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: 7964430
• 负责人: Victor Lobanenkov
• 金额: $ 57.84万
• 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/7964430
• 关键词: 16q22; Architecture; Area; B-Cell Development; B-Lymphocytes; Binding; Binding Sites; Biological; Biological Assay; Biology; CCCTC-binding factor; CTCF Pathway; Cell Lineage; Cell Proliferation; Cell physiology; Cells; Centromere; Cervix carcinoma; Chromatin; Chromatin Loop; Chromatin Structure; Chromosomal Stability; Chromosome Segregation; Chromosomes; DNA; DNA Binding; DNA Binding Domain; DNA Methylation; Data; Defect; Deoxyribonucleases; Development; Dimerization; Distant; Down-Regulation; Drosophila genome; Drosophila genus; EP300 gene; Elements; Enhancers; Ensure; Epigenetic Process; Essential Genes; Exons; Fingers; Gene Expression; Gene Expression Regulation; Gene Order; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Hela Cells; Heterochromatin; Hormones; Housekeeping; Human; Human Cell Line; Human Genome; Immune response; Individual; Insulator Elements; Investigation; K-562; Knockout Mice; Literature; Lymphocyte Function; Lymphoid Cell; Malignant Neoplasms; Maps; Mediating; Meiosis; Mitosis; Mono-S; Mus; Nuclear; Nucleosomes; PAX5 gene; Pathologic; Pathway interactions; Pattern; Plasmids; Play; Point Mutation; Positioning Attribute; Proteins; Publications; Recurrence; Regulation; Regulator Genes; Repression; Research; Role; Site; Somatic Cell; Specificity; TERT gene; Telomerase; Time; Transcriptional Regulation; Transgenes; Transgenic Organisms; Tumor Suppressor Genes; Vertebrates; Work; Zinc Fingers; cancer cell; cell immortalization; cell transformation; cell type; cohesin; derepression; design; dosage; early embryonic stage; embryonic stem cell; follow-up; gene repression; genome wide association study; genome-wide; genome-wide analysis; histone modification; human disease; imprint; in vivo; leukemia; lymphoblast; mammalian genome; novel; nucleophosmin; prevent; programs; promoter; theories; transcription factor; tumor; vertebrate genome

CTCF is a highly conserved, multi-functional nuclear factor involved both in global genome architecture and in many aspects of gene regulation, latter ranging from the direct gene repression/activation to enhancer blocking and hormone-facilitated silencing. CTCFs from evolutionary distant species all contain a central highly conserved 11 Zn-finger DNA-binding domain, which mediates the multiple sequence specificity of its DNA binding activity. Dimerization activity of DNA-bound CTCF may potentially be at the core of its activity as a versatile chromatin-bridging and chromatin-looping agent in most cell types, underlying its chromatin-insulator and heterochromatin-boundary functions. In the context of specific genes, CTCF may also functionally modulate transcriptional enhancers via chromatin-looping. Genome-wide mapping of tens of thousands of CTCF target sites (CTS) showed that CTCF can position nucleosomes around DNAse hypersensitive sites that landmark insulators, enhancers, and other regulatory sequences. By virtue of having so many vital functions CTCF became an essential gene in vertebrates, as CTCF-knockout mice are non-viable (lethality at the early embryonic stages). With respect to human disease, CTCF is a well-established tumor suppressor gene (TSG); several functional point mutations in the 11ZF DBD of CTCF have been characterized in primary cancers, in tumors initially characterized by the LOH of the CTCF locus. (1) In the past year, genome-wide analyses have led to significantly deeper understanding of the global role of CTCF in genomes of vertebrates. It is now clear that CTCF controls at least three major pathways: the global architecture of the genome, the chromatin-structure-mediated (loops formation, enhancer-blocker activity) regulation of gene expression (of both imprinted and imprinting-independent genes), and the direct regulation of gene expression (through interface with other transcription factors). We previously analyzed genome-wide CTCF targets for the first time (Cell 2007, vol. 128, pp1231-1245), and the fundamental roles of CTCF in cellular functions were validated by a strong correlation of CTCF target sites (CTS) with gene positions in human genome. Therefore, we developed a comprehensive program to expand genome-wide studies, in order to identify positive correlations and, eventually, functional significances of CTCF (and BORIS) binding at the individual chromosomal loci. We performed ChIP-chip analysis to map CTS, as well as the p300 coactivator, and compared them to histone modification patterns in 5 human cell lines: cervical carcinoma (HeLa), immortalized lymphoblast GM06690 (GM), leukaemia K562, embryonic stem cells (ES), and BMP4-induced ES cells (dES). We used the ENCODE microarrays (1% of the human genome) to identify putative CTCF-bound sites for these cell types and observed highly reproducible CTCF