Distinct Modes of Gene Regulation by KDM5

描述(由申请人提供)： KDM5家族转录调控因子越来越多地被认为是发育和人类疾病之间的关键角色。哺乳动物细胞编码四个KDM5同源基因(KDM5A-D)，其中三个具有临床意义：KDM5A或KDM5B在乳腺癌、结直肠癌和黑色素瘤等多种癌症中过表达，KDM5C突变约占X连锁智力残疾患者的3%。缺乏关于KDM5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用果蝇剖析KDM5蛋白的基因调控功能，因为在这种生物体中存在单一的KDM5蛋白，从而绕过了哺乳动物细胞的功能冗余问题。以往的研究表明，KDM5蛋白通过组蛋白去甲基酶活性抑制转录，通过改变组蛋白乙酰化来激活基因表达。在这项提议中，新的数据首次证明KDM5也通过改变转录因子对其目标启动子的招募来影响基因的表达。具体地说，KDM5招募致癌转录因子Myc为细胞生长基因启动子，这需要KDM5的染色质结合PhD基序。KDM5也招募FOXO为氧化应激抵抗基因启动子，但这是以一种不依赖于PHD的方式发生的。基于这些数据，这一建议的第一个假设是KDM5通过不止一个机制影响转录因子招募，并且这涉及KDM5的不同结构域。初步数据还显示，在携带与人类严重智力残疾相关的等位基因(Kdm5L854F)的苍蝇品系中，KDM5-FOXO共同调节靶标的表达减少。由于与智能障碍相关的其余12个KDM5C错义突变也发生在进化上保守的残基中，第二个假设是Fly KDM5中的相应突变将显示转录缺陷。这些假说将通过追求三个具体目标来验证：1)确定KDM5招募Myc到细胞生长基因的机制；2)确定KDM5招募FOXO到氧化应激靶基因的机制；以及3)确定类似于人类智力残疾相关突变的kdm5等位基因的转录和表型缺陷。这些分析具有重要意义，因为定义KDM5如何以上下文相关的方式发挥作用将导致治疗恶性肿瘤和由人类KDM5家族蛋白调节失调引起的认知表型的新策略。这项提议是创新的，因为它偏离了目前对赖氨酸脱甲基酶(KDM)蛋白的酶功能的关注，描述了KDM5激活基因的两种新机制，这两种机制独立于其酶活性。另一项创新是分析果蝇中的kdm5错义等位基因，这些等位基因类似于在智力残疾患者中发现的突变，以此作为剖析KDM5基因调节功能的一种手段。

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.