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: 8745378
• 负责人: Victor Lobanenkov
• 金额: $ 76.58万
• 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/8745378
• 关键词: 2,4-Dinitrophenol; Affect; Alleles; Amino Acids; Apoptosis; Architecture; Attention; Binding; Binding Sites; Biological; Biological Process; Biology; Birds; Brothers; CCCTC-binding factor; Cell Cycle; Cell Proliferation; Cell physiology; Cells; Centrosome; ChIP-seq; Chromatin; Chromatin Loop; Chromosomal Stability; Chromosome Pairing; Chromosome Segregation; Chromosomes; Code; Collaborations; Communication; Complement; Complementary DNA; Complex; Coupled; Coupling; DNA; DNA Binding; DNA Binding Domain; DNA Methylation; DNA Polymerase II; DNA Repair; DNA Sequence; DNA biosynthesis; Data; Defect; Development; Dimerization; Diploid Cells; Drosophila genus; Enhancers; Ensure; Epigenetic Process; Essential Genes; Evolution; Excision; Exons; Fingers; Functional RNA; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Germ; Germ Cells; Haploid Cells; Hormones; Housekeeping; Human; Immune response; In Vitro; Interphase; Introns; Lead; Legal patent; Length; Link; Location; Malignant Neoplasms; Malignant neoplasm of testis; Maps; Mediating; Meiosis; Messenger RNA; Methylation; Mitosis; Mitotic Chromosome; Modification; Mono-S; Multigene Family; Mus; NPM1 gene; Nature; Nuclear; Nuclear Proteins; Pathologic; Pathway interactions; Pattern; Phosphorylation; Play; Point Mutation; Post-Translational Protein Processing; Process; Promoter Regions; Proteins; Publishing; RNA Binding; RNA Splicing; Reading; Regulation; Regulator Genes; Regulatory Element; Repression; Role; Sensory Receptors; Signal Transduction; Sin3A protein; Site; Somatic Cell; Specificity; Stem cells; Structure; Surveys; System; Tandem Repeat Sequences; Time; Transcript; Transcription Initiation; Transcription Initiation Site; Transcriptional Activation; Transcriptional Regulation; Tumor Suppressor Genes; Undifferentiated; Vertebrates; Work; X Inactivation; base; cancer cell; cancer therapy; cell type; cohesin; dosage; early embryonic stage; embryonic stem cell; genome-wide; human disease; imprint; in vivo; novel; novel strategies; nucleophosmin; paralogous gene; promoter; spatiotemporal; stem; tumor

The first US patent issued for CTCF in October 1999 included full length cDNAs sequences coding for exceptionally conserved gene ubiquitously expressed in somatic cells from a wide range of evolutionary divergent species, including Drosophila, birds, mice, and humans. CTCF is unique multifunctional nuclear factor with multiple sequence-specificity which controls global genome architecture and many aspects of allele-specific epigenetic regulation including direct repression and/or activation of transcription, pausing-associated splicing, replication timing, long-range enhancer-promoter communications and hormone-induced silencing. Initially demonstrated by us (MCB 2004) in vitro dimerization between distinct CTCF/DNA complexes was recently shown to mediate universal site-specific intergenic, intra-chromosomal, and inter-chromosomal linking-looping in vivo through interactions between distinct classes of regulatory elements containing varying DNA sequences of CTCF-target-sites (CTSes). We discovered that sequence-specific DNA-bound CTCF-complexes are capable of dimerization in vitro which could serve in vivo to bridge different CTCF-bound sites across nuclear chromatin. Next, we have first demonstrated that upon binding to dissimilar DNA-sites CTCF can thereby function through two major mechanisms: either by direct regulation of a gene downstream of CTSes or by indirect regulation via the formation of chromatin loops stabilized upon CTCF/DNA-dimerization. that affects allele-specific relationships between the promoter, enhancer, and/or an imprinted control region (ICR) on either same or different chromosome. Dimerization of DNA-bound CTCF may potentially be at the core of its activity as a versatile chromatin-bridging and chromatin-looping agent, underlying its fundamental biological functions. The loop-forming activity of CTCF can be naturally extended to include formation of localized somatic inter-chromosome pairing sites that may acquire potential for epigenetic co-regulation through allele-specific transcription factories, DNA replication factories, 5-mC/5-hmC excisions, and DNA repair foci. Many other chromatin-anchored functions, such as the establishment of imprinting marks and their reading, X-chromosome inactivation, and apoptosis, are also regulated by CTCF. CTCF has emerged as a key facilitator of 3D organization of interphase chromatin, as well as a major player in cell proliferation control. In some cases, the loop-forming activity of CTCF was found to be accompanied/complemented by the more direct regulation of a particular gene. This mixed mode regulation is likely the most appropriate representation of a native gene regulation framework. In addition to phosphorylation and poly(ADP)rybozylation of CTCF, we also identified