IOCAS-IR
刺参肠道再生的DNA甲基化调控解析及再生候选基因的功能分析
李晓妮
第一作者单位中国科学院海洋研究所
学位类型博士
导师杨红生
2018-05-08
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位专业海洋生态学
关键词刺参 再生 Wgbs Dna 甲基化 Wnta
摘要

再生作为 Science 公布的 125 个最具科学挑战的科学问题中的第 7 个问题,
一直是最受关注的科学热点。目前再生研究主要集中在果蝇、涡虫、蝾螈等模式
动物与人类器官再生研究。刺参作为棘皮动物,为无脊索动物中分类地位最高等
的类群,具有超强的再生能力,在应答刺激会排出内脏,并很快再生出一套完整
的内脏器官,正逐渐成为研究再生的新模型。DNA 甲基化是最早发现的修饰之
一,能引起染色质结构、DNA 构象与稳定性及 DNA 与蛋白质相互作用方式的改
变,从而控制基因表达,其控制细胞重编程与干细胞分化,对器官再生起到重要
的调控作用。由于研究手段与基础数据缺乏等原因,无脊椎动物中特别是棘皮动
物中的 DNA 甲基化对动物特殊生理行为的调控研究严重滞后,本人所在团队的
刺参基因组的解析为 DNA 甲基化修饰对刺参肠道再生的调控研究带来了良好的
契机。综上所述,研究 DNA 甲基化修饰和候选再生基因在刺参肠道再生中的作
用不仅为海参肠道再生的分子机制研究提供参考,而且可以为人类再生医学研究
提供新思路,同时可以为 DNA 甲基化再生药物研制和再生途径研究提供理论指
导。
本研究利用 WGBS(Whole Genome Bisulfite Sequencing)、RNA-Seq、
RT-qPCR、Western-blot 和免疫组化等技术对 DNA 甲基化修饰以及候选基因在刺参肠道再生中的作用展开研究,分析刺参肠道再生过程中 DNA 甲基化酶和 DNA去甲基化酶的表达特征和调控机制。利用 DNA 甲基化抑制剂分析 DNA 甲基化对刺参肠道再生的影响。构建刺参肠道再生全基因组 DNA 甲基化图谱,解析刺参肠道再生过程的 DNA 甲基化调控特征。并进一步与转录组关联分析,解析DNA 甲基化对刺参肠道再生的调控机制。查明关键再生基因的时空表达特征,
分析其调控作用。研究结果如下:
1.DNA  甲基化酶与 DNA  去甲基化酶对刺参肠道再生的调控作用
刺参 DNA 甲基化调控系统相关基因的 mRNA 表达量在再生过程中显著上
调,说明 DNA 甲基化修饰参与刺参肠道再生。克隆得到 DNA 甲基化关键调控
酶 Dnmt3b、TDG 和 Tet2 的开放阅读框区序列,体内体外转染构建的三个关键调控酶过表达载体和干扰片段后,增殖相关基因转录上调表达。
2 .DNA  甲基化对刺参肠道再生的影响
成功构建了刺参肠道原代细胞培养体系,高浓度抑制剂抑制细胞存活,体内
体外抑制 DNA 甲基化阻碍刺参原代细胞的增殖和肠道再生,说明 DNA 甲基化
修饰在刺参肠道再生中的关键作用。DNA 甲基化抑制过程中,大部分增殖相关
基因的 mRNA 上调表达。
3.刺参肠道再生 DNA  甲基化 图 谱构建
再生过程中差异 DNA 甲基化相关基因以 CG 甲基化类型为主,主要分布在
基因编码区中间和后半部位的外显子区域。KEGG 分析表明差异 DNA 甲基化基
因显著富集在神经、免疫、代谢相关通路和泛素化、磷酸化修饰等基因分类中。
同时,验证了 RIPK1、FGFR3、KRAB1、Notch1 和 ROBO4 基因在肠道再生过程中的 DNA 甲基化,刺参肠道再生 2 h,RIPK1 和 KRAB1 DNA 甲基化程度低,FGFR3 DNA 甲基化程度较高;再生 3 d,Notch1 和 ROBO4 DNA 甲基化程度也较高。
4. 刺参肠道再生初级阶段转录组 表 达谱的构建
构建了刺参再生初级阶段转录表达图谱,结果显示再生初级阶段显著上调表
达基因主要富集在免疫和神经相关通路中,与 DNA 甲基化聚类结果一致。DNA
甲基化和转录组联合分析结果表明,不同基因 DNA 甲基化调控表达模式不同,
大部分基因 DNA 甲基化抑制基因转录表达,DNA 去甲基化促进基因转录表达。
肠道再生2 h磷酸化相关基因大部分在基因本体区发生DNA甲基化且下调表达,
3 d 磷酸化相关基因大部分在启动子区发生 DNA 甲基化且上调表达,其他分类
基因表达模式因基因不同而有差异。
5. 再生候选基因在刺参肠道再生中的调控机制
(1) 获得刺参 DNA 甲基化调控不同类别基因 KRAB1、FGFR3、ROBO4 和
RIPK1 基因开放阅读框序列,发现其在刺参肠道再生过程中上调表达。体外细胞
转染构建的 KRAB1、FGFR3、ROBO4 和 RIPK1 过表达载体后发现 KRAB1 抑制
细胞增殖,FGFR3、ROBO4 和 RIPK1 促进细胞增殖及其相关基因的表达。
(2) 获得刺参 WntA 基因全长序列并分析其表达特征。结果表明 WntA 在再
生过程中逐渐上调表达,WntA 蛋白表达定位在表皮细胞、肠组织的肌肉和粘膜
下层中和细胞增殖定位表达模式相似。
(3) 经典 Wnt 信号通路的 Cateninβ和 DVL3,与非经典 Wnt/Ca+通路的 PKC
以及其他 Wnt 信号通路相关基因都在肠道再生的 21 d 显著上调表达。

其他摘要

Regeneration, the seventh of the 125 most scientifically-challenged scientific
issues published by Science, has been the most concerned scientific hotspot. At
