PML通过转录调控CTCF影响C-Myc表达的机制研究

该研究探讨了PML(promyelocytic leukemia protein)对多功能转录因子CTCF(CCCTCbinding factor)的转录调控及其下游靶基因C-Myc的功能影响。通过荧光定量PCR方法分析儿童急性淋巴细胞白血病样本中PML和CTCF基因的表达水平,并分析二者之间的相关性。构建了PML与CTCF真核表达载体,在共转染细胞中,利用荧光定量PCR方法和Western blot分析PML对CTCF表达水平的影响。通过双荧光素报告基因系统分析了PML对CTCF启动子区的调控,并研究了其对CTCF下游靶基因C-Myc的表达水平的影响。结果显示,在临床样本中PML与CTCF表达水平存在负相关性,PML通过抑制CTCF的转录降低CTCF的表达水平,干扰CTCF对下游靶基因C-Myc的调控功能。同时,在CTCF的启动子区域可能存在着PML的结合区域,从而导致CTCF的启动子活性被抑制。     

利用RNA干扰抑制HeLa细胞中CTCF基因的表达

目的:CTCF是一种多功能的转录因子,参与启动子调控、绝缘子功能和遗传印记等过程。利用RNA干扰技术抑制CTCF在体细胞中的表达。方法:以CTCF为靶基因,利用ShortCutRNaseⅢ切割体外转录的长双链RNA制备esiRNA,转染HeLa细胞,用RTReal-TimePCR检测CTCF的mRNA水平,用免疫印迹检测CTCF水平。结果:转染CTCFesiRNA后,细胞内的CTCFmRNA被抑制了70%左右,CTCF水平明显下降。结论:CTCF的esiRNA可以明显抑制CTCF在HeLa细胞中的表达,从而为进一步研究CTCF的未知功能打下了基础。     

CCCTC结合因子在HeLa细胞中调控X染色体连锁的凋亡抑制蛋白的表达

目的:确定HeLa细胞的CCCTC结合因子(CTCF)表达水平是否与细胞的抗凋亡能力相关,并研究其具体分子机制。方法:用顺铂和阿糖胞苷分别诱导HeLa细胞和CTCF敲降的HeLa-CTCF-II-11细胞凋亡,比较两者的凋亡率;用基因表达芯片检测CTCF敲降后HeLa细胞的表达谱变化,寻找并验证受CTCF调控的与凋亡相关的蛋白。结果:用顺铂和阿糖胞苷诱导后,HeLa-CTCF-II-11细胞的凋亡率显著高于HeLa细胞;HeLa细胞的CTCF敲降后,X染色体连锁的凋亡抑制蛋白(XIAP)的表达显著下降。结论:CTCF敲降使HeLa细胞的抗凋亡能力下降,CTCF对XIAP基因的表达调控在这个过程中起了重要作用。     

CCCTC-结合因子与核周蛋白α4存在分子间相互作用并调控其基因表达

目的:研究CCCTC-结合因子(CTCF)是否与核周蛋白α4(KPNA4)有蛋白间相互作用并调控其表达。方法:用GSTpull-down实验研究CTCF是否与KPNA4存在蛋白间相互作用;用逆转录半定量PCR和免疫印迹检测CTCF敲降对KPNA4基因表达的影响。结果:GSTpull-down实验表明CTCF与KPNA4之间存在相互作用;当CTCF敲降时,KPNA4基因的表达随之下降。结论:CTCF与KPNA4之间存在相互作用并调控其基因表达。     

利用CRISPR/Cas9技术构建CTCF蛋白降解细胞系

CTCF是脊椎动物关键的绝缘子蛋白,在细胞生命过程中发挥重要作用,敲除CTCF基因会导致小鼠胚胎死亡。为进一步探讨CTCF的功能,本文利用CRISPR/Cas9介导的同源重组,在内源性CTCF表达框上游敲入一个有丝分裂期降解结构域(Mitosis-special degradation domain, MD),该结构域可以带动CTCF融合蛋白在M期降解。作为对照,将MD结构域的第42位的精氨酸突变为丙氨酸,形成无降解活性的MD^*,可使MD^*-CTCF融合蛋白始终稳定存在。将嘌呤霉素与融合蛋白同时表达,即可利用抗生素筛选,高效地筛选到纯合克隆。利用蛋白印迹技术和免疫荧光检测3种细胞在不同细胞周期的CTCF蛋白变化情况,发现MD-CTCF细胞系CTCF蛋白含量约为野生型细胞的10%,MD^*-CTCF细胞系的CTCF含量与野生型没有显著差别;通过流式细胞术观测降解CTCF对细胞的影响,发现MD-CTCF细胞系G1期明显延长。总之,利用CRISPR/Cas9技术在CTCF表达框上游高效地插入MD,首个CTCF特异性降解的人类细胞系获得成功构建。     

