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: 7592248
• 负责人: VICTOR LOBANENKOV
• 金额: $ 80.49万
• 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/7592248
• 关键词: 16q22; 20q13; Affinity; Alleles; Animal Model; Antibodies; Apoptosis; Binding; Binding Sites; Biological Assay; Birds; CCCTC-binding factor; CTAG1 gene; Caenorhabditis elegans; Cell Nucleus; Cell Proliferation; Cells; Chemosensitization; Chromatin; Chromosomes; Class; Classification; Cloning; Complementary DNA; Complex; Consensus; DNA; DNA Binding Domain; DNA Methylation; Data; Depth; Development; Distal; Down-Regulation; Drosophila genus; Ectopic Expression; Electrophoretic Mobility Shift Assay; Enhancers; Epigenetic Process; Exons; Failure; Fibroblasts; Fingers; Gene Activation; Gene Cluster; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Genome; Genome Mappings; Genomics; Germ; Globin; Goals; H19 gene; Hand; Higher Order Chromatin Structure; Hormones; Hot Spot; Human; Human Genome; In Vitro; Inhibition of Cell Proliferation; Kidney; Lesion; Libraries; Link; Localized; Loss of Heterozygosity; Malignant Neoplasms; Malignant neoplasm of testis; Mammals; Maps; Masks; Matrix Attachment Regions; Mediating; Methods; Methylation; Modeling; Molecular; Molecular Conformation; Moon; Mus; Mutation; Nephroblastoma; Nuclear; Nucleic Acid Regulatory Sequences; Numbers; Pathologic; Pattern; Point Mutation; Polymerase; Polymerase Chain Reaction; Process; Proliferating; Promoter Regions; Proteins; Publishing; RNA Polymerase II; Range; Rattus; Regulation; Reporter; Reporter Genes; Reporting; Repression; Residual state; Resources; Rest; Role; Site; Spermatogenesis; Stem cells; TERT gene; Taq Polymerase; Telomerase; Testis; Tumor Suppressor Genes; Tumor Tissue; WT1 gene; Work; Zebrafish; base; beta Globin; cancer cell; cell immortalization; cell type; crosslink; demethylation; gene function; gene repression; imprint; in vivo; knock-down; melanoma-associated antigen-A1; mutant; novel; nuclease; prevent; programs; promoter; size; tissue culture; transcription factor; tumor; tumorigenesis; vertebrate genome

From 10-01-06 to 9-01-07, we continued our studies of CTCF - a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of chromatin-insulator function and gene imprinting. Although many of these functions rely on CTCF-DNA interactions, there is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. We demonstrated that a subpopulation of CTCF in chromatin interacts directly with the RNA polymerase II (Pol II) complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly co-localizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. "Double" ChIP analyses revealed that both CTCF and LS Pol II were present at the beta-globin insulator in proliferating HD3 cells but not in differentiated globin-synthesizing cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-Chip hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. Next, we performed a genome-wide characterization of CTCF sites in human genome. We were able to describe 13,804 novel CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. These results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of a particular class of CTCF sites. Also, these data raise an important question about the overall validity of the ChIP-to-chip approach for CTCF, because many of CTCF-binding sites, especially those in gene-promoters, have been certainly missed by using it. Apparently, the main problems are associated with: 1) the type of anti-CTCF antibodies (because different classes of CTCF-DNA-complexes in chromatin are differentially masked by interactions with different co-factors); 2) method of cross-linking CTCF to DNA before the IP step (because