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类型 基础研究 预答辩日期 2018-05-07
开始(开题)日期 2014-05-30 论文结束日期 2018-01-22
地点 南京四牌楼2号东南大学李文正楼北225 论文选题来源 国家自然科学基金项目     论文字数 5.82 (万字)
题目 CtBP调控PcG/trxG靶基因转录激活作用的分子机制研究
主题词 CtBP,PcG/trxG,果蝇,转录调控,组蛋白修饰
摘要 基因转录调控在众多生物学进程中具有决定性的作用。序列特异的转录因子/共转录因子以及不依赖于序列特异性的染色质修饰因子 (例如PcG与trxG蛋白家族) 在发育基因的转录调控中均发挥重要的功能。然而,转录因子与染色质修饰因子之间如何相互协调,共同决定特定细胞中基因的表达模式迄今尚不清楚。羧基末端结合蛋白 (C-terminal binding protein, CtBP) 是一种重要的共转录因子,目前的研究主要集中于其转录共阻遏功能。相关研究表明CtBP与染色质修饰因子PcG家族成员之间存在相互作用,但其分子机制和生理功能仍不完全清楚。我们在研究中意外发现,CtBP在遗传学上拮抗PcG基因功能,提示CtBP在表观遗传调控中可能具有转录激活的功能。据此,我们对CtBP在表观遗传中的功能进行了深入的研究,主要结果阐述如下。 在果蝇遗传学水平,我们通过观察统计PcG杂合突变所产生的性梳与翅膀表型,发现CtBP在遗传学上拮抗PcG的功能。为了探讨其分子机制,我们分别在足与翅膀的成虫盘中对PcG的靶基因Scr与Ubx进行免疫染色,发现CtBP确实拮抗PcG对其靶基因的抑制作用,提示CtBP对PcG的遗传学拮抗作用可能是直接的。同时,我们也检测了CtBP与trxG的遗传学相互作用,通过对腹节表型的统计分析发现CtBP具有促进trxG的功能,从而也间接支持CtBP对PcG的拮抗作用。 在细胞水平,我们对CtBP的转录调控功能进行了专门的研究。在果蝇Kc细胞中,PcG家族成员的敲低会导致其靶基因显著的转录去阻遏现象,该现象可作为研究基因转录激活的模型。因此我们通过该模型,并结合高通量转录组测序(RNA-seq) 和生物信息学分析,发现有381个基因在PcG成员ph-p与E(z)同时敲低后出现显著的转录激活现象。其中153个基因的转录激活在敲低CtBP后明显减弱,表明CtBP是这些基因转录激活所必须的。我们进一步对这些基因进行功能富集 (GO) 分析,发现这些基因主要参与转录调控与胚胎发育等生物学进程,且主要为同源异形基因 (HOX),表明CtBP是胚胎发育相关的基因的转录激活所必须的。 为了更精细地探究CtBP对PcG所结合的靶基因的调控,我们将这381个受到PcG抑制的基因与之前报道的PcG靶基因进行了维恩分析,发现其中的73个基因是PcG直接结合的靶基因,且90%以上 (66个基因) 的基因受CtBP的激活调控。对这66个CtBP共激活靶基因的GO分析结果显示,这些基因的功能富集与之前的153个CtBP共激活调控的基因非常一致,也主要为HOX基因。为了验证RNA-seq及生物学信息学分析的可靠性,我们从这66个CtBP共激活的靶基因中挑选出4个典型的靶基因 (Ubx, Antp, abd-A和Abd-B) 进行了RT-qPCR的验证实验,发现这些靶基因在同时敲低PcG成员 (ph-p和E(z)) 后均上调40倍以上,且该上调表达在CtBP敲低后明显消失;同时,我们在另一个PcG核心成员Pc单独敲低的情况下也观察到了一致的结果。表明CtBP能促进PcG靶基因 (尤其是HOX基因) 的转录激活。 为了更深入地探讨CtBP对PcG靶基因转录激活作用的分子机制,我们对PcG/trxG及其所催化的组蛋白修饰进行了染色质免疫共沉淀分析 (ChIP)。我们发现在PcG成员敲低后,Pc蛋白在PRE区的结合显著降低,而去除CtBP可基本消除这一现象,表明CtBP具有拮抗PcG染色质募集的功能;另一方面,PcG敲低后,trxG成员CBP与UTX的DNA结合显著升高,而该升高可被CtBP的敲低所抑制,表明CtBP参与trxG成员的染色质募集。我们也在PcG敲低的情况下,通过ChIP的方法检测了组蛋白修饰与RNA聚合酶II的结合情况,发现CtBP是启动子区与PRE区H3K27me3向H3K27ac转换所必须的,同时也是启动子区RNA聚合酶II结合所必须的,证实CtBP可能直接参与PcG靶基因的转录激活调控。ChIP-seq数据比对结果也支持CtBP的激活功能。最后,我们通过免疫共沉淀技术 (Co-IP) 证实CtBP与这些激活的染色质调控因子之间存在物理上的相互作用。 综上所述,我们通过表型分析发现CtBP在遗传学上存在拮抗PcG但促进trxG的功能,并通过免疫染色实验从基因表达的层面对该表型做出了一个解释;在Kc细胞中,通过RNAi结合RNA-seq以及生物信息学分析,我们发现CtBP参与部分PcG靶基因的转录激活,且这部分基因主要为同源异形基因;随后,我们通过ChIP实验证实CtBP拮抗PcG但促进trxG的DNA募集,并参与了组蛋白H3K27me3向H3K27ac的转换;最后我们通过ModENCODE数据库的ChIP-Seq数据比对,发现CtBP与众多激活的染色质修饰因子存在广泛的共定位现象,并通过Co-IP证实CtBP与这些激活的染色质修饰因子均存在相互作用。以上这些证据均支持这样的分子作用的模型,即CtBP存在广泛的转录激活功能。 因此,本文揭示了CtBP参与PcG/trxG靶基因转录激活的新现象,并阐明了这其中的表观遗传调控机制。此外,相关研究成果均间接支持CtBP及PcG蛋白与乳腺癌、结肠癌、胃癌等癌症相关联,因此,解析CtBP对PcG靶基因转录调控的分子机制将为进一步探讨胚胎发育过程中的基因表达模式,及为进一步探究其是否与癌症的形成相关联提供必要的理论基础。
英文题目 Molecular Mechanism Study on the Activation Role of CtBP in PcG/trxG Targets Regulation
英文主题词 CtBP; PcG/trxG; Drosophila; Transcription; Histone modifications
英文摘要 Both sequence-specific transcriptional factors/cofactors and the chromatin structure modifiers PcG/trxG proteins play crucial roles in the developmental gene regulation. However, it remains to be elusive on how do these two categories of transcriptional regulators cooperate with each other to define the precise gene expression profiles for certain cell-lineages. The evolutionarily conserved C-terminal binding protein (CtBP) is a well-characterized context-dependent transcriptional cofactor in developmental processes. The prevailing model of CtBP actions is gene repression, and CtBP is reported to interact with PcG members. However, the mechanism of CtBP involved in PcG targets regulation