CTCF和cohesin参与人成纤维细胞中HOXA基因簇染色质高级构象的组织

位于人体不同部位的成纤维细胞具有细胞特异性的HOX基因表达模式,可以作为区分不同成纤维细胞的依据之一.在个体发育的过程中,建立或维持不同HOX基因表达模式的机制始终是引人关注的问题.本实验室前期工作在NT2/D1人畸胎瘤细胞中证明了CTCF/cohesin介导的染色质高级构象在维甲酸诱导的HOXA基因共线性开启过程中发挥了重要作用.为了进一步研究原代细胞中CTCF/cohesin对HOXA基因的调控作用,本研究选取了来自体轴不同部位并且HOXA基因表达模式互补的人胚肺和包皮成纤维细胞,对HOXA基因簇中CTCF和cohesin的结合水平以及相关的染色质高级构象进行了检测.与人胚肺成纤维细胞相比,包皮成纤维细胞中的cohesin结合水平较低,相关的染色质高级构象比较"开放",并且主要表达5′端的HOXA基因.本研究还发现CTCF结合位点CBSA56处于HOXA基因簇染色质高级构象中的核心位置,并且该位点参与的染色质相互作用在两种成纤维细胞中呈现出明显的差异,说明CBSA56是一个关键的CTCF结合位点.以上结果表明,CTCF和cohesin参与了人原代成纤维细胞中HOXA基因簇染色质高级构象的组织和HOXA基因的表达调控,并且提示细胞类型特异性的染色质高级构象与HOXA基因的空间共线性表达模式之间存在协同关系.     

串联反向CTCF位点的系列删除揭示增强子调控HOXD基因簇表达的平衡

三维基因组染色质架构蛋白CTCF(CCCTC-bindingfactor)能够介导增强子与基因启动子的远距离染色质相互作用,也可以结合调控区域的绝缘子发挥增强子绝缘功能,对发育中的基因表达调控具有重要作用。同源框基因家族(Homeoboxgenefamily,Hox)编码一类控制动物发育的关键转录因子,在发育中主要沿胚胎首尾轴(head-to-tail axis)呈时空线性表达。在哺乳动物中,Hox基因分为HoxA、HoxB、HoxC和HoxD四个基因簇,在中枢神经系统、骨骼和四肢发育中发挥重要功能。HoxD基因簇主要调控四肢发育,受位于其两侧调控域内的增强子调节,沿肢体近远轴(proximal-distal axis)呈时空线性表达。在人类基因组中,HOXD基因簇及其两侧的调控区域分布有串联排列的CTCF结合位点(简称CTCF位点),参与9个HOXD基因的表达调控。本研究以HOXD基因簇为模式基因,探究CTCF对发育基因(developmentalgenes)转录调控的影响。利用CRISPRDNA片段编辑技术在人HEK293T细胞中获得一系列的串联反向CTCF位点删除的单细胞克隆株。RNA-seq实验揭示CTCF位点删除后HOXD基因表达下降。定量高分辨率染色体构象捕获实验显示,HOXD与上游增强子簇的远距离染色质相互作用增强,与下游增强子簇的远距离染色质相互作用减弱。综上所述,串联反向的CTCF位点通过其绝缘子功能维持上下游增强子簇对HOXD基因簇表达调控的平衡,为探究动物发育过程中Hox基因表达的精准调控机制提供参考。     

CCCTC-结合因子的表达水平影响其在细胞内的分布

目的:研究CCCTC-结合因子(CTCF)的表达水平与其在HeLa和HepG2细胞内分布的关系。方法:利用小干扰RNA敲降CTCF的表达;利用免疫荧光染色检测CTCF在细胞内的分布。结果:在HeLa和HepG2细胞中下调CTCF的表达,检测到CTCF在细胞核内的分布比例减少,而在细胞质内的分布比例相应增加。结论:CTCF的表达水平会影响其在细胞内的分布。     

敲低CTCF腺病毒表达载体的构建及效果检测

目的:获得敲低效果较好的CCCTC结合因子(CTCF)的RNA干扰腺病毒载体,以便于研究其在肿瘤发生发展中的作用。方法:从已发表文献中获得CTCF敲低靶序列,合成2对含有小发卡结构的寡核苷酸序列,将其进行退火磷酸化后,分别克隆到腺病毒包装载体上;将重组质粒转染人胚肾293A细胞,收获腺病毒;将收获的腺病毒分别感染人胚肾HEK293细胞和靶细胞人肺腺癌细胞A549,通过RT-PCR和Western印迹鉴定相关基因的表达变化。结果与结论:RT-PCR和Western印迹鉴定显示构建的表达CTCF短发夹RNA(shRNA)的腺病毒载体能够有效抑制CTCF转录和蛋白水平,为后续CTCF的生物学功能和机制研究奠定了基础。     

