Distinct Modes of Gene Regulation by KDM5

描述（由申请人提供）： KDM 5家族转录调节因子越来越被认为是发育和人类疾病界面的关键参与者。哺乳动物细胞编码四种KDM 5旁系同源物（KDM 5A-D），其中三种具有临床意义：KDM 5A或KDM 5 B在许多癌症中过表达，包括乳腺癌，结直肠癌和黑色素瘤，KDM 5C突变占X连锁智力残疾患者的约3%。缺乏关于KDM 5作用机制的基本知识阻碍了治疗这些疾病的疗法的发展。因此，这些研究的长期目标是使用果蝇剖析KDM 5蛋白的基因调控功能，因为在该生物体中存在单个KDM 5蛋白绕过了哺乳动物细胞中功能冗余的问题。先前的数据表明，KDM 5蛋白可以通过其组蛋白去甲基化酶活性抑制转录，并可以通过改变组蛋白乙酰化来激活基因表达。在这项研究中，新的数据首次证明KDM 5还通过改变转录因子向其靶启动子的募集来影响基因表达。具体而言，KDM 5招募致癌转录因子Myc到细胞生长基因启动子，这需要KDM 5的染色质结合PHD基序。KDM 5还将Foxo募集到抗氧化应激基因启动子，然而这以PHD独立的方式发生。基于这些数据，该提议的第一个假设是KDM 5通过一种以上的机制影响转录因子募集，并且这涉及KDM 5的不同结构域。初步数据还显示，KDM 5-Foxo共调节靶标的表达在携带与人类严重智力残疾相关的等位基因的果蝇品系（kdm 5L 854 F）中降低。由于KDM 5C中与智力残疾相关的其余12个错义突变也发生在进化上保守的残基中，第二个假设是苍蝇KDM 5中的相应突变将显示转录缺陷。这些假设将通过追求三个具体目标进行测试：1）确定KDM 5将Myc招募到细胞生长基因的机制; 2）定义KDM 5将Foxo招募到氧化应激靶基因的机制; 3）确定类似于人类智力残疾相关突变的kdm 5等位基因的转录和表型缺陷。这些分析是重要的，因为定义KDM 5如何以上下文依赖的方式发挥作用将导致治疗人类KDM 5家族蛋白失调引起的恶性肿瘤和认知表型的新策略。该提案是创新的，因为它偏离了目前关注的赖氨酸脱甲基酶（KDM）蛋白质的酶功能，通过描述两种新的机制，基因激活KDM 5是独立的酶活性。另一个创新是分析果蝇中的kdm 5错义等位基因，其类似于在智力残疾患者中发现的突变，作为剖析KDM 5的基因调控功能的手段。

DESCRIPTION (provided by applicant): KDM5 family transcriptional regulators are increasingly recognized as critical players at the interface of development and human disease. Mammalian cells encode four KDM5 paralogs (KDM5A-D), three of which are clinically significant: KDM5A or KDM5B are overexpressed in a number of cancers including breast, colorectal and melanoma, and mutations in KDM5C account for ~3% of X-linked intellectual disability patients. A lack of basic knowledge regarding the mechanisms of KDM5 action has hindered the development of therapies to treat these diseases. The long-term goal of these studies is therefore to dissect the gene- regulatory functions of KDM5 proteins using Drosophila, since the presence of a single KDM5 protein in this organism bypasses the issue of functional redundancy in mammalian cells. Previous data showed that KDM5 proteins can repress transcription via their histone demethylase activity and can activate gene expression by altering histone acetylation. In this proposal, new data demonstrate for the first time that KDM5 also influences gene expression by altering the recruitment of transcription factors to their target promoters. Specifically, KDM5 recruits the oncogenic transcription factor Myc to cell growth gene promoters, and this requires the chromatin-binding PHD motif of KDM5. KDM5 also recruits Foxo to oxidative stress resistance gene promoters, however this occurs in a PHD-independent manner. Based on these data, the first hypothesis of this proposal is that KDM5 affects transcription factor recruitment by more than one mechanism, and that this involves distinct domains of KDM5. Preliminary data also show that the expression of KDM5-Foxo co- regulated targets is reduced in a fly strain harboring an allele associated with severe intellectual disability in humans (kdm5L854F). Because the remaining 12 missense mutations in KDM5C associated with intellectual disability also occur in evolutionarily conserved residues, the second hypothesis is that the corresponding mutations in fly KDM5 will show transcriptional defects. These hypotheses will be tested by pursuing three specific aims: 1) Determine the mechanism by which KDM5 recruits Myc to cell growth genes; 2) Define the mechanism by which KDM5 recruits Foxo to oxidative stress target genes; and 3) Determine the transcriptional and phenotypic defects of kdm5 alleles analogous to human intellectual disability-associated mutations. These analyses are significant because defining how KDM5 functions in context-dependent manner will lead to new strategies for treating malignancies and cognitive phenotypes caused by dysregulation of KDM5 family proteins in humans. The proposal is innovative because it deviates from the current focus on the enzymatic function of lysine demethylase (KDM) proteins by describing two new mechanisms of gene activation by KDM5 that are independent of its enzymatic activity. An additional innovation is the analyses of kdm5 missense alleles in flies that are analogous to mutations found in patients with intellectual disability as a means to dissect the gene- regulatory functions of KDM5.