is still unclear. In this study, we unexpectedly found that CtBP genetically antagonized PcG, suggesting activation function might be more pronounced for the role of CtBP in an epigenetic context. Thus, we furthered our activation research of CtBP and obtained the following results. At the genetic level, by observation of the sex comb and wing phenotype of heterozygotic PcG, we found that CtBP genetically antagonize PcG function in Drosophila. To explore the molecular mechanism of the antagonism between CtBP and Pc, immunostaining was carried out in the third instar leg/wing imaginal disc, which found that CtBP was required for the activation of Scr/Ubx. Thus, CtBP might directly antagonize PcG repression. And heterozygotic mutants of the trxG conponents such as trx, Ash1 and brm bring about an abdominal tergite defect phenotype, the propotion of which was evidently increased by heterozygotic CtBP mutant or deficiency. Above all, CtBP could enhance trxG gene function, which also indirectly support the antagonism between CtBP and PcG. We furtherly carried out more experiments in cells. In Drosophila Kc cells, Knock down of PcG members resulted in a great depression of the targets, a good model to study the activation regulation of target genes. Thus, the PcG conponents ph-p and E(z) were simutanously knocked down and sustained for transcriptome sequencing. Through the subsequent bioinformatic analysis, 381 genes were greatly depressed, among which, 153 genes were not able to derepress with the co-knock down of CtBP, suggesting that CtBP is required for activation of these genes. Further Gene Ontology analysis showed that these targets are mainly involved in transcriptional regulation and development, most of which are HOX genes. Taken together, CtBP is required for activation of the PcG targets relative to embryonic development. To explore the function of CtBP in typical PcG targets regulation, 73 of the 381 genes were conducted to be PcG targets through Venn diagram analysis. Over 90 percent (66 genes) of the targets were activated by CtBP. GO analysis of the 66 targets showed the same enrichment pattern with the former 153 CtBP co-activated genes, most of which were HOX genes. To verify the results of the RNA-seq and GO analysis, four PcG targets (Ubx, Antp, abd-A and Abd-B) were selected for further qPCR verifications. All four targets were derepressed for at least 40 folds in ph-p and E(z) knocked down cells, which were remarkably receded by CtBP co-knock down. The same results were also observed in Pc and CtBP RNAi-ed cells. Thus, CtBP could promote the activation of PcG targets in Kc cells, especially HOX genes. To further explore the molecular mechanism of targets activation by CtBP, ChIP analysis was carried out to detect the DNA recruitments of the PcG and trxG components as well as the histone modificaitons in different PcG and CtBP RNAi-ed Kc cells. The binding of Pc was greatly reduced after PcG RNAi, which was recovered in CtBP and PcG simutaneously RNAi-ed cells, indicating CtBP antagonize Pc DNA binding. On the other hand, the DNA bindings of trxG components UTX and CBP were sharply increased by PcG RNAi, which was removed by the addition of CtBP RNAi. These results suggesting that CtBP help recruit the trxG to DNA to activate the target genes. Besides, we also detected the DNA bindings of