小鼠M1细胞中ctcf基因的敲降及其对c-myb基因表达的影响

c-Myb转录因子能够调控造血细胞的分化和增殖,其转录调控受到多种机制影响。本实验室前期研究发现在小鼠急性髓系白血病M1 (Mouse myeloid leukemia)细胞中CTCF结合在c-myb基因上游调控区域,但是具体机制仍然不清楚。为了探索CTCF对c-myb基因转录调控的影响,本研究设计了针对小鼠ctcf基因的shRNA (Small hairpin RNA)干扰序列,并将其连接到载体pLKO.1中,得到ctcf-shRNA慢病毒干扰载体,测序验证后,与慢病毒包装质粒pCMV-DR8.91、pCMV-VSVG共转染至HEK293T细胞中,经HEK293T细胞包装病毒感染小鼠M1细胞,通过RT-qPCR和Western blotting实验分别检测ctcf基因被干扰后,c-myb基因在mRNA水平和蛋白水平的表达变化。RT-qPCR实验结果表明,含有ctcf-shRNA序列的慢病毒感染M1细胞后,ctcf基因mRNA表达量与对照组相比明显下降,同时检测到ctcf基因被敲降之后,c-myb基因的mRNA表达随之降低,Western blotting实验结果表明,当ctcf基因的表达被干扰之后,c-Myb蛋白的表达也明显下降。本研究发现c-myb基因的表达受到CTCF的调控,为进一步研究c-myb基因的表达调控机制提供实验依据。     

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.     

腺相关病毒介导的RNA干扰抑制HeLa细胞中CTCF的表达

目的:利用RNA干扰(RNAi)稳定抑制HeLa细胞中CTCF的表达。方法:构建能够抑制转录因子CTCF表达的短发夹RNA(shRNA)的腺相关病毒载体,重组病毒感染HeLa细胞后挑取单克隆,用Western印迹法和实时荧光定量PCR检测CTCF的表达水平。结果:获得3株CTCF被显著抑制的HeLa细胞株,Western印迹法和实时荧光定量PCR结果均显示CTCF的表达水平被抑制,其中最明显的被下调76%。结论:shRNA病毒表达载体构建成功,HeLa细胞中CTCF的表达可被长期稳定地抑制。     

Perinuclear positioning of the inactive human cystic fibrosis gene depends on CTCF, A-type lamins and an active histone deacetylase

The nuclear positioning of mammalian genes often correlates with their functional state. For instance, the human cystic fibrosis transmembrane conductance regulator (CFTR) gene associates with the nuclear periphery in its inactive state, but occupies interior positions when active. It is not understood how nuclear gene positioning is determined. Here, we investigated trichostatin A (TSA)-induced repositioning of CFTR in order to address molecular mechanisms controlling gene positioning. Treatment with the histone deacetylase (HDAC) inhibitor TSA induced increased histone acetylation and CFTR repositioning towards the interior within 20 min. When CFTR localized in the nuclear interior (either after TSA treatment or when the gene was active) consistent histone H3 hyperacetylation was observed at a CTCF site close to the CFTR promoter. Knockdown experiments revealed that CTCF was essential for perinuclear CFTR positioning and both, CTCF knockdown as well as TSA treatment had similar and CFTR-specific effects on radial positioning. Furthermore, knockdown experiments revealed that also A-type lamins were required for the perinuclear positioning of CFTR. Together, the results showed that CTCF, A-type lamins and an active HDAC were essential for perinuclear positioning of CFTR and these components acted on a CTCF site adjacent to the CFTR promoter. The results are consistent with the idea that CTCF bound close to the CFTR promoter, A-type lamins and an active HDAC form a complex at the nuclear periphery, which becomes disrupted upon inhibition of the HDAC, leading to the observed release of CFTR.     

The role of CTCF in coordinating the expression of single gene loci

The CCCTC-binding factor (CTCF), which binds insulator elements in vertebrates, also facilitates coordinated gene expression at several gene clusters, including the β-globin, Igf2/H19 (insulin like growth factor 2/H19 noncoding RNA), and major histocompatibility complex (MHC) class II loci. CTCF controls expression of these genes both by enabling insulator function and facilitating higher order chromatin interactions. While the role of CTCF in gene regulation is best studied at these multi-gene loci, there is also evidence that CTCF contributes to the regulated expression of single genes. Here, we discuss how CTCF participates in coordinating gene expression at the CFTR (cystic fibrosis transmembrane conductance regulator) and IFNG (interferon-gamma) loci. We consider the structural similarities between the loci with regard to CTCF-binding elements, the possible interaction between nuclear receptors and CTCF, and the role of CTCF in chromatin looping at these genes. These comparisons reveal a functional model that may be applicable to other single-gene loci that require CTCF for coordinated gene expression.