Normal and Pathologic Functions of CTCF and Its Distinct Classes of DNA-targets

CTCF 的正常和病理功能及其不同类型的 DNA 靶标

• 批准号: 7732551
• 负责人: VICTOR LOBANENKOV
• 金额: $ 73.07万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732551
• 关键词: 16q22; 20q13; Adult; Alleles; Apoptosis; Binding; C-terminal; CCCTC-binding factor; Cell Proliferation; Cells; Chromatin; Chromatin Loop; Chromosome Mapping; Chromosomes; Class; Code; Complex; DNA; DNA Binding; DNA Binding Domain; DNA Methylation; Defect; Development; Drosophila genus; Ectopic Expression; Embryo; Embryonic Development; Enhancers; Exons; Fingers; Functional RNA; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Hormones; Hot Spot; Human; Knock-out; Malignant Neoplasms; Malignant neoplasm of testis; Maps; Mediating; Meiosis; Messenger RNA; Methylation; Microinjections; Mitolactol; Mitotic; Modification; Mono-S; Mus; Mutation; Normal Cell; Nuclear; Nucleic Acids; Nucleosomes; Numbers; Oocytes; Pan Genus; Pathologic; Pathway interactions; Play; Point Mutation; Positioning Attribute; Property; Proteins; RNA Interference; Range; Recurrence; Regulation; Reporter; Repression; Role; Site; Somatic Cell; Specificity; Staging; System; Technology; Time; Tissues; Transcript; Transcriptional Activation; Transgenic Organisms; Tumor Suppressor Genes; Up-Regulation; Ursidae Family; Virus; Work; base; blastocyst; cohesin; demethylation; derepression; egg; in vivo; nuclease; nucleophosmin; pp 135; promoter; research study; size; tumor

CTCF is a highly conserved, multi-functional nuclear factor involved in many aspects of gene regulation ranging from gene activation or repression to enhancer blocking and hormone regulated silencing. CTCF-proteins in many species, including Drosophila, have similar size and contain central highly conserved 11 Zn-finger DNA-binding domain that mediates multiple sequence-specificity of DNA binding flanked by N- and C-terminal fragments of roughly equal size. By making inter- and intra-chromosomal loops through selective interactions between various DNA-bound CTCF molecules at different genomic targets, CTCF can act as a universal and versatile component of chromatin-insulators and boundaries for spreading of nucleosome modifications, of non-coding intergenic transcripts, and of bi- and/or mono- allelic regional DNA methylation. It can also function as a promoter-proximal repressor or activator. Depending on the context, CTCF may also couple DNA-looping with activity of transcriptional enhancers. Genome-wide in-vivo mapping of tens of thousands of DNA/CTCF-complexes showed that CTCF can position nucleosomes around nuclease hypersensitive sites that landmark insulators, enhancers, and other regulatory sequences. The featuring of so many functions suggested that CTCF has important interacting partners (ranging from B23/nucleophosmin to YY1 and cohesins) and that it is an essential factor. Indeed, CTCF knockout in mice is lethal at early stages of embryo development. However, expression of ectopic CTCF results in a profound negative regulation of proliferation, indicating that CTCF may be a tumor suppressor gene (TSG). Human CTCF maps within 16q22 region of recurrent LOH in many tumors. Accordingly, several functional point mutations in the 11ZF DBD of CTCF have been characterized in tumors selected for the loss of the second CTCF allele. Given the importance of CTCF for development, cell proliferation, etc., we not only analyzed genome wide CTCF targets for the first time (Kim TH, Abdullaev ZK, Smith AD, et al., Cell 2007, vol. 128, pp1231-1245) but also studied the in vivo roles of CTCF in adult tissues and during embryonic development. We depleted maternal stores of CTCF from growing mouse oocytes using transgenic RNAi technology, and identified hundreds of misregulated genes. Moreover, our analysis suggests that CTCF predominantly activates or derepresses transcription in oocytes. CTCF depletion causes meiotic defects in the egg, and mitotic defects in the embryo that are accompanied by defects in zygotic gene expression, and culminate in apoptosis. Maternal pronuclear transfer and CTCF mRNA microinjection experiments indicate that CTCF is a mammalian maternal effect gene, and that persistent transcriptional defects rather than persistent chromosomal defects perturb early embryonic development. This is the first study detailing a global and essential role for CTCF in mouse oocytes and preimplantation embryos (Wan LB, Pan H, Hannenhalli S, et.al., Development 2008 vol.135, pp. 2729-2738). We also detailed hTERT up-regulation by methylation of the first exon CTCF methylation sensitive repressive site. We showed that in many cancers as well as in the reporter system methylation of exon 1 CTCF site results in activation of hTERT gene.This observation was rather unexpected as usually DNA methylation is associated with gene inactivation (Renaud S, Loukinov D, Abdullaev Z, et. al., Nucleic Acids Res 2007 vol. 35, pp.1245-56). Finally, we discovered role of CTCF as repressor of all three promoters of human BORIS gene. We showed that blocking of CTCF in normal cells results in demethylation and derepression of BORIS promoters. We identified a number of CTCF sites in 5non-coding region of BORIS gene. Our results provide a basis for understanding of functional connection between a lessening of the strictness of BORIS silencing in somatic cells with haploinsufficiency of CTCF observed in cancers. (Renaud S, Pugacheva EM, Delgado MD, et.al. Nucleic Acids Res, 2007, vol. 35, pp.7372-7388).

