|Alternative Title||Functional interplay and targeting dynamics between polycomb proteins CBX2 and CBX7 in mammalian cells|
|Place of Conferral||兰州|
|Keyword||多梳抑制复合体 CBX蛋白 染色质免疫共沉淀 生物信息学|
多梳蛋白家族PcG是一类在染色质水平上通过表观遗传修饰调控靶基因的转录因子，主要以多梳蛋白抑制复合物1和2（PRC1和 PRC2）的形式存在。CBX蛋白家族是PRC1的核心组分之一，在哺乳动物中有CBX2，4，6，7，8五个同源蛋白。在小鼠胚胎干细胞（ESC）中已知CBX7是主要发挥作用的CBX蛋白且含有CBX7的PRC1能够不同程度地反馈抑制其他CBX同源蛋白编码基因的表达。本实验室前期工作中对CBX蛋白在ESC的表达及功能进行系统检测时发现在ESC中有一定程度的CBX2表达；进一步ChIP-qPCR结果显示CBX2与CBX7共同靶向到诸多已知的PRC1的靶基因上，但在单分子靶基因位点上二者是否共同协同工作以及这种工作方式的生物学意义目前未知。本论文中我们主要采用生物信息学和基因GO注释的方法对ESC中CBX2与CBX7共同调控的靶基因进行功能学分类，并进一步实验检测和比较了在小鼠胚胎干细胞E14TG2a和小鼠睾丸畸胎瘤F9细胞中二者靶向性的异同；利用免疫共沉淀法和连续染色质免疫共沉淀法探究二者在ESC中的分子相互作用和共调控靶基因的机制。研究结果显示Cbx2基因在ESC中被CBX7蛋白部分抑制的情况下仍然发挥功能，CBX2与CBX7在全基因组范围内的靶基因群有着较高的重合度，其中包括大量多能性维持相关基因。免疫共沉淀结果显示CBX2与CBX7在F9细胞中存在相互作用 E14TG2a和F9细胞中的靶向性存在差异，揭示了胚胎分化过程中包含不同CBX同源蛋白的PRC1复合体的靶向识别具备特异性。同时，本论文也利用CRISPR/Cas9基因编辑技术敲除Cbx7基因的实验体系进行了摸索；在体外诱导ESC形成类胚体的实验系统中利用形态学观察和qPCR检测方法探究二者在类胚体形成过程中的动态变化规律；以及检测了Cbx7基因的多种转录剪切体的表达及制备了特异性的抗体。这些研究结果为进一步深入探究多梳蛋白在干细胞中的复杂调控机制奠定了一定基础。
Polycomb group protein (PcG) is a series of epigenetic regulatory proteins that regulates transcription of target genes, which mainly functions in the form of protein complexes named Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2). CBX protein family is one of core components of PRC1 and there are five homologous proteins in mammals including CBX2, 4, 6, 7, and 8. PRC1 is known to play an important role in maintaining the stemness of mouse embryonic stem cells (mESC) and CBX7 is a dominant component in this process since PRC1 containing CBX7 is able to bind to and repress other Cbx genes. However, our preliminary results indicated that significant amount of CBX2 was also detectable in mESC although its coding gene was partially repressed by CBX7. Our ChIP-qPCR data suggested that CBX2 shared many targets with CBX7, while further investigation is needed to check whether and how these two homologous proteins bind and work together at a given target gene.
In this study, we analyzed ChIP-Seq data of CBX2 and CBX7 in mESC, classified their target genes into three groups (common targets of CBX2 and CBX7, CBX2 specific targets, and CBX7 specific targets) and performed Gene Ontology (GO) analyses of genes in each class. The results showed that CBX2 and CBX7 shared a large number of target genes genome-wide in mESC, including many pluripotency-related genes, indicating that CBX2 did play an important role in mESC. We further compared the targeting specificity of CBX2 and CBX7 between mouse ESC (E14TG2a cell line) and mouse testicular teratoma cells (F9 cell line) and did see the differences, indicating that the PRC1 targets were dynamically changing upon embryonic development. The molecular interaction of CBX2 and CBX7 was detected by Co-ImmunoPrecipitation (Co-IP) in F9 cells and Sequential Chromatin ImmunoPrecipitation was carried out in mESC. In addition, we initiated the experiment of knocking out Cbx7 gene using CRISPR/Cas9 based gene editing technique. Morphological observation and qPCR detection were performed to detect expression changes of Cbx genes during the in vitro differentiation of mESC into Embryoid bodies. We also analyzed the expression of various transcriptional splicing variants of mouse Cbx7 gene and generated isoform specific antibodies. These findings provide a basis for further study the regulatory mechanisms of PRC1 complex in stem cells.
|卢汉文. 多梳蛋白CBX2和CBX7在哺乳动物细胞中的功能互作及靶向动态性分析[D]. 兰州. 兰州大学,2017.|
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