CTCF-interacting partner proteins, Sin3A and YB-1, which have recently gained increased attention in course of ongoing interpretation of the NGS data generated by MPS (in collaboration with Dr. B. Ren) with regard to the genomewide CTCF and CTCFL occupancy, Pol 2 initiation, transcription pausing coupled with splicing, and rewiring regulatory elements of CTCFL positive cancer cells by mobile retrotransposones with variable length tandem repeats (VNTR) affected often by a gain of CTCF site specific SNPs. Moreover, we expanded our previous studies of important CTCF activity that directly links CTCF to transcriptional machinery: the binding of CTCF to the Pol II. This novel pathway is sensitive to external signals that affect post-translational modifications of critical CTCF amino acids and provides either a mechanism for opening loop-independent transcription start sites downstream of the promoter-determined +1 site (at intron/exon sequences)and it may have specifically evolved to induce non-coding transcripts throughout the genome depending on the presence of BORIS in a particular cell type under study. Mechanistically regulated recruitment and the subsequent release of Pol II from a DNA-bound CTCF complex indicates that the CTCF site itself could act as an attenuator and/or promoter in some locations in the genome. At imprinted genes, CTCF likely works together with BORIS (Brother of the Regulator of Imprinted States), the testis/cancer-specific CTCF-paralog that we discovered and characterized (PNAS, 2002). In addition, initiated earlier (Cell 2007) genomewide mapping of CTCF targets by ChIP-Seq with our 9 CTCF Mabs lead to understanding fundamental role of CTCF in spatiotemporal coordination of major cellular functions (Nature, 2011; 2012). By virtue of having so many vital functions CTCF became an essential gene in vertebrates, as CTCF KO mice are non-viable, and early lethality occurs at the very early embryonic stages (PloS One 2012). With respect to human disease, CTCF is a candidate tumor suppressor gene (TSG); several functional point mutations in the 11ZF DBD of CTCF have been characterized in primary cancers, in combination with the LOH of the CTCF locus. In the past year, we studied several genes and their associated regulatory sequences in an effort to elucidate the contributions of CTCF and CTCF binding sites to the regulation of gene expression. These studies included genes important for immune responses, mono-allelic multigene families of sensory receptors, as well as genes with a potential for a breakthrough in the development of new approaches for cancer treatment. Unlike somatic cells, testicular germ cells undergo meiosis rather that mitosis, - whereby CTCF is normally likely to work together with BORIS. Our recent results have identified BORIS as an anti-silencing component of undifferentiated ES and cancer cells that directly interacts with CTCF. We found earlier that functionally important dimerization between two different CTCF/DNA complexes is based on the capability of CTCF to interact with itself that requires Zn-fingers (MCB, 2004), and showed that literally same fingers are uniquely duplicated and preserved in evolution of mouse and human BORIS proteins (PNAS, 2002). Since formation of homodimeric CTCF/CTCF complexes on DNA underlie site-specific long-range interactions that serve for chromatin linking-folding in normal somatic cells (which do not express BORIS), we expect that formation of heterodimeric CTCF/BORIS complexes observed in chromatin of germ/stem and in cancer cells is likely to have many important structure-functional implications for understanding functional consequences of abnormal CTCF/BORIS dimerization, including an altered chromatin packaging mode normally driven by CTCF alone. While CTCF is mostly known as a regulator of gene expression, our data point to its potential functions in nuclear and nucleolar compartmentalization and heterochromatinization, as well as in in formation of centrosomes. We have also characterized an unusual form of CTCF protein in condensed mitotic chromosomes pointing to its roles in mitosis and meiosis, and thereby suggesting a significant housekeeping role of CTCF in spatiotemporal coordination of genome organization and chromosome segregation in dividing diploid and haploid cells. CTCF was previously shown to undergo a variety of post-translational modifications and we expanded these studies to characterize novel modifications. Another pathological aspect of the misregulated CTCF occupancy on promoter target sites is aberrant alteration of DNA methylation pattern at CTCF sites that can loose protection by DNA-bound CTCF in cancer. Both of these novel biological roles of CTCF are subjects of ongoing studies in the MPS. We also have obtained direct in vivo evidence that at least one role of BORIS is to facilitate DNA re-methylation and Pol 2 recruitment at certain intergenic CTS-sequences found in intrones and promoters of two cancer/testis genes as we published recently.

