本文選題：CTCF + 染色質(zhì)相互作用　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2015年博士論文

【摘要】：CCCTC序列結(jié)合因子,即CTCF,是一種廣泛表達(dá)的多功能鋅指蛋白,其序列高度保守,目前被認(rèn)為是脊椎動物中唯一的絕緣子調(diào)控蛋白。CTCF最早被發(fā)現(xiàn)為雞c-myc基因的轉(zhuǎn)錄阻遏物,后續(xù)研究不斷揭示CTCF參與多種生物學(xué)過程,具有不同功能,主要包括:基因轉(zhuǎn)錄的抑制與活化、絕緣子調(diào)控、印跡基因調(diào)控、X染色體失活、影響m RNA可變剪接、影響核小體重排、影響DNA復(fù)制與重組等。CTCF表達(dá)異常往往與多種疾病、腫瘤的發(fā)生發(fā)展等密切相關(guān)。通過自身11個鋅指結(jié)構(gòu)的不同組合,CTCF能夠選擇性識別數(shù)萬個靶位點,其靶位點在基因組內(nèi)廣泛分布,并且CTCF與靶位點的結(jié)合跟DNA甲基化狀態(tài)存在動態(tài)調(diào)控機(jī)制,進(jìn)而形成細(xì)胞類型特異性結(jié)合位點,同時,不同位點間還存在不同的保守性和序列特征。除了具有眾多的結(jié)合位點,通過自身蛋白序列N-端和C-端的內(nèi)在無序結(jié)構(gòu)特點以及多種翻譯后修飾等方式,CTCF還可以與眾多不同蛋白、甚至RNA直接相互作用。另外,CTCF還被證實能夠結(jié)合于細(xì)胞有絲分裂期染色體,可能作為有絲分裂書簽因子發(fā)揮作用,但具體分子機(jī)制及潛在功能仍有待探索。作為染色質(zhì)三維高級結(jié)構(gòu)的主要組織者,依靠眾多的結(jié)合位點以及其它蛋白、甚至RNA的幫助,CTCF能夠介導(dǎo)廣泛的染色質(zhì)內(nèi)、染色質(zhì)間相互作用,幫助建立和維持染色質(zhì)高級構(gòu)象,進(jìn)而發(fā)揮其多種多樣的功能。但目前為止,對于CTCF結(jié)合染色質(zhì)上的不同類型結(jié)合位點、介導(dǎo)不同類型染色質(zhì)相互作用、發(fā)揮多種多樣的不同類型生理功能三者之間的關(guān)系還停留在個別現(xiàn)象或有限局部數(shù)據(jù)分析上,其內(nèi)在的對應(yīng)關(guān)系、生物學(xué)意義及背后的生物學(xué)邏輯仍有待全面、系統(tǒng)地深入研究。本文利用包括人類基因組CTCF結(jié)合位點在內(nèi)的來源于ENCODE等的Ch IP數(shù)據(jù)、介導(dǎo)染色質(zhì)相互作用的Ch IA-PET數(shù)據(jù),來源于mod ENCODE等的果蠅基因組d CTCF結(jié)合位點及其隨有絲分裂周期變化的Ch IP數(shù)據(jù),以及其它轉(zhuǎn)錄因子、組蛋白表觀修飾譜等多層次、多維度的組學(xué)數(shù)據(jù),結(jié)合多種基因組信息數(shù)據(jù)庫,通過多種生物信息學(xué)分析工具及算法,從“結(jié)合位點”、“相互作用”、“有絲分裂周期傳遞”三方面對CTCF不同類型的全基因組圖譜進(jìn)行了細(xì)致而全面的系統(tǒng)生物學(xué)分析。第一部分,我們從ENCODE數(shù)據(jù)庫中整理得到人類基因組70個細(xì)胞系共162,209個CTCF結(jié)合位點,分析提取得到其三段式結(jié)合基序(three-part motif),并且發(fā)現(xiàn)結(jié)合位點的細(xì)胞類型特異性、基序motif顯著性、序列保守性三者之間存在一定的相關(guān)性,即細(xì)胞類型非特異性越高的位點,其motif顯著性越強(qiáng),其保守性越高。我們認(rèn)為:在大多數(shù)細(xì)胞中共有的非特異性結(jié)合位點,即組成性位點,可能作為基因組內(nèi)的關(guān)鍵性節(jié)點,在進(jìn)化選擇壓力下,最終維持高保守性,同時,顯著的motif能夠提供更高的親和力,從而與CTCF蛋白更穩(wěn)定地結(jié)合。隨后,對于CTCF靶基因的功能學(xué)分析結(jié)果顯示:細(xì)胞類型特異性CTCF結(jié)合位點可能與細(xì)胞類型特異性基因表達(dá)調(diào)控相關(guān)聯(lián),非特異性位點靶基因則對應(yīng)于所有細(xì)胞都必需的基本生物學(xué)功能。同時,我們從新的角度,對160個能夠與CTCF結(jié)合位點共定位的轉(zhuǎn)錄因子進(jìn)行聚類分析,首次將其分為四種不同類型,提示CTCF能夠在不同類型結(jié)合位點募集到不同類型的轉(zhuǎn)錄因子,進(jìn)而發(fā)揮不同的生物學(xué)功能。第二部分內(nèi)容著眼于人類基因組內(nèi)CTCF參與介導(dǎo)的染色質(zhì)相互作用。我們首先總結(jié)出CTCF結(jié)合位點的細(xì)胞類型非特異性(組成性)是其參與介導(dǎo)染色質(zhì)相互作用的充要條件,即非特異性的結(jié)合位點傾向于參與相互作用,而且參與相互作用的位點也大多是細(xì)胞類型高度非特異性的。進(jìn)一步地,我們從拓?fù)鋵W(xué)的角度,發(fā)現(xiàn)細(xì)胞類型高度非特異性的CTCF結(jié)合位點往往處于染色質(zhì)相互作用網(wǎng)絡(luò)的中心位置,在維持網(wǎng)絡(luò)的連通性、穩(wěn)定性、魯棒性等過程中發(fā)揮關(guān)鍵性作用,我們認(rèn)為這類型結(jié)合位點可能充當(dāng)染色質(zhì)三維結(jié)構(gòu)的底盤或骨架節(jié)點。此外,我們首次發(fā)現(xiàn)CTCF能夠建立一種特殊的“多對多”相互作用結(jié)構(gòu),該結(jié)構(gòu)可能參與構(gòu)建和維持小鼠物種特異性染色質(zhì)區(qū)域的三維結(jié)構(gòu),但是在人類基因組中的作用和意義還有待進(jìn)一步探索。另一方面,通過整合不同轉(zhuǎn)錄因子共定位、組蛋白表觀修飾譜、染色體狀態(tài)等數(shù)據(jù),我們將CTCF介導(dǎo)的染色質(zhì)相互作用分為不同類型,同時發(fā)現(xiàn)cohesin復(fù)合物和ZNF143分子可能在幫助CTCF建立和維持穩(wěn)定的染色質(zhì)相互作用中發(fā)揮重要功能,其中cohesin已有前人相關(guān)文獻(xiàn)報道,而ZNF143的具體功能機(jī)制還有待進(jìn)一步研究。綜合以上結(jié)果,我們認(rèn)為CTCF在其介導(dǎo)的染色質(zhì)相互作用網(wǎng)絡(luò)中,能夠起到底盤式的功能,選擇性識別特定靶位點,建立染色質(zhì)相互作用關(guān)系,并在cohesin復(fù)合物和ZNF143的幫助下,形成穩(wěn)定的、不同類型的相互作用,其中一部分作為染色質(zhì)三維結(jié)構(gòu)的骨架,維持染色質(zhì)構(gòu)象的穩(wěn)定性,另一部分進(jìn)一步募集其他轉(zhuǎn)錄因子,產(chǎn)生不同的基因轉(zhuǎn)錄調(diào)控機(jī)制。第三部分著眼于考察CTCF與染色質(zhì)的結(jié)合在細(xì)胞身份(cell identity)的建立與維持中的重要作用。我們以有絲分裂為切入點,以模式生物果蠅為研究對象,分類討論細(xì)胞有絲分裂周期不同階段,d CTCF在果蠅基因組上結(jié)合情況的變化及相應(yīng)的生物學(xué)意義。我們首先將d CTCF位點分為間期—分裂期共有(IM)、間期特有(IO)、分裂期特有(MO)三類。通過motif、GC含量、保守性等序列特征分析,發(fā)現(xiàn)MO位點與IM、IO存在不同序列特征,我們推測是由于染色體在有絲分裂期大量凝縮,形態(tài)環(huán)境、物理學(xué)特性等可能與間期完全不同所致,d CTCF結(jié)合于分裂期染色體特有的那部分結(jié)合位點即MO位點可能基于一種新的分子機(jī)制。