本文選題：粗糙脈孢菌 + frequency　； 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：生物鐘是地球上的生物在體內(nèi)形成的能夠測量時間的分子機器,其運行機制十分保守。粗糙脈孢菌是研究生物鐘的理想模式生物之一,生物鐘基因frequency (frq)的周期性轉(zhuǎn)錄對于粗糙脈孢菌生物鐘的運行十分重要,但目前對其具體轉(zhuǎn)錄調(diào)控機制還不是十分清楚。為了探究表觀遺傳修飾對生物鐘的影響,我們在粗糙脈孢菌中對可能參與生物鐘調(diào)控的相關(guān)因子進行了遺傳篩選,發(fā)現(xiàn)組蛋白H3K36的甲基轉(zhuǎn)移酶SET-2參與生物鐘的調(diào)控。首先,無論是SET-2的缺失還是H3K36R點突變均導(dǎo)致frq基因不能周期性轉(zhuǎn)錄。其次,SET-2介導(dǎo)的H3K36me3在frq基因ORF 3'端的富集具有周期性。進一步的研究發(fā)現(xiàn),SET-2對加基因的調(diào)控需要組蛋白去乙�；窻PD-3的幫助。RPD-3是組蛋白去乙�；笍�(fù)合體Rpd3S中的重要組分,該復(fù)合體中的EAF-3可以識別H3K36的甲基化并將Rpd3S復(fù)合體募集到相應(yīng)的位置。在set-2KO和pd-3KO中,frq基因ORF區(qū)域組蛋白的乙酰化水平大幅升高。同時,我們在set-2KO、 rpd-3KO、eaf-3KO中均可以檢測到不依賴于WC復(fù)合體的frq表達,它可以通過影響WC復(fù)合體的活性干擾正常frq基因的轉(zhuǎn)錄,從而導(dǎo)致粗糙脈孢菌生物鐘紊亂。為了探究組蛋白哪些位點的乙�；揎梾⑴cfrq基因的轉(zhuǎn)錄調(diào)控,我們對RPD-3潛在的識別位點進行了篩選。從賴氨酸(K)到谷氨酰胺(Q)的點突變可以模擬該位點的乙酰化修飾,我們發(fā)現(xiàn)H3K9QK14QK18Q突變體的生物鐘表型與rpd-3KO十分相似。同時,在H3K9QK14QK18Q中也能檢測到不依賴于WC復(fù)合體的frq表達,從而證明RPD-3對生物鐘的調(diào)控主要是通過影響組蛋白H3K9、K14、K18位點的乙�；揎棇崿F(xiàn)的。SET-2對frq基因的轉(zhuǎn)錄調(diào)控除了需要組蛋白去乙�；窻PD-3的幫助外,還需要染色質(zhì)重塑蛋白的參與。我們的數(shù)據(jù)表明,SET-2可以與染色質(zhì)重塑蛋白CHD-1一起調(diào)控frq基因ORF區(qū)域新舊組蛋白的交換。因為細(xì)胞中游離的組蛋白一般都處于高乙�；癄顟B(tài),SET-2和CHD-1可以抑制轉(zhuǎn)錄過程中因過多新組蛋白的摻入造成的高乙�；Ｍㄟ^遺傳篩選,我們還發(fā)現(xiàn)組蛋白去乙�；窰DA-2也參與生物鐘的調(diào)控。與組蛋白去乙酰化酶RPD-3不同,HDA-2主要從兩方面調(diào)控加基因的轉(zhuǎn)錄。其一是通過組蛋白去乙�；富钚哉{(diào)控生物鐘的振幅。在hda-2KO中,frq基因的轉(zhuǎn)錄水平升高,但并不影響其周期。另一方面,HDA-2在一定條件下又可以激活不依賴于WC復(fù)合體的frq轉(zhuǎn)錄。已知在chd-1KO wc-IRIP中有一定水平的不依賴于WC復(fù)合體的frq轉(zhuǎn)錄,而在chd-1KO wc-1RIP hda-2KO三突變體中frq基因不能轉(zhuǎn)錄。我們的研究解析了組蛋白修飾對生物鐘基因frq的轉(zhuǎn)錄調(diào)控機制,這為揭示粗糙脈孢菌生物鐘的運行機制提供了重要依據(jù),同時對其它物種中相關(guān)的研究有一定的指導(dǎo)意義。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q78

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