CTCF是一种高度保守的多功能核因子，参与基因调控的许多方面，从基因激活或抑制到增强子阻断和激素调节的沉默。包括果蝇在内的许多物种中的CTCF蛋白具有相似的大小，并且含有中心高度保守的11个Zn指DNA结合结构域，其介导DNA结合的多个序列特异性，两侧是大致相等大小的N-和C-末端片段。通过在不同基因组靶标处的各种DNA结合的CTCF分子之间的选择性相互作用来形成染色体间和染色体内环，CTCF可以充当染色质绝缘体和边界的通用和多功能组分，用于核小体修饰、非编码基因间转录物和双和/或单等位基因区域DNA甲基化的扩散。它也可以作为启动子近端阻遏物或激活物发挥作用。根据上下文，CTCF还可以将DNA环与转录增强子的活性偶联。数万个DNA/CTCF复合物的全基因组体内图谱显示，CTCF可以将核小体定位在核酸酶超敏位点周围，这些位点标志着绝缘子、增强子和其他调节序列。CTCF具有多种功能，提示其与B23/核磷蛋白、YY 1和cohesins等分子有重要的相互作用，是一个重要的调节因子。事实上，CTCF基因敲除小鼠在胚胎发育的早期阶段是致命的。然而，异位CTCF的表达导致增殖的深刻的负调控，表明CTCF可能是肿瘤抑制基因（TSG）。人CTCF定位于许多肿瘤复发性洛缺失的16 q22区域。因此，CTCF的11 ZF DBD中的几个功能性点突变已经在选择为第二CTCF等位基因缺失的肿瘤中表征。鉴于CTCF对发育、细胞增殖等的重要性，我们不仅首次分析了全基因组CTCF靶（KimTH，AbdullaevZK，SmithAD，等，Cell 2007，第128卷，第1231 -1245页），而且还研究了CTCF在成体组织和胚胎发育期间的体内作用。我们使用转基因RNAi技术从生长中的小鼠卵母细胞中耗尽了CTCF的母体储存，并鉴定了数百个失调基因。此外，我们的分析表明，CTCF主要激活或解抑制卵母细胞中的转录。 CTCF缺失导致卵中的减数分裂缺陷，以及伴随合子基因表达缺陷的胚胎中的有丝分裂缺陷，并最终导致细胞凋亡。母体原核转移和CTCF mRNA显微注射实验表明，CTCF是哺乳动物的母体效应基因，持续的转录缺陷，而不是持续的染色体缺陷扰乱早期胚胎发育。这是第一项详细说明CTCF在小鼠卵母细胞和植入前胚胎中的全局和重要作用的研究（Wan LB，Pan H，Hannenhalli S，et.al.，《2008》第135卷，页。2729-2738）。我们还详细描述了第一外显子CTCF甲基化敏感抑制位点的甲基化对hTERT的上调作用。我们发现，在许多癌症中以及在报道系统中，外显子1CTCF位点的甲基化导致hTERT基因的活化。例如，Nucleic Acids Res 2007第35卷，第1245 -56页）。 最后，我们发现CTCF作为人BORIS基因的所有三个启动子的阻遏物的作用。我们发现，在正常细胞中阻断CTCF会导致BORIS启动子的去甲基化和去抑制。在BORIS基因的5个非编码区中发现了多个CTCF位点。我们的研究结果为理解体细胞中BORIS沉默的严格性降低与癌症中观察到的CTCF单倍不足之间的功能联系提供了基础。（Renaud S，Pugacheva EM，Delgado MD等人，Nucleic Acids Res，2007，第35卷，第7372 -7388页）。

项目成果

CTCF binds the proximal exonic region of hTERT and inhibits its transcription.

• DOI: 10.1093/nar/gki989
• 发表时间: 2005
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Renaud S;Loukinov D;Bosman FT;Lobanenkov V;Benhattar J
• 通讯作者: Benhattar J

Dynamic association of the mammalian insulator protein CTCF with centrosomes and the midbody.

哺乳动物绝缘体蛋白 CTCF 与中心体和中体的动态关联。

• DOI: 10.1016/j.yexcr.2003.11.015
• 发表时间: 2004
• 期刊: Experimental cell research
• 影响因子: 3.7
• 作者: Zhang,Ru;Burke,LesJ;Rasko,JohnEJ;Lobanenkov,Victor;Renkawitz,Rainer
• 通讯作者: Renkawitz,Rainer

Expression of the CTCF-paralogous cancer-testis gene, brother of the regulator of imprinted sites (BORIS), is regulated by three alternative promoters modulated by CpG methylation and by CTCF and p53 transcription factors.

CTCF - 多个癌症测试基因的表达是印迹位点调节剂（Boris）的兄弟，受到CPG甲基化调节的三个替代启动子的调节，由CPG甲基化和CTCF和p53转录因子调节。

• DOI: 10.1093/nar/gkm896
• 发表时间: 2007
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Renaud, Stephanie;Pugacheva, Elena M;Delgado, M Dolores;Braunschweig, Richard;Abdullaev, Ziedulla;Loukinov, Dmitri;Benhattar, Jean;Lobanenkov, Victor
• 通讯作者: Lobanenkov, Victor

Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene.

• DOI: 10.1093/nar/gkl1125
• 发表时间: 2007
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Renaud, S.;Loukinov, D.;Abdullaev, Z.;Guilleret, I.;Bosman, F. T.;Lobanenkov, V.;Benhattar, J.
• 通讯作者: Benhattar, J.