同時還發(fā)現(xiàn)IM位點具有明顯高于其它類型位點的motif強(qiáng)度、Ch IP信號強(qiáng)度及保守性,且相關(guān)靶基因涉及到多種生物學(xué)過程,既跟細(xì)胞基本生理活動相關(guān),又與有絲分裂進(jìn)程密切聯(lián)系,充分說明d CTCF穩(wěn)定結(jié)合于IM位點對于維持正常的細(xì)胞活動,特別是細(xì)胞身份的建立與維持等具有重要意義。隨后我們進(jìn)一步發(fā)現(xiàn),相同類型的d CTCF位點呈現(xiàn)相互聚集傾向,并且富集有d CTCF結(jié)合位點的TAD結(jié)構(gòu)域邊界以及果蠅基因組保守性結(jié)構(gòu)域邊界,同樣也具有這種“相同類型聚集”(物以類聚)的傾向。我們認(rèn)為d CTCF在有絲分裂周期不同階段可能參與建立不同的TAD結(jié)構(gòu),同時還幫助維持基因組保守性區(qū)域的高級構(gòu)象。基于上述結(jié)果,我們推測CTCF在有絲分裂期結(jié)合于染色體的可能生物學(xué)意義主要有以下幾個方面:一方面,CTCF通過與那些需要在有絲分裂期發(fā)揮特定功能的靶基因的相應(yīng)結(jié)合位點結(jié)合,參與這些特殊基因的轉(zhuǎn)錄調(diào)控,同時CTCF還可以在有絲分裂M/G1期過渡后快速建立起新的轉(zhuǎn)錄調(diào)控網(wǎng)絡(luò)以利于細(xì)胞活動;另一方面,CTCF在分裂期可能參與建立分裂期特異性的染色質(zhì)構(gòu)象,并在細(xì)胞進(jìn)入間期后幫助染色質(zhì)迅速重建新的三維結(jié)構(gòu)。綜合上述三部分內(nèi)容,本文通過從橫向的有絲分裂間期的點(結(jié)合位點)到面(相互作用網(wǎng)絡(luò)),再到縱向的整個細(xì)胞周期動態(tài)變化過程的完整研究模式,系統(tǒng)構(gòu)建基因組內(nèi)CTCF全方位、多層次圖譜,深度解析CTCF的功能及其背后的生物學(xué)意義,對CTCF的相關(guān)研究提供了重要的實驗資源和理論依據(jù)。
[Abstract]:CCCTC sequence binding factor, CTCF, is a widely expressed multifunction zinc finger protein. Its sequence is highly conserved. At present, it is considered that the only insulators regulated protein.CTCF in vertebrates is first discovered as a transcriptional repressor of the chicken c-myc gene. Follow up studies continue to reveal that CTCF is involved in a variety of biological processes and has different functions. The inhibition and activation of gene transcription, the regulation of insulators, the regulation of the imprinted gene, the inactivation of the X chromosome, the influence of the variable splicing of the m RNA, the influence of the nuclear small body weight, and the influence of the.CTCF expression on the replication and recombination of DNA are closely related to the occurrence and development of a variety of diseases and tumors. Through the different combinations of the 11 zinc finger structures of their own, CTCF can The selective identification of tens of thousands of target loci, the target loci are widely distributed in the genome, and the combination of CTCF with the target site and the DNA methylation state has a dynamic regulation mechanism, and then forms the specific binding site of the cell type. At the same time, there are different conservatism and sequence characteristics between different loci. Besides, there are numerous binding sites. CTCF can interact directly with many different proteins, even RNA, over the intrinsic disorder structure characteristics of the N- and C- ends of the autologous protein sequence and a variety of post-translational modifications. In addition, CTCF has also been proved to be able to bind to cell mitotic chromosomes and may play a role as a mitotic bookmark factor, but the specific molecular machine As the main organizer of the three-dimensional structure of chromatin, the main organizer of the three dimensional structure of chromatin, with the help of many binding sites and other proteins, and even RNA, CTCF can mediate a wide range of chromatin, interchromatin interaction, help to establish and maintain the advanced conformation of chromatin, and then play a variety of functions. So far, the relationship between the different types of chromatin interaction between CTCF and different types of chromatin interaction, and the relationship between the various types of different types of physiological functions, and the internal relationship, the biological significance and the biological logic behind the relationship between the different types of chromatin interaction and the analysis of three different types of physiological functions In this paper, we use Ch IP data, including the CTCF binding site of the human genome, and other Ch IP data, to mediate the Ch IA-PET data of