occupancy (in contrast to largely cell-type specific histone modifications and p300 binding), confirming our theory that the bulk of CTCF functions in the cell is cell-type independent. At the same time, a subset of the CTS that appeared to be cell-type-dependent, confirmes that in addition to its global function in the human genome, CTCF may be directly involved in the regulation of cell lineage, possibly through acting at few specific enhancers or at alternative (intragene) promoters. (2) We also conducted genome-wide DNA-binding analysis of Drosophila CTCF (DrCTCF), which we previously cloned and characterized. We identified more than 3561 strong DrCTS (as well as 8872 weaker ones, with two-fold lower enrichment) genome-wide. Whole-genome analysis showed that DrCTCF in general was often found to bind between genes that are closely positioned but differentially regulated. However, DrCTS were also highly enriched to the 5′ of genes, which did not have closely-positioned neighboring promoters. In contrast, distribution of predicted Su(Hw) insulator sites did not display any bias towards promoters. Therefore, it is likely that, in addition to its insulator function, DrCTCF binding upstream of promoters might have a more general role: either in direct regulation of transcription or/and in global genome organization of Drosophila genome. In a specific case, as a result of this genome-wide analysis, the Fab-6 insulator element from the Abd-B locus was identified as a new strong drCTCF binding site (with two CTS), and was shown (using specially designed transgenic and plasmid EB assays) to be a new CTCF-controlled EB element. Follow-up results indicate that DrCTCF is essential for the enhancer blocking activity of the Fab-6, in addition to the Fab-8 insulator, and that CTCF likely plays an important role in organizing the Abd-B locus. (3) A specific case of CTCF function as a direct regulator of gene expression and its interface with other (more specialized) transcription factors was revealed upon continuing analysis of CTCFs role in the regulation of human telomerase gene (hTERT). In our previous publications, we demonstrated that CTCF was essential for the repression of hTERT transcription in a variety of normal somatic cells, while CTCF downregulation (in specifically designed assays and in cancer cells) resulted in hTERT expression activation, facilitating cell immortalization. The repressor activity of CTCF was not promoter-specific but was mediated by its binding to the first exons of the hTERT gene. In our recent work, we investigated what mechanism is involved in the atypical enhanced expression of hTERT in lymphoid cells. We uncovered that a transcription factor PAX5, which is specific for B-cell development and is essential for B-lymphocyte function, binds downstream of two CTCF-binding sites in hTERT and enables the derepression of the gene, overpowering the CTCF repressor activity (apparently without CTCF displacement). These results identify hTERT as a novel target of PAX5, which thus participates in cellular mechanisms underlying cell immortality. Furthermore, this finding reveals a novel pathway of CTCF involvement in the direct regulation of gene expression, possibly employing its interactions with a range of other cellular factors, including some that are cell-type specific. (4) Even more daring area of research was targeted with our investigation of the functions of CTCF outside of gene expression regulation. This subject is largely ignored in the CTCF literature, even though it is apparent that the bulk of cellular CTCF is bound to repeated/noncoding DNA. Nevertheless, such a localization pattern might indicate that the CTCFs function as a critical factor ensuring genome integrity and chromosome stability is largely mediated by these genomic loci. Indeed, our previous data on potential CTCFs functions in heterochromatin, as well as its roles in mitosis and meiosis, suggested a significant housekeeping role of CTCF in the organization of mammalian genome and in chromosome segregation. We focused our studies at one particular region of chromosomes the centromeric gamma satellite repeats residing in the heterochromatic regions flanking human centromeres. It was known that in hematopoetic cells these repeats are bound by Ikaros, a carrier of the heterochromatin barrier (or anti-silencing) activity of gamma-satellites. It was not understood, however, what proteins take the place of Ikaros binding in gamma-satellites in other cell types. Our data indicated that gamma-satellites could be bound by CTCF in vivo, and thus this binding could contribute to centromere function. Our data showing that CTCF-bound gamma-satellites serve as a heterochromatin barrier (protecting a transgene from silencing), indicate that the biological role of of human gamma-satellite chromatin may be to prevent spreading of pericentric hheterochromatin into gene-containing chromosomal zones.