1999年10月，为CTCF颁发的第一份美国专利包括编码的全长cDNA序列，该序列编码为从广泛的进化发散物种的体细胞中表达的异常保守的基因，包括果蝇，鸟类，小鼠，小鼠和人类。 CTCF是具有多种序列特异性的独特多功能核因子，它控制着全球基因组结构以及等位基因特异性表观遗传调控的许多方面，包括直接抑制和/或激活转录，暂停相关的拼接，复制时间，远距离增强剂增强器 - 增强器 - 促进剂 - 促进剂通讯和激素诱导的沉积物。 最近，美国（MCB 2004）在不同的CTCF/DNA复合物之间的体外二聚变最初证明，最近证明可以通过包含不同类别的ctcf-s-s-s-s-s-s-ctcf-target（CTCFTARGET）的不同类别的相互作用（通过含有不同类别的类别的相互作用）（通过体内相互作用）（通过体内相互作用）（染色体链接 - 在体内）中介导普遍的染色体链接循环。我们发现，序列特异性DNA结合的CTCF复合物能够在体外二聚化，可以在体内用于跨核染色质的不同CTCF结合的位点。接下来，我们首先证明，在与不同的DNA位点结合后，CTCF可以通过两种主要机制进行功能：通过直接调节CTSES下游基因，或通过通过在CTCF/DNA降解上稳定的染色质循环而形成的染色质循环通过间接调节。 这会影响启动子，增强子和/或在相同或不同染色体上的印迹控制区（ICR）之间的等位基因特异性关系。 DNA结合的CTCF的二聚化可能是其活性的核心，它是一种多功能染色质桥和染色质循环剂，其基本的基本生物学功能是基本的。 CTCF的循环形成活性可以自然扩展到包括形成局部体细胞间染色体配对位点，这些位点可能会通过等位基因特异性转录工厂，DNA复制工厂，5-MC/5-HMC ICCINITY和DNA维修灶。 CTCF也调节了许多其他染色质锚定功能，例如建立印迹痕迹及其阅读，X染色体灭活和凋亡。 CTCF已成为3D组织相间染色质组织的关键促进者，也是细胞增殖控制的主要参与者。在某些情况下，发现CTCF的循环形成活性伴随/补充了特定基因的更直接调节。这种混合模式调节可能是天然基因调节框架的最合适的表示。 In addition to phosphorylation and poly(ADP)rybozylation of CTCF, we also identified CTCF-interacting partner proteins, Sin3A and YB-1, which have recently gained increased attention in course of ongoing interpretation of the NGS data generated by MPS (in collaboration with Dr. B. Ren) with regard to the genomewide CTCF and CTCFL occupancy, Pol 2 initiation, transcription pausing coupled通过移动逆转录座酮的剪接和重新布线CTCFL阳性癌细胞的调节元件，其长度串联重复序列（VNTR）经常受到CTCF位点特定SNP的增益的影响。此外，我们扩展了对重要CTCF活性的先前研究，该研究将CTCF直接连接到转录机械：CTCF与POL II的结合。