chromatin interaction, derived from the D CTCF binding site of the Drosophila genome, and its Ch IP data with the mitotic cycle, as well as other transcripts, as well as other transcripts. Factor, histone apparent modification spectrum, multilevel, multidimensional group data, combined with a variety of genomic information databases, through a variety of bioinformatics analysis tools and algorithms, from "binding sites", "interaction", "mitosis cycle transfer" three parties to the whole genome of different types of CTCF in detail and comprehensive. In the first part, we collate 162209 CTCF binding sites in the 70 human genome of the human genome from the ENCODE database, and analyze and extract the third segment binding sequence (three-part motif), and find the cell type specificity of the binding site, the motif significance of the sequence motif, and the sequence conservatism between the three. In a certain correlation, the higher the non specificity of the cell type, the stronger the motif, the higher the conservativeness. We think that the non specific binding sites in most cells, that is, the constituent loci, may be the key nodes within the genome, and ultimately maintain high conservatism under the pressure of evolutionary selection, at the same time, The motif can provide a higher affinity and more stable binding with CTCF protein. Subsequently, the functional analysis of the CTCF target gene shows that the cell type specific CTCF binding site may be associated with the regulation of cell type specific gene expression, and the non specific site target genes correspond to the essential bases for all cells. At the same time, we cluster analysis of 160 transcriptional factors that can co loci with CTCF binding sites from a new perspective. For the first time, we divide them into four different types, suggesting that CTCF can raise different types of transcription factors at different types of binding sites and play different biological functions. Second parts of the contents are used. Focusing on the interaction of chromatin mediated mediated by CTCF in the human genome, we first conclude that the non specificity (composition) of the cell types of the CTCF binding site is the necessary and sufficient condition for its involvement in mediating chromatin interaction, that is, non specific binding sites tend to participate in interaction, and most of the sites involved in interaction are also involved. Further, from the topological point of view, we find that the highly non specific CTCF binding sites of cell types are often at the center of the chromatin interaction network and play a key role in maintaining connectivity, stability, and robustness in the network, and we consider this type of binding site. The point may act as a chassis or skeleton node for the three-dimensional structure of chromatin. In addition, we have discovered for the first time that CTCF can build a special "multi to many" interaction structure that may be involved in building and maintaining the three-dimensional structure of the specific chromatin region of the mouse species, but the role and significance in the human genome is still to be advanced. On the other hand, we divide the CTCF mediated chromatin interaction into different types by integrating different transcription factors co location, histone epigenetic modification spectrum, chromosome state and other data, and find that cohesin complex and ZNF143 molecules may play an important role in helping CTCF to establish and maintain stable chromatin interactions. Function, in which cohesin has been reported in previous literature, and the specific functional mechanism