Transcription factor and epigenetic regulation of hypoxic gene expression

说明（由申请人提供）：所有细胞都需要氧气才能正常工作。对低氧水平（缺氧）的协调转录反应通过短期和长期适应机制维持体内平衡。两个结构相似的转录因子，缺氧诱导因子1和2 (HIF-1和HIF-2)，被认为是缺氧诱导基因表达的主要介质。hif在正常发育、控制红细胞生成中起关键作用；伤口愈合和对缺血性疾病的反应。不幸的是，它们在促进肿瘤生长方面也起着重要作用。越来越多的证据表明HIF-1和HIF-2在功能上并不相同，尽管这些差异的机制基础尚不清楚。利用一种新兴技术ChIP-chip，我们全面鉴定了基因组中与HIF-1结合的所有区域，并将这些数据与基因表达谱相结合，建立了HIF-1结合与基因转激活之间的功能关系。在本提案的第一个具体目标中，我们将使用相同的方法来确定HIF-2结合位点和基因转激活。我们将确定HIF-1和HIF-2特异性缺失对基因表达的影响，并将寻找HIF-1和HIF-2在DNA结合和基因转激活方面差异的机制。整合这些不同的数据集，我们将建立一个关于HIF-1和HIF-2如何相似以及它们作为转录激活因子的主要功能如何不同的综合观点。基因表达也很大程度上受到染色质局部结构的影响。在第二个特定目标中，我们将使用ChIP-chip来绘制基线和缺氧诱导的特定组蛋白修饰的变化，包括调节染色质结构的乙酰化和甲基化。当结合HIF-1结合、HIF-2结合和基因表达谱时，这些全球性和基因特异性的研究将建立对缺氧反应中调节基因表达的反式作用和表观遗传机制网络的理解。特别是，我们希望定义表观遗传学如何调节HIF与DNA的结合，表观遗传学如何通过HIF影响基因表达，以及反过来缺氧/HIF如何改变表观遗传学。这方面的知识是至关重要的策略，以增加或减少缺氧基因表达进展到临床试验。

DESCRIPTION (provided by applicant): All cells require oxygen for proper functioning. A coordinated transcriptional response to decreased oxygen levels (hypoxia) maintains homeostasis through short-term and long-term adaptive mechanisms. Two structurally similar transcription factors, Hypoxia-Inducible Factor 1 and 2 (HIF-1 and HIF-2), are thought to be the primary mediators of hypoxia-induced gene expression. The HIFs play critical roles in normal development, control of red blood cell production; wound healing, and response to ischemic diseases. Unfortunately, they also play important roles in promoting tumor growth. Mounting evidence suggests that HIF-1 and HIF-2 are not identical in function, although the mechanistic basis for these differences is not known. Using an emerging technology, ChIP-chip, we have comprehensively identified all regions of the genome that are bound by HIF-1, and have integrated these data with gene expression profiling to establish a functional relationship between HIF-1 binding and gene transactivation. In the first Specific Aim of this proposal, we will use the same approaches to identify HIF-2 binding sites and gene transactivation. We will determine the effects of specific loss of HIF-1 and HIF-2 on gene expression, and will search for mechanisms that dictate differences between HIF-1 and HIF-2 in their DNA binding and gene transactivation. Integrating these diverse datasets, we will establish a comprehensive view of how HIF-1 and HIF-2 are similar and how they differ in their primary function as transcriptional activators. Gene expression is also greatly influenced by the localized structure of chromatin. In the second Specific Aim, we will use ChIP-chip to map the baseline and hypoxia-induced changes in specific histone modifications, including acetylation and methylation, which regulate chromatin structure. When integrated with HIF-1 binding, HIF-2 binding, and gene expression profiles, these global and gene-specific studies will establish an understanding of the network of trans-acting and epigenetic mechanisms regulating gene expression in response to hypoxia. In particular, we wish to define how epigenetics modulate HIF binding to DNA, how epigenetics impact gene expression by HIF, and in turn how hypoxia/HIF alters epigenetics. This knowledge is critical as strategies to increase or decrease hypoxic gene expression progress into clinical testing.