组蛋白H3K4甲基转移酶dSet1在果蝇原始生殖细胞命运及行为调控中的功能研究

In eukaryote cells，the gene expression involves both genetic and epigenetic regulation. It has been a hot topic that epigenetic regulation functions in developmental processes via regulating the target gene expression. Histone H3 lysine 4 (H3K4) trimethylation correlates with active transcription and this post-transcription modification catalyzed by the transmethylase dSet1, and the enzymatic activity of dSet1 is modulated through a multi-subunit protein complex known as COMPASS. Based on the existing results that dSet1 play important roles in a variety of cellular and developmental processes, we have initiated studies on the role of transmethylase dSet1 in regulating the cell fate and behavior of the Drosophila primordial germ cells (PGCs). Our results showed that dSet1 not only essential for histone H3K4 tri-methylation in gonad cells, but also defective dSet1 function perturbs the PGC differentiation and proliferation in developing female larval gonads. To further understand how dSet1-mediated epigenetic regulation functions in PGC cell fate and proliferation, we will determine the specific regulatory processes controlled by the dSet1, as well as the underlying mechanism. Meanwhile, we will screen and functionally identify the potential target genes of the dSet1 in gonad development. We expect that studies in this proposal will not only reveal epigenetic mechanism that underlie the PGC fate and proliferation regulation, based on the target specificities, but also greatly stimulate the further research of adult gonad development in higher organisms.

真核基因的表达在遗传和表观遗传两个层面受到调控。表观遗传介导的基因表达调控在发育中的功能已引起广泛关注。组蛋白H3K4甲基化修饰与基因转录活性密切关联，该翻译后修饰主要由Set1甲基转移酶以复合物形式催化完成的。我们及同行实验室研究证实，Set1在多种细胞与发育过程中起重要作用。为拓展对该表观遗传因子的研究，我们以果蝇原始生殖细胞PGC的命运及行为调控为工作平台，先期开展了相关实验。结果显示，dSet1在雌蝇幼虫性腺细胞组蛋白H3K4三甲基化修饰中不可或缺，敲减其功能致使性腺发育中的PGC在分化模式与细胞增殖上发生异常。基于这一发现，本次申请项目拟进一步解析dSet1的具体作用环节及机理，同时，高通量筛查并鉴定dSet1的功能性靶基因，旨在阐述其中的表观遗传调控机制。本项目的研究不仅有望揭示组蛋白H3K4甲基转移酶dSet1新的功能及机理，还将在一定程度上促进高等生物性腺发育研究的开展。

果蝇卵巢幼虫期正常发育保证了果蝇进入成虫能持续不断地产生配子。幼虫期性腺包含生殖细胞和体细胞两大谱系，原始生殖细胞(primordial germ cells, PGCs)是成体生殖干细胞的前体，其在幼虫期经历增殖与有序分化等过程。随着性腺的发育，体细胞系会逐步分化为不同功能的体细胞类群，其中端丝细胞（terminal filament cells, TFCs）和混合细胞（intermingled cells，ICs）是重要的两大类体细胞。PGC微环境体细胞表达BMP信号配体并以旁分泌的方式激活与其相邻PGC中BMP信号活性，保证这部分PGC保持不分化状态，而远离微环境体细胞的生殖细胞开始分化。表观遗传调控参与到众多的细胞与发育过程中，本项目较为系统的研究了表观遗传调控因子组蛋白H3K4甲基转移酶dSet1，染色质重塑因子Brahma（Brm）和microRNA-33在果蝇幼虫性腺发育中的调控功能。.甲基转移酶dSet1调控H3K4二甲基化和三甲基化。我们研究发现，dSet1以细胞自治和非自治两种方式参与调控三龄幼虫性腺PGC有序分化过程。不仅如此，dSet1在PGC与混合细胞（IC）之间的互作中也发挥着重要作用，dSet1下调的IC中，不仅IC的数目减少了，而且PGC的数目增多了。这样的结果表明，甲基转移酶dSet1对性腺正常发育的调控是多层次，多方面的。.染色质重塑BRM复合物中具有ATP酶活性的亚基Brahma（Brm）广泛表达于果蝇幼虫性腺两大细胞谱系的细胞核中，而且Brm以细胞自治和非自治两种方式参与调控PGC有序分化过程。不仅如此，Brm调控TF命运获得。我们推测Brm通过调控TF命运获得与分化从而影响PGC有序分化。.MicroRNAs （miRNAs）为一类由21-24个核苷酸组成的短非编码RNA分子，特异性靶向mRNA的降解或抑制其翻译下调靶基因的表达。本项目采用microRNA微阵列芯片（Affymetrix miRNA array 4.0）分析筛选参与调控果蝇性腺发育的microRNA。并得到miR-33参与雌性果蝇幼虫性腺发育的实验证据。.本研究首次证明组蛋白甲基转移酶dSet1、染色质重塑因子Brahma（Brm）和microRNA-33在雌性果蝇幼虫性腺发育中具有调控功能。本项目必将为更深层次的研究提供可靠的参考。