present, researches on regeneration mainly focuses on the regeneration of animals
such as Drosophila, planaria, and pupa and human organs. As the echinoderm, A.
japonicus(Apostichopus japonicus) is the group with the highest classification status
in invertebrate animals and has superior regenerative ability. It will excrete internal
organs in response to the stimulus, and quickly regenerate a complete set of internal
organs It is gradually becoming a new model for regeneration research. DNA
methylation is one of the earliest found modifications that can cause chromatin
structure, DNA conformation and stability, and changes in the way DNA interacts
with proteins, thereby controls genes expression, cell reprogramming and stem cell
differentiation, and Plays an important regulatory role in organ regeneration. Due to
the lack of research methods and basic data, DNA methylation researches lags behind
in the regulation of animal specific physiological behaviors in invertebrates,
especially in echinoderms. Genome researches that finished by our group has brought
a good opportunity for the study about DNA methylation modification role on the
regulation of intestinal regeneration of A. japonicus. In summary, studying the role of
DNA methylation modification and candidate regeneration genes in intestinal
regeneration of sea cucumber not only can provide a reference for the molecular
mechanism of intestinal regeneration of A. japonicus, but also provide new ideas for
research on human regenerative medicine. At the same time, it will provide theoretical
guidance for the research of DNA methylation regenerative drug and regeneration
pathways.
In this study, WGBS (Whole Genome Bisulfite Sequencing), RNA-Seq,
RT-qPCR, Western-blot, and immunohistochemistry were used to study the role of
DNA methylation modification and candidate genes in intestinal regeneration of A.japonicus and study expression characteristics and regulatory mechanism of DNA
methylase and DNA demethylase during intestinal regeneration of A. japonicus. DNA
methylation inhibitors were used to analyze the effect of DNA methylation on
intestinal regeneration of A. japonicus. We construct the genome-wide DNA
methylation map of intestinal regeneration of sea cucumber, and analyze the DNA
methylation regulation characteristics of A. japonicus's intestinal regeneration process.
