去甲基化酶PHF8介导非组蛋白去甲基化的功能研究及其在前列腺癌中的潜在应用

The PHD-finger protein 8 (PHF8) is a histone demethylase with both PHD- and JmjC-domains. Mutations to the human PHF8 gene are associated with Siderius X-linked mental retardation (XLMR) syndrome. The symptoms of Siderius XLMR syndrome include, in addition to mild MR, facial dysmorphism and cleft lip/palate. Cleft lip, with or without cleft palate, and isolated cleft palate are serious birth defects which affect approximately 1 in every 600 newborn babies worldwide. In addition, the expression level of PHF8 is significantly higher in clinical prostate cancer samples compared to normal and benign samples. Strong PHF8 expression correlates significantly with high Gleason grade and was borderline significant for poor prognosis. Our previous study showed that PHF8 can demethylate H3K9me1/2, H3K27me2 and H4K20me1 on histones. Although lysine methylation of histones has been studied extensively as an essential mechanism for epigenetic regulation of chromatin, our understanding of this modification on non-histone proteins has just begun. Particularly, demethylation of non-histone substrates by PHF8 and its biological functions have never been reported so far. Our preliminary study revealed that PHF8 physically interacts with a number of transcription factors, which could serve as substrates for PHF8. Furthermore, our preliminary data suggested methylation and demethylation events occur on these non-histone proteins seem to be functional important, including transcriptional regulation and cell cycle progression. Therefore, we hypothesize that study of the functional importance and underlying molecular mechanism of PHF8-mediated demethylation events on non-histone proteins will shed light on the screening, synthesis and optimization of small molecule inhibitors specifically targeting PHF8, therefore resulting in promising leads for the treatment of XLMR and prostate cancer. In this proposal, we aim to reveal the potential function of the demethylation events on non-histone substrates and the underlying molecular mechanism through biochemical, molecular, cellular and genome-wide sequencing tools. Furthermore, selective inhibitors targeting PHF8 will be synthesized applying peptidomimetic approach or identified through chemical compound library screening. Promising hits will then be tested through both in vitro biochemical assays and in vivo cellular assays. The results from this proposed study will enhance our understanding of how PHF8 functions in gene transcription, cell cycle control and cancer biology besides its activity towards histone methylation, which will serve as guidance for designing and optimizing small molecular inhibitors selectively targeting PHF8, and provide possible cures for the diseases PHF8 associated with. Furthermore, the proposed study will reveal new paradigms through which lysine demethylases regulate cellular processes, such as transcriptional control, developmental control, cell-fate decisions and disease.

PHF8是一种具有PHD和JmjC结构域的组蛋白去甲基化酶。人PHF8基因突变与X-连锁智力迟缓综合征（XLMR）和前列腺癌密切相关。本课题组已发表成果表明PHF8可以催化组蛋白去甲基化。同时，我们也发现PHF8能够通过与非组蛋白相互作用，介导其去甲基化，并在基因转录和细胞周期调控中发挥重要功能。由此我们推测对PHF8介导的非组蛋白去甲基化的功能和分子机制的研究将有助于探寻以PHF8为靶点小分子抑制剂，从而有助于获得治疗与其相关的疾病的靶向药物。本项目将通过通过生物化学、分子、细胞以及全基因组测序的方法研究非组蛋白底物去甲基化的潜在功能和分子机制。进而，利用多肽模拟，合成或通过药物化合物库的筛选靶向PHF8的选择性抑制剂。最后通过体外和体内实验研究先导化合物的特异性和有效性。本项目将为其它表观遗传学调控子的作用分子机制和功能提供新的研究思路以及寻找治疗癌症和其它疾病药物靶点提供指导意义。

PHF8是一个经典的含有jmjc结构域的去甲基化酶，能够去甲基化组蛋白的多个位点，其通过与基因启动子区域结合而调控基因的转录活性，其突变、缺失或者高表达与与机体的神经系统发育和多种癌症的发生发展密切相关。本课题组曾报道过PHF8的去甲基化组蛋白H4赖氨酸20的酶活性，并揭示其在细胞周期调控中的关键作用及其详细阐述了相应的分子机制。该项目研究发现PHF8能与细胞内重要的转录因子YY1相互作用并调控其翻译后修饰，从而调控其与染色质DNA的结合及其介导的基因转录，最终参与细胞的正常功能。进一步研究发现赖氨酸甲基化转移酶SET7/9能够甲基化YY1，并且该甲基化对YY1的DNA结合活性，YY1参与调控的基因转录，YY1介导的细胞生长和肿瘤形成至关重要。YY1的同源体YY2也被发现受SET7/9介导的甲基化修饰调控。PHF8被发现在前列腺癌中高表达，我们进一步发现其参与前列腺癌细胞的生长，并揭示该功能和其调控雄性激素诱导的基因转录调控密切相关。最后，我们通过计算机模拟进行了大规模筛选靶向PHF8的小分子抑制剂并对其活性进行检测。本项目通过生物化学、分子、细胞、高通量测序和基因编辑的方法深入系统研究PHF8对其相互作用的蛋白YY1的调控作用和分子机制，及其在癌症中的应用。靶向PHF8的小分子抑制剂将为治疗与其相关的疾病提供新的希望，同时，本项目的研究将为其它表观遗传调控子的作用分子机制和功能研究提供新的思路，以及寻找治疗癌症和其它疾病靶点提供重要的指导意义。