这种新的途径对影响关键CTCF氨基酸的翻译后修饰的外部信号敏感，并且提供了一种机制，是开放循环独立的转录起始站点下游的启动子+1位点（在内含子/外显子序列上）下游的机制（内含子/外显子序列），并且可以在整个基础上诱导非编码的转录组，以诱导非编码的转录组。 机械调节的募集以及随后从DNA结合的CTCF复合物中释放POL II表明CTCF位点本身可以​​在基因组的某些位置充当衰减剂和/或启动子。在印迹基因上，CTCF可能与鲍里斯（印刷状态的调节者的兄弟）一起工作，即我们发现和表征的睾丸/癌症特异性CTCF-PARALOG（PNAS，2002）。此外，通过我们的9个CTCF mAbs对CTCF靶标的较早（Cell 2007）范围（Cell 2007）绘制了CTCF靶标，从而使CTCF在主要细胞功能的时空配位中的基本作用中理解了基本作用（自然，2011年; 2012年）。 由于具有如此多的重要功能，CTCF成为脊椎动物中的必不可少的基因，因为CTCF KO小鼠是不可行的，并且早期致死性发生在非常早期的胚胎阶段（PLOS One 2012）。 关于人类疾病，CTCF是候选肿瘤抑制基因（TSG）。 CTCF的11ZF DBD中的几个功能点突变已在初级癌症中与CTCF基因座的LOH结合使用。在过去的一年中，我们研究了几个基因及其相关的调节序列，以阐明CTCF和CTCF结合位点对基因表达调节的贡献。这些研究包括对免疫反应，感官受体的单相多基因家族重要的基因，以及具有突破性新方法的癌症治疗方法的潜力的基因。 与躯体细胞不同，睾丸生殖细胞经历减数分裂，而不是有丝分裂，因此CTCF通常很可能与鲍里斯一起工作。我们最近的结果将Boris确定为直接与CTCF相互作用的未分化ES和癌细胞的抗沉淀成分。 我们早些时候发现，两个不同的CTCF/DNA复合物之间在功能上重要的二聚化是基于CTCF与需要Zn-Fingers相互作用的能力（MCB，2004），并表明实际上相同的手指在小鼠和人类硼蛋白的演化中具有独特的重复和保存（PNAS，PNAS，2002年）。 由于在DNA基础基础位点特异性的长距离相互作用上形成同二聚体CTCF/CTCF复合物，这些相互作用用于正常体细胞中的染色质链接（不表达骨），因此我们期望在癌细胞中观察到的异性CTCF/鲍里斯复合物的杂种含量可能是许多重要的结构。 CTCF/Boris二聚化，包括通常仅由CTCF驱动的改变的染色质包装模式。虽然CTCF通常被称为基因表达的调节剂，但我们的数据表明其在核和核仁分区化和异染色性化以及中心体形成中的潜在功能。我们还表征了CTCF蛋白在凝结有丝分裂染色体中的一种异常形式，该染色体指向其在有丝分裂和减数症中的作用，从而表明CTCF在基因组组织的时空配位中具有重要的家政作用，并在基因组组织和染色体分离中在二倍体二倍体和染色体分离中发挥了重要作用。 先前显示CTCF经历了各种翻译后修饰，我们扩大了这些研究以表征新颖的修饰。启动子目标部位上CTCF占用率不正常的另一个病理方面是CTCF部位的DNA甲基化模式的异常改变，可以通过癌症中DNA结合CTCF的保护放宽保护。 CTCF的这两个新型生物学作用都是MPS中正在进行的研究的主题。我们还获得了直接的体内证据，表明鲍里斯的至少一种作用是在某些基因间CTS序列中促进DNA再甲基化和POL 2募集，如我们最近发表的两个癌症/睾丸基因的启动子和启动子中发现的。