本文選題：胚胎干細(xì)胞 + DNA甲基化標(biāo)記　； 參考：《哈爾濱工業(yè)大學(xué)》2015年博士論文

【摘要】：胚胎干細(xì)胞(ESC)具有多向分化潛能和發(fā)育的全能性。DNA甲基化在胚胎干細(xì)胞自我更新和分化過程中扮演重要角色。因此胚胎干細(xì)胞特異的甲基化新標(biāo)記的識(shí)別和功能分析對于理解決定細(xì)胞命運(yùn)的復(fù)雜而精細(xì)的調(diào)控網(wǎng)絡(luò)具有重要意義。因此,整合基于重亞硫酸鹽測序技術(shù)測定的發(fā)育過程中的高通量單堿基水平的DNA甲基化圖譜,開發(fā)面向?qū)嶒?yàn)科學(xué)家的精確高效的數(shù)據(jù)庫平臺(tái)和生物信息學(xué)軟件,系統(tǒng)地識(shí)別和表征胚胎干細(xì)胞特有的DNA甲基化新標(biāo)記,對于深入理解胚胎干細(xì)胞的多能性維持和定向分化具有重要意義。本論文基于信息熵理論開發(fā)了定量甲基化特異性的算法,并通過隨機(jī)數(shù)據(jù)和真實(shí)數(shù)據(jù)對該方法的精確性、樣本適用性和資源占用等特性進(jìn)行了系統(tǒng)評估;基于基因組相鄰Cp G位點(diǎn)間距離依賴的甲基化相似性開發(fā)了基因組片段化的算法,進(jìn)一步基于t檢驗(yàn)開發(fā)了甲基化標(biāo)記識(shí)別的統(tǒng)計(jì)學(xué)方法,利用Python實(shí)現(xiàn)了以上算法并編制了甲基化特異性分析報(bào)告軟件SMART。通過與已有甲基化分析軟件的比較表明了SMART在基于大樣本甲基化組從頭識(shí)別基因組甲基化功能區(qū)域的作用。通過整合高通量DNA甲基化數(shù)據(jù),以胚胎干細(xì)胞為中心,以發(fā)育時(shí)間為主線,以方便實(shí)驗(yàn)科學(xué)家為目標(biāo),開發(fā)了小鼠發(fā)育甲基化數(shù)據(jù)庫Dev Mouse。并基于信息熵理論開發(fā)集成了全自動(dòng)化的在線分析和可視化工具。同時(shí)整合人類的高通量DNA甲基化數(shù)據(jù),構(gòu)建了以胚胎干細(xì)胞為中心的人類甲基化數(shù)據(jù)庫Human MethyDB。兩個(gè)發(fā)育甲基化數(shù)據(jù)庫的構(gòu)建不僅為本論文的后續(xù)研究奠定了數(shù)據(jù)基礎(chǔ),也有利于實(shí)驗(yàn)科學(xué)家方便快捷地從事發(fā)育相關(guān)DNA甲基化的生物信息學(xué)分析�；诒疚拈_發(fā)的信息熵算法分析Dev Mouse中收錄的小鼠由胚胎干細(xì)胞向腦發(fā)育過程中DNA甲基化等表觀基因組學(xué)數(shù)據(jù),結(jié)果發(fā)現(xiàn)了DNA甲基化與組蛋白修飾H3K27me3的共同變化。進(jìn)一步識(shí)別了小鼠腦發(fā)育過程中1 341個(gè)差異甲基化的Cp G島,發(fā)現(xiàn)了其與差異H3K27me3修飾Cp G島的顯著重疊。對429個(gè)差異表達(dá)基因的分析證實(shí)了DNA甲基化與其他組蛋白修飾對發(fā)育相關(guān)基因差異表達(dá)的協(xié)同調(diào)控,特別是對重編程轉(zhuǎn)錄因子基因和印記基因的動(dòng)態(tài)調(diào)控。并揭示了基因間區(qū)Cp G島作為新基因標(biāo)記及重要調(diào)控元件的潛能。將SMART應(yīng)用到Human MethyDB收集的人類DNA甲基化組,從頭識(shí)別了757 887個(gè)功能片段。其中75%的片段在所有細(xì)胞類型中一致甲基化,表明了人類基因組甲基化的穩(wěn)定性,分析發(fā)現(xiàn)一致超高甲基化片段多位于重復(fù)元件上,而一致超低甲基化片段多為基因啟動(dòng)子Cp G島。對高特異甲基化片段的分析則發(fā)現(xiàn)胚胎干細(xì)胞特異超低甲基化不僅可以作為干細(xì)胞的穩(wěn)定標(biāo)記,還參與調(diào)控重要的發(fā)育基因。并利用SMART識(shí)別了人類胚胎干細(xì)胞中的3 758個(gè)DNA甲基化標(biāo)記,且發(fā)現(xiàn)各多能性干細(xì)胞間共享更多的超高甲基化標(biāo)記。利用高通量的表觀基因組數(shù)據(jù)對胚胎干細(xì)胞甲基化標(biāo)記進(jìn)行了系統(tǒng)的表征。結(jié)果發(fā)現(xiàn)人類胚胎干細(xì)胞超低甲基化標(biāo)記顯著富集在發(fā)育相關(guān)功能,對長度大于3500 bp的甲基化標(biāo)記的分析揭示了胚胎干細(xì)胞的特異性甲基化模式。通過組學(xué)分析發(fā)現(xiàn)了胚胎干細(xì)胞超低甲基化標(biāo)記以細(xì)胞特異性的方式顯著富集了超級(jí)增強(qiáng)子標(biāo)記(H3K27ac和轉(zhuǎn)錄因子結(jié)合位點(diǎn))。發(fā)現(xiàn)了胚胎干細(xì)胞中超低甲基化標(biāo)記和超級(jí)增強(qiáng)子的顯著重疊,識(shí)別了二者重疊的超低甲基化標(biāo)記以及71個(gè)相關(guān)的多能性基因,功能富集分析揭示了這些基因的敲除可導(dǎo)致發(fā)育異常。人鼠間的比較分析則揭示了胚胎干細(xì)胞甲基化標(biāo)記的物種保守性。綜上所述,本文基于信息熵開發(fā)了甲基化分析的軟件和數(shù)據(jù)庫,有助于實(shí)驗(yàn)人員進(jìn)行胚胎干細(xì)胞甲基化新標(biāo)記的篩選和功能分析。對小鼠和人類胚胎干細(xì)胞的DNA甲基化新標(biāo)記進(jìn)行了系統(tǒng)的識(shí)別和表征,為深入理解胚胎干細(xì)胞的多能性維持和定向分化機(jī)制提供了新的重要參考。
[Abstract]:Embryonic stem cells (ESC) have multiple differentiation potential and developing omnipotent.DNA methylation plays an important role in the self renewal and differentiation of embryonic stem cells. Therefore, the identification and functional analysis of the specific new methylation markers in embryonic stem cells are of great significance for understanding the complex and fine regulatory network determining cell fate. Therefore, the DNA methylation Atlas of high throughput single base level in the development process based on sulfite sequencing technology is integrated, the accurate and efficient database platform and bioinformatics software for experimental scientists are developed to systematically identify and characterize the new DNA methylation markers specific to embryonic stem cells, and to understand the embryos in depth. The pluripotent maintenance and directional differentiation of stem cells is of great significance. This paper developed a quantitative methylation specific algorithm based on the information entropy theory, and systematically evaluated the accuracy of the method, sample applicability and resource occupancy through random data and real data; based on the distance dependence of the Cp G loci between the genome adjacent to the genome The methylation similarity of Lai developed a genomic fragmentation algorithm, further developed a statistical method for methylation identification based on t test, implemented the above algorithm by using Python and compiled a methylation