组蛋白修饰调控粗糙脉孢菌生物钟基因frequency的分子机制

发布时间：2018-06-04 10:11

本文选题：粗糙脉孢菌 + frequency　；参考：《中国农业大学》2016年博士论文

【摘要】：生物钟是地球上的生物在体内形成的能够测量时间的分子机器,其运行机制十分保守。粗糙脉孢菌是研究生物钟的理想模式生物之一,生物钟基因frequency (frq)的周期性转录对于粗糙脉孢菌生物钟的运行十分重要,但目前对其具体转录调控机制还不是十分清楚。为了探究表观遗传修饰对生物钟的影响,我们在粗糙脉孢菌中对可能参与生物钟调控的相关因子进行了遗传筛选,发现组蛋白H3K36的甲基转移酶SET-2参与生物钟的调控。首先,无论是SET-2的缺失还是H3K36R点突变均导致frq基因不能周期性转录。其次,SET-2介导的H3K36me3在frq基因ORF 3'端的富集具有周期性。进一步的研究发现,SET-2对加基因的调控需要组蛋白去乙酰化酶RPD-3的帮助。RPD-3是组蛋白去乙酰化酶复合体Rpd3S中的重要组分,该复合体中的EAF-3可以识别H3K36的甲基化并将Rpd3S复合体募集到相应的位置。在set-2KO和pd-3KO中,frq基因ORF区域组蛋白的乙酰化水平大幅升高。同时,我们在set-2KO、 rpd-3KO、eaf-3KO中均可以检测到不依赖于WC复合体的frq表达,它可以通过影响WC复合体的活性干扰正常frq基因的转录,从而导致粗糙脉孢菌生物钟紊乱。为了探究组蛋白哪些位点的乙酰化修饰参与frq基因的转录调控,我们对RPD-3潜在的识别位点进行了筛选。从赖氨酸(K)到谷氨酰胺(Q)的点突变可以模拟该位点的乙酰化修饰,我们发现H3K9QK14QK18Q突变体的生物钟表型与rpd-3KO十分相似。同时,在H3K9QK14QK18Q中也能检测到不依赖于WC复合体的frq表达,从而证明RPD-3对生物钟的调控主要是通过影响组蛋白H3K9、K14、K18位点的乙酰化修饰实现的。SET-2对frq基因的转录调控除了需要组蛋白去乙酰化酶RPD-3的帮助外,还需要染色质重塑蛋白的参与。我们的数据表明,SET-2可以与染色质重塑蛋白CHD-1一起调控frq基因ORF区域新旧组蛋白的交换。因为细胞中游离的组蛋白一般都处于高乙酰化状态,SET-2和CHD-1可以抑制转录过程中因过多新组蛋白的掺入造成的高乙酰化。通过遗传筛选,我们还发现组蛋白去乙酰化酶HDA-2也参与生物钟的调控。与组蛋白去乙酰化酶RPD-3不同,HDA-2主要从两方面调控加基因的转录。其一是通过组蛋白去乙酰化酶活性调控生物钟的振幅。在hda-2KO中,frq基因的转录水平升高,但并不影响其周期。另一方面,HDA-2在一定条件下又可以激活不依赖于WC复合体的frq转录。已知在chd-1KO wc-IRIP中有一定水平的不依赖于WC复合体的frq转录,而在chd-1KO wc-1RIP hda-2KO三突变体中frq基因不能转录。我们的研究解析了组蛋白修饰对生物钟基因frq的转录调控机制,这为揭示粗糙脉孢菌生物钟的运行机制提供了重要依据,同时对其它物种中相关的研究有一定的指导意义。
[Abstract]:Biological clock is a molecular machine formed in the body of organisms on earth to measure time, and its working mechanism is very conservative. C. crassa is one of the ideal model organisms to study the biological clock. The periodic transcription of the biological clock gene frequency / frqis is very important for the operation of the biological clock, but the specific transcriptional regulation mechanism is not very clear. In order to investigate the effect of epigenetic modification on the biological clock, we screened the related factors which may be involved in the regulation of the biological clock in C. crassa, and found that methyltransferase SET-2 of histone H3K36 was involved in the regulation of the biological clock. Firstly, both the deletion of SET-2 and the point mutation of H3K36R lead to frq gene not being cyclically transcribed. Secondly, SET-2 mediated H3K36me3 enrichment at the 3 'end of frq gene ORF has periodicity. Further studies have found that the regulation of histone deacetylase (RPD-3) by SET-2 requires the help of histone deacetylase (RPD-3). RPD-3 is an important component of histone deacetylase complex (Rpd3S). The EAF-3 in the complex can recognize the methylation of H3K36 and raise the Rpd3S complex to the corresponding position. The acetylation level of ORF region histone of frq gene was significantly increased in set-2KO and pd-3KO. At the same time, we can detect the frq expression independent of WC complex in set-2KOO, rpd-3KOeaf-3KO, which can interfere with the transcription of normal frq gene by affecting the activity of WC complex, which leads to the disorder of circadian clock of C. crassicolor. In order to investigate which sites of histone acetylation are involved in the transcriptional regulation of frq gene, we have screened the potential recognition sites of RPD-3. The point mutation from Lysine K to Glutamine Q) can mimic the acetylation modification at this site. We found that the biological clock phenotype of H3K9QK14QK18Q mutant is very similar to that of rpd-3KO. At the same time, frq expression independent of WC complex could also be detected in H3K9QK14QK18Q. It is proved that the regulation of RPD-3 on biological clock is mainly realized by affecting the acetylation modification of histone H3K9K14K18 site. Besides the help of histone deacetylase RPD-3, the transcriptional regulation of frq gene by SET-2 also needs the participation of chromatin remodeling protein. Our data suggest that SET-2, together with chromatin remodeling protein CHD-1, regulates the exchange of new and old histone in the ORF region of the frq gene. Because the free histone in the cell is generally in a hyperacetylated state, SET-2 and CHD-1 can inhibit the hyperacetylation caused by excessive incorporation of new histone in the transcriptional process. Through genetic screening, we also found that histone deacetylase HDA-2 is also involved in the regulation of biological clock. Different from histone deacetylase (RPD-3), HDA-2 mainly regulates the transcription of additive gene in two ways. One is to regulate the amplitude of the circadian clock by histone deacetylase activity. The transcription level of frq gene increased in hda-2KO, but did not affect its cycle. On the other hand, HDA-2 can activate frq transcription independent of WC complex under certain conditions. It is known that there is a certain level of frq transcription independent of WC complex in chd-1KO wc-IRIP, but frq gene can not be transcribed in chd-1KO wc-1RIP hda-2KO triple mutants. Our work has elucidated the transcriptional regulation mechanism of histone modification on the biological clock gene frq, which provides an important basis for revealing the mechanism of circadia circadian clock, and also has certain guiding significance for other species related research.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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