct transcriptional repression of cell lineage commitment and differentiation regulatory genes. We hypothesize that Gem does this: 1. by directly cooperating with PcG complexes to repress the expression of common target loci. In support of this, we recently defined Gem-repressed target genes, and found these overlapped strikingly with direct targets of PcG repression to block cell lineage commitment. 2. Gem also acts in neuronal progenitor cells to regulate differentiation timing. For this activity, we hypothesize that Gem antagonizes target gene transactivation by the coordinated activities of SWI/SNF and Neurogenin2 and NeuroD, neural bHLH transcription factors required for neuronal fate commitment and differentiation. Here, in Aim 1 we will use an innovative, high-throughput approach to identify enhancers that are bound and regulated by Neurogenin2 and NeuroD in their direct target genes, and negatively regulated by Gem. This will fill an existing gap in our knowledge by defining transcriptional regulatory mechanisms and networks through which Neurogenin2 and NeuroD perform essential roles in neurogenesis and will provide a necessary sequence context for analyzing Gem's role in this regulation. In Aims 2 and 3, we will analyze how Gem, SWI/SNF and PcG are recruited to and mechanistically control expression of Gem target genes and how disrupting functional interplay between these activities perturbs target gene transactivation at the chromatin level during neuronal differentiation. Together, these studies will elucidate mechanisms and logic of a core transcriptional regulatory switch required to maintain embryonic stem and progenitor cells and to regulate differentiation. Information and tools that we develop here will provide a foundation for diagnostic and therapeutic manipulations of this regulatory switch in both normal stem and progenitor cells and in disease contexts such as malignancy. PUBLIC HEALTH RELEVANCE: The proposed work will determine mechanisms of action of a central regulatory switch controlling gene expression in embryonic stem cells and dysregulated in many aggressive, therapy-resistant forms of cancer. Information and tools that we develop here will provide a critical foundation for manipulating this regulatory switch for both diagnostic and therapeutic purposes in normal stem cell contexts and in treating human malignancies.