CTCF是一种高度保守的多功能核因子，涉及全球基因组结构和基因调控的许多方面，后者的范围从直接基因抑制/激活到增强子阻断和激素促进的沉默。来自进化遥远物种的 CTCF 均含有一个高度保守的中心 11 锌指 DNA 结合结构域，该结构域介导其 DNA 结合活性的多序列特异性。 DNA 结合 CTCF 的二聚活性可能是其在大多数细胞类型中作为多功能染色质桥接剂和染色质环剂的活性核心，是其染色质绝缘体和异染色质边界功能的基础。在特定基因的背景下，CTCF 还可以通过染色质环功能调节转录增强子。对数万个 CTCF 靶位点 (CTS) 的全基因组图谱表明，CTCF 可以将核小体定位在 DNAse 超敏感位点周围，这些位点是绝缘子、增强子和其他调控序列的标志。由于 CTCF 具有如此多的重要功能，CTCF 成为脊椎动物的必需基因，因为 CTCF 敲除小鼠无法存活（在早期胚胎阶段具有致死性）。对于人类疾病来说，CTCF是一个公认的抑癌基因（TSG）； CTCF 11ZF DBD 中的几个功能性点突变已在原发性癌症中得到表征，在最初以 CTCF 基因座的 LOH 为特征的肿瘤中。 (1) 在过去的一年中，全基因组分析使人们对 CTCF 在脊椎动物基因组中的整体作用有了更深入的了解。现在已经清楚，CTCF 控制至少三个主要途径：基因组的整体结构、染色质结构介导的（环形成、增强子阻断剂活性）基因表达调节（印记和印记无关基因）、以及基因表达的直接调节（通过与其他转录因子的接口）。我们之前首次分析了全基因组 CTCF 靶标（Cell 2007，第 128 卷，第 1231-1245 页），并且通过 CTCF 靶位点 (CTS) 与基因位置的强相关性验证了 CTCF 在细胞功能中的基本作用在人类基因组中。因此，我们开发了一个综合计划来扩展全基因组研究，以确定 CTCF（和 BORIS）在各个染色体位点结合的正相关性，并最终确定其功能意义。我们进行了 ChIP 芯片分析来绘制 CTS 以及 p300 共激活子图谱，并将它们与 5 种人类细胞系中的组蛋白修饰模式进行比较：宫颈癌 (HeLa)、永生化淋巴母细胞 GM06690 (GM)、白血病 K562、胚胎干细胞 ( ES）和BMP4诱导的ES细胞（dES）。我们使用 ENCODE 微阵列（人类基因组的 1%）来识别这些细胞类型的假定 CTCF 结合位点，并观察到高度可重复的 CTCF 占据（与主要细胞类型特异性组蛋白修饰和 p300 结合相反），证实了我们的理论：细胞中的大部分 CTCF 功能与细胞类型无关。与此同时，CTS 的一个子集似乎与细胞类型相关，证实除了其在人类基因组中的全局功能外，CTCF 可能直接参与细胞谱系的调节，可能通过作用于少数特定的增强子或替代（基因内）启动子。 (2) 我们还对之前克隆并表征的果蝇 CTCF (DrCTCF) 进行了全基因组 DNA 结合分析。我们在全基因组范围内鉴定了超过 3561 个强 DrCTS（以及 8872 个较弱的 DrCTS，富集度低两倍）。全基因组分析表明，DrCTCF 通常被发现与位置接近但差异调节的基因结合。