of ZNF143 remains to be further studied. Combining the above results, we think that CTCF can function as a chassis type in its mediated chromatin interaction network, selectively identify specific target points, establish chromatin interaction, and recover in cohesin. With the help of compound and ZNF143, a stable, different type of interaction is formed, one part of which serves as the skeleton of the three-dimensional structure of chromatin, maintains the stability of the chromatin conformation, and the other further raises other transcription factors to produce different gene transcriptional regulation mechanisms. The third part focuses on the combination of CTCF and chromatin. The important role in the establishment and maintenance of cell identity (cell identity). We take the mitosis as the breakthrough point, take the model biological Drosophila as the research object, and discuss the changes of the D CTCF binding in the Drosophila genome and the corresponding biological significance. We first divide the D CTCF site into interphase. IM, interphase specific (IO), split period endemic (MO) three classes. Through the analysis of motif, GC content, conservatism and other sequence characteristics, we found that MO loci and IM, IO have different sequence characteristics, we speculate that a large amount of condensation, morphological environment, physical properties, etc., in the mitosis period, may be completely different from the interval, D CTCF. The MO locus, which is specific to the division of chromosomes at the split stage, may be based on a new molecular mechanism. It is also found that the IM site is significantly higher than the motif intensity of other types of loci, the intensity and conservatism of the Ch IP signal, and the related target genes are involved in a variety of biological processes, which are related to the basic physiological activities of the cells. In close connection with the mitosis process, it is important to demonstrate that D CTCF is stable binding to the IM site for maintaining normal cell activity, especially the establishment and maintenance of cell identity. Then we further found that the same type of D CTCF loci showed mutual aggregation tendency and enriched the TAD domain with D CTCF binding sites. We believe that D CTCF may participate in the establishment of different TAD structures at different stages of the mitosis cycle, while also helping to maintain the advanced conformation of the conserved region of the genome. The possible biological significance of binding to chromosomes at mitosis stage is mainly in the following aspects: on the one hand, CTCF is involved in the transcription regulation of these special genes by combining with the corresponding binding sites that require specific function of the target genes during mitosis, and CTCF can also be fast after the mitotic M/G1 phase transition. On the other hand, CTCF may participate in the establishment of a split phase specific chromatin conformation at the split stage and help chromatin to quickly reconstruct a new three-dimensional structure at the interval. The three parts of the content are synthesized through a crosswise mitotic point (binding). The complete research pattern of the site (interaction network) and the dynamic process of the whole cell cycle in the longitudinal direction, and the systematic construction of CTCF omnibearing, multilevel atlas, depth analysis of the function of CTCF and the biological significance behind it, provide important experimental resources and theoretical basis for the related research of CTCF.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q343