histone modifications and different forms of RNA Pol II. In PcG RNAi-ed Kc cells, the H3K27me3 levels were greatly decreased in both of the Promoter and PRE regions, where the H3K27ac levels were generally increased, which was disappeared in PcG and CtBP double RNAi-ed cells, suggesting that CtBP is required for the swith of histone modifications in PcG reduced background. Besides, knock down of PcG caused an increased promoter binding of RNA Pol II, all of which was removed by simutanously knock down of CtBP and PcG, demonstrating that CtBP is required for transcriptional activation and elongation. ChIP-seq data alignment between CtBP and many acitive hisone marks from ModENCODE showed the genomewide coocupation of CtBP with many acitve marks. We also carried out co-IP experiments, which showed that CtBP indeed physically interacts with the acitve histone marks. Above all, we unexpectedly found that CtBP genetically antagonize PcG while enhance trxG function through phenotype analysis, which was furtherly confirmed by immunostaining of the targets in imaginal discs. In Kc cells, by RNAi based RNA-seq and bioinformatics analysis, we have shown that CtBP might also be required for the derepression of PcG target genes, such as homeotic genes. To get further insight into the mechanism underlying the transcriptional activation role of CtBP, we carried out ChIP analyses and found that CtBP might help recruiting proteins with activation roles, including UTX, CBP and RNA Pol II. Besides, CtBP may be an essential player in the histone modification dynamics. As such, CtBP is required for the switch of the methylated H3K27 into acetylated one. In addition, based on the alignment of publically available ChIP seq data, we found that CtBP might colocalized with many chromatin regulators. Also, our Co-IP assays indicated that CtBP might indeed physically interact with these proteins. All the results support a model that CtBP is widely required for transcriptional activation. Taken together, we have revealed a new aspect of CtBP in contributing epigenetic regulation of gene transcription. In addition, a series of researches indirectly support the relevence between CtBP or PcG and cancers such as breast, colon and stomach cancers. Thus, analyzing the mechanism of CtBP in PcG target gene transcriptional regulation will help provide the necessary theoretical basis for developmental gene expression as well as the relevance between CtBP and cancers.
学术讨论
主办单位时间地点报告人报告主题
东南大学 2014.09.21 东南大学李文正楼北211 毕彩丽 CtBP 参与PcG/trxG表观调控的研究
东南大学 2016.05. 27 东南大学李文正楼北211 王萍 Nlg3的转录调控研究
东南大学 2017.09.12 东南大学李文正楼北211 毕彩丽 CtBP参与表观遗传调控与信号通路的研究
东南大学 2012.09.25 东南大学李文正楼南213 毕彩丽 Genetic Interaction between CtBP and PcG
东南大学 2013.09.20 东南大学李文正楼南213 马璐 Fsh的作用机制研究
东南大学 2014.03.15 东南大学李文正楼北211 汪锐 NKD的入核研究
东南大学 2016.05.21 东南大学李文正楼南213 成琳 CtBP的脱氢酶活性研究
东南大学 2017.05.03 东南大学李文正楼北211 毕彩丽 CtBP参与Wnt与Dpp信号通路的研究
     
学术会议
会议名称时间地点本人报告本人报告题目
第二届中国果蝇会议 2013.04.26 湖南长沙 CtBP在表观及信号通路中的机制研究
第二届亚太果蝇会议 2013.05.13 韩国首尔 The role of CtBP in the regulation of PcG target genes
第三届亚太果蝇会议 2015.05.11 中国北京
第四届亚太果蝇会议 2017.05.08 日本大阪
     
代表作
论文名称
CtBP represses Dpp signaling as a dimer.
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
翁杰敏 正高 教授 博导 华东师范大学
赵权 正高 教授 博导 南京大学
谢维 正高 教授 博导 东南大学
韩俊海 正高 教授 博导 东南大学
王苏 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
谢浩 其他 讲师 东南大学