And further we associated DNA methylation with transcriptome analysis results to
analysis the control mechanism of A. japonicus intestinal regeneration. We identify
the spatio-temporal expression characteristics of key regeneration genes and analyze
their regulatory roles. The results of the study are as follows:
1. Research on the Regulatory Mechanism of DNA Methylase in Intestinal
Regeneration of A. japonicus
The mRNAexpression levels of related genes in the DNA methylation regulatory
system of A. japonicus were significantly upregulated during the regeneration process,
suggesting that DNA methylation modification participates in the intestinal
regeneration of A. japonicus. The open reading frame regions of DNA methylation
key regulatory enzymes Dnmt3b, TDG and Tet2 were obtained by cloning. After the
constructed three key regulatory enzymes overexpression vectors and interfering
fragments in vitro were transfected, the expression of proliferation related genes were
upregulated.
2. DNAmethylation can affect Intestinal Regeneration of A. japonicus
The primary culture system of intestinal cells of A. japonicus was successfully
constructed. High-level inhibitors inhibited cell survival. Inhibition of DNA
methylation inhibited the proliferation of primary cells of A. japonicus and intestinal
regeneration in vitro and in vivo, indicating that the key role of DNA methylation
modification in intestinal regeneration of A. japonicus. In the process of DNA
methylation inhibition, most mRNAs of proliferation-related genes were up-regulated.
3. Construction of DNAmethylation map in intestine of A. japonicus
During the regeneration process, the differential DNA methylation-related genes
are mainly CG methylation types, and are mainly distributed in the middle and the latter half of exon region of the coding region of the gene. KEGG analysis showed
that the differential DNA methylation genes were significantly enriched in neural,
immunological, metabolic pathways, and ubiquitylation and phosphorylation
modifications. At the same time, DNA methylation of RIPK1, FGFR3, KRAB1,
Notch1, and ROBO4 genes during intestinal regeneration was verified.. DNA
methylation level of RIPK1 and KRAB1 were lower, and DNA methylation level of
FGFR3 were Higher at 2 h in intestinal regeneration; DNA methylation level of
Notch1 and ROBO4 were also higher at 3 d.
4. Construction of transcription expression profile at early stage of
intestinal regeneration of A. japonicus
The transcriptional expression profile of the primary stage of A. japonicus
regeneration was constructed. The results showed that the significantly up-regulated
genes in the primary regeneration stage were mainly enriched in the immune and
neural related pathways, and were consistent with the DNA methylation genes
clustering results. The results of DNA methylation and transcriptome combined
analysis showed that the expression pattern of DNA methylation regulation in
different genes was different. Most methylated genes inhibited genes transcription and
DNA demethylation genes promoted genes transcription and expression. Most related
genes to phosphorylati were DNA methylated in the gene's body regions at 2 h and
their expressions were down-regulated. Most of the phosphorylation-related genes had
DNA methylation in the promoter region and their expressions were up-regulated at 3
d. Expression patterns of other classified genes were differences in different genes.
5. Regulatory Mechanism of Regeneration Candidate Genes in Intestinal
Regeneration of A. japonicus
(1) The open reading frame sequences of KRAB1, FGFR3, ROBO4, and RIPK1
genes that regulated by DNA methylation were detected, and they were up-regulated
expression during the intestinal regeneration. After transfecting constructed KRAB1,
FGFR3, ROBO4 and RIPK1 overexpression vectors in vitro, we found KRAB1
inhibited cell proliferation, and FGFR3, ROBO4 and RIPK1 promoted cell
proliferation and related genes expression. (2) We obtained the full-length sequence of WntA from A. japonicus and
analyzed its expression characteristics. The results showed that WntA expression
gradually up-regulated during regeneration, WntA protein localized in the epidermal
cells, intestinal muscle and submucosa, and expression patterns are similar with cell
proliferation localization.
(3) Cateninβ and DVL3 in the canonical Wnt signaling pathway, and PKC in the
non-canonical Wnt/Ca+ pathway and other Wnt signaling pathway-related genes
expression were significantly up-regulated on the 21st day of intestinal regeneration.
Key words: Apostichopus japonicus, Regeneration, WGBS, DNA methylation,
WntA

语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154478
专题中国科学院海洋研究所
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李晓妮. 刺参肠道再生的DNA甲基化调控解析及再生候选基因的功能分析[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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