【相似文獻(xiàn)】

相關(guān)期刊論文 前9條

1 邱平,易祿康;大鼠老化過程大腦皮層及小腦神經(jīng)元染色質(zhì)構(gòu)象分析[J];生物化學(xué)雜志;1990年05期

2 邱平,易祿康;大鼠老化過程大腦皮層細(xì)胞核、染色質(zhì)轉(zhuǎn)錄研究[J];生物化學(xué)雜志;1990年04期

3 亓合媛;張昭軍;李雅娟;方向東;;染色質(zhì)構(gòu)象調(diào)控真核基因的表達(dá)[J];遺傳;2011年12期

4 陳石根,周潤琦,鄭升;正常及白血病小鼠白細(xì)胞染色質(zhì)和DNA的酶切分析[J];生物化學(xué)雜志;1987年03期

5 周國嶺;宋崴;付湘輝;辛立;唐曉彬;馮冬曉;劉光;劉德培;;轉(zhuǎn)基因鼠中人α類珠蛋白基因簇染色質(zhì)構(gòu)象調(diào)控變化[J];中國醫(yī)學(xué)科學(xué)院學(xué)報;2007年03期

6 劉宏德;羅坤;馬昕;翟金城;謝建明;孫嘯;萬亞坤;;核小體及染色質(zhì)修飾的基因組分布模式和染色質(zhì)狀態(tài)[J];生物化學(xué)與生物物理進(jìn)展;2013年11期

7 楊華;樊紅;;基于分子結(jié)構(gòu)的lncRNA分子功能的研究進(jìn)展[J];東南大學(xué)學(xué)報(醫(yī)學(xué)版);2014年01期

8 高雅;齊錦生;栗彥寧;;ChIA-PET技術(shù)[J];生命的化學(xué);2011年01期

9 ;[J];;年期

相關(guān)博士學(xué)位論文 前3條

1 周國嶺;細(xì)胞分裂中染色質(zhì)活性和轉(zhuǎn)錄狀態(tài)記憶機(jī)制的研究[D];中國協(xié)和醫(yī)科大學(xué);2006年

2 沈文龍;CTCF介導(dǎo)不同類型染色質(zhì)相互作用的研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2015年

3 孔衛(wèi)青;家蠶含染色質(zhì)域基因的鑒定與MSL-3同源體和Bm-MOF基因的功能研究[D];西南大學(xué);2007年

相關(guān)碩士學(xué)位論文 前1條

1 馮想想;HeLa細(xì)胞染色質(zhì)結(jié)構(gòu)在細(xì)胞間期的動態(tài)變化[D];東北師范大學(xué);2013年

，

本文編號：1963508