specific analysis report software SMART. by comparison with the existing methylation analysis software to show that SMART is based on large sample armour. Based on the integration of high throughput DNA methylation data, integrated high-throughput DNA methylation data, embryonic stem cells as the center and development time as the main line, the mouse developmental methylation database Dev Mouse. was developed and integrated with the information entropy theory to develop a fully automated online system based on the information entropy theory. Analysis and visualization tools, simultaneously integrating human high throughput DNA methylation data, constructs two developmental methylation databases for human methylation database Human MethyDB. centered on embryonic stem cells, which not only lays a data base for the follow-up study of this paper, but also facilitates the rapid and rapid development of experimental scientists. Bioinformatics analysis of related DNA methylation. Based on the information entropy algorithm developed in this paper, the epigenetic data of DNA methylation in mouse embryonic stem cells from Dev Mouse to brain development were analyzed. The results found that DNA methylation and histone modified H3K27me3 were common changes. Further identification of brain development in mice was made. 1341 Cp G islands with differential methylation showed a significant overlap with the differential H3K27me3 modified Cp G island. Analysis of 429 differentially expressed genes confirmed the synergistic regulation of DNA methylation and other histone modification on differential expression of developmental genes, especially the dynamic regulation of reprogramming transcription factor gene and imprinting genes. The potential of Cp G island in the intergenic region was revealed as a new gene marker and important regulatory element. SMART was applied to the DNA methylation group of Human MethyDB to identify 757887 functional segments from scratch. 75% of the fragments were methylation in all cell types, indicating the stability of the methylation of human genome. The hypermethylation fragment is mostly on the repeating element, while the unanimous hyper methylation fragment is mostly the gene promoter Cp G island. The analysis of the highly specific methylation fragments shows that the specific hyper methylation of embryonic stem cells can not only be used as a stable marker for stem cells, but also participate in the regulation of the important developmental genes. And the use of SMART to identify human beings 3758 DNA methylation markers in embryonic stem cells were identified and more hypermethylation markers were shared among various pluripotent stem cells. The methylation markers of embryonic stem cells were systematically characterized by high throughput epigenetic data. The results showed that ultra low methylation markers in human embryonic stem cells were significantly enriched in development related work. The specific methylation patterns of embryonic stem cells were revealed by the analysis of the methylation markers longer than 3500 BP. The superhyper methylated markers in embryonic stem cells were found to significantly enrich the superenhancer markers (H3K27ac and transcriptional binding sites) in a cell specific way. The hyper methylation and 71 related pluripotent genes overlapped by the two were identified, and 71 related pluripotent genes were identified. Functional enrichment analysis revealed that the knockout of these genes could lead to abnormal development. In this paper, based on the information entropy, the software and database of methylation analysis are developed, which help the experimenters to screen and analyze the new markers for the methylation of embryonic stem cells. A systematic identification and characterization of the new DNA methylation markers for mouse and human embryonic stem cells are carried out in order to understand the pluripotent maintenance of embryonic stem cells. The mechanism of directional differentiation provides a new and important reference.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R321
，

本文編號(hào)：1956947