然而，DrCTS 也高度富集到基因的 5' 端，该基因没有紧密定位的相邻启动子。相反，预测的 Su(Hw) 绝缘体位点的分布没有表现出对启动子的任何偏向。因此，除了其绝缘体功能之外，启动子上游的 DrCTCF 结合可能具有更普遍的作用：直接调节转录或/和果蝇基因组的全局基因组组织。在一个具体案例中，作为全基因组分析的结果，来自 Abd-B 基因座的 Fab-6 绝缘子元件被鉴定为新的强 drCTCF 结合位点（具有两个 CTS），并被展示（使用专门设计的转基因和质粒 EB 检测）成为新的 CTCF 控制的 EB 元件。后续结果表明，除了 Fab-8 绝缘子之外，DrCTCF 对于 Fab-6 的增强子阻断活性至关重要，并且 CTCF 可能在组织 Abd-B 基因座中发挥重要作用。 (3) 通过继续分析 CTCF 在人端粒酶基因 (hTERT) 调节中的作用，揭示了 CTCF 作为基因表达的直接调节因子及其与其他（更专门的）转录因子的界面的具体情况。在我们之前的出版物中，我们证明了 CTCF 对于抑制多种正常体细胞中 hTERT 转录至关重要，而 CTCF 下调（在专门设计的测定中和在癌细胞中）导致 hTERT 表达激活，促进细胞永生化。 CTCF 的阻遏活性不是启动子特异性的，而是通过其与 hTERT 基因第一个外显子的结合介导的。在我们最近的工作中，我们研究了淋巴细胞中 hTERT 表达非典型增强所涉及的机制。我们发现转录因子 PAX5 对 B 细胞发育具有特异性，对 B 淋巴细胞功能至关重要，它与 hTERT 中两个 CTCF 结合位点的下游结合，使该基因去阻遏，从而压倒 CTCF 阻遏物活性（显然无 CTCF 位移）。这些结果将 hTERT 确定为 PAX5 的新靶标，从而参与细胞永生的细胞机制。此外，这一发现揭示了 CTCF 参与直接调控基因表达的新途径，可能利用其与一系列其他细胞因子的相互作用，包括一些细胞类型特异性的因子。 (4) 更大胆的研究领域是我们对 CTCF 在基因表达调控之外的功能的研究。尽管很明显大部分细胞 CTCF 与重复/非编码 DNA 结合，但 CTCF 文献在很大程度上忽略了这一主题。然而，这种定位模式可能表明 CTCF 作为确保基因组完整性和染色体稳定性的关键因素，很大程度上是由这些基因组位点介导的。事实上，我们之前关于 CTCF 在异染色质中的潜在功能及其在有丝分裂和减数分裂中的作用的数据表明，CTCF 在哺乳动物基因组的组织和染色体分离中具有重要的看家作用。我们的研究集中在染色体的一个特定区域，着丝粒伽马卫星重复位于人类着丝粒侧翼的异染色质区域。众所周知，在造血细胞中，这些重复序列与 Ikaros 结合，Ikaros 是 γ 卫星异染色质屏障（或抗沉默）活性的载体。然而，目前尚不清楚是什么蛋白质取代了其他细胞类型中伽马卫星中 Ikaros 的结合。我们的数据表明，γ-卫星可以在体内被 CTCF 结合，因此这种结合可能有助于着丝粒功能。我们的数据显示，CTCF 结合的伽马卫星充当异染色质屏障（保护转基因免于沉默），表明人类伽马卫星染色质的生物学作用可能是防止中心周异染色质扩散到含有基因的染色体区域。