Regulation of gene expression by bHLHPAS protein transcriptional complexes

我是一名分子生物学家，希望通过细胞多蛋白机器确定基因调控的分子机制。我们鉴定了一个转录复合体，包括缺氧诱导因子1 （HIF）、芳烃受体核转运子（ARNT）、甲状腺激素受体/视网膜母细胞瘤蛋白相互作用蛋白-230 （TRIP230）和视网膜母细胞瘤蛋白（Rb），这些转录复合体参与了负责发育、细胞转化适应低氧和组织损伤的特定基因组的调控。我们在人类细胞系中消融了Rb，发现调节组织发育、细胞运动和适应性反应的遗传程序以缺氧依赖的方式对Rb的损失很敏感。这些基因中有很大一部分聚集在染色体上，表明该复合体调节染色质组织，从而影响特定的遗传程序。我建议通过关注缺氧诱导的多蛋白复合物如何调节基因转录并影响适应性细胞反应和恶性转化的结果来扩展我们之前的发现。具体来说，我的短期目标是阐明缺氧诱导因子-/ (HIF1-ARNT)共调节复合体指导特定遗传程序的表达和调节染色质组织的手段。本建议的具体目的是：(1)鉴定和表征HIF-ARNT_TRIP-Rb （HATR）复合物活性的介导蛋白。Rb招募的因子减弱了缺氧反应，这是调控细胞分化和适应环境应激的遗传程序的新靶点。(2)。分析Rb丢失后长期缺氧依赖性基因沉默和去抑制。阐明Rb调控的HIF适应性反应的分子机制将使我们能够确定修复Rb损失的可延展性靶标。(3)确定HATR复合体的环端（DNA结合位点）。该策略将确定由HATR复合体调控的生物反应的下游效应物及其控制染色质组织的方式。这些目标是新颖的，因为它们定义了已知参与细胞发育生物学，对环境刺激的适应性反应和许多病理条件的转录复合物的新作用。按照这一战略，我们将在五年内实现；(i)对HATR复合体如何指导特定的遗传程序来调节适应性反应的现实理解；（ii）确定调节HATR复合体、其功能和反应的蛋白质靶点。这些靶点可能在未来被操纵，以开发一种更合理和有针对性的治疗方法来治疗包括那些涉及细胞转化的许多病理。

I am a molecular biologist with a desire to determine the molecular mechanisms of gene regulation by cellular multi-protein machines. We have characterized a transcriptional complex consisting of hypoxia-inducible factor1 (HIF), the aryl hydrocarbon receptor nuclear translocator (ARNT), the thyroid hormone receptor/retinoblastoma protein interacting protein-230 (TRIP230), and the retinoblastoma protein (Rb) involved in the regulation of specific sets of genes responsible for development, cellular transformation adaptation to low oxygen, and tissue damage. We have ablated Rb in human cell lines and found that genetic programs regulating tissue development, cellular motility and adaptive responses are sensitive to loss of Rb in a hypoxia-dependent fashion. A significant proportion of these genes are clustered on chromosomes suggesting that this complex regulates chromatin organization, thereby affecting specific genetic programs.I propose to expand our previous findings by focusing on how the hypoxia-inducible multi-protein complex regulates gene transcription and affects the outcomes of adaptive cellular responses and malignant transformation. Specifically, my short-term goal is to elucidate the means by which the hypoxia-inducible factor-/ (HIF1-ARNT) co-regulatory complex directs the expression of specific genetic programs and regulate chromatin organization. The specific aims of this proposal are;(1) To identify and characterize proteins mediating the HIF-ARNT_TRIP-Rb (HATR) complex activity. Factors recruited by Rb to attenuate the hypoxic response represent novel targets to manipulate genetic programs that regulate cell differentiation and adaptation to environmental stress. (2). To analyze long-term hypoxia-dependent gene silencing and de-repression after loss of Rb. Elucidation of the molecular mechanisms underlying Rb regulated HIF adaptive responses will enable us to identify malleable targets to remediate Rb loss. (3) To define the cistrome (DNA binding sites) of the HATR complex.This strategy will identify down-stream effectors of the biological responses regulated by the HATR complex and the manner in which it controls chromatin organization. These aims are novel in that they define a new role for a transcriptional complex known to be involved in the biology of cell development, adaptive responses to environmental stimuli and many pathological conditions. Following this strategy, we will in five years, have; (i) a realistic understanding of how the HATR complex directs specific genetic programs to regulate adaptive responses, and: (ii) identified protein targets regulating the HATR complex, its function and responses. These targets may be manipulated in the future to develop a more rational and targeted therapeutic approach to a number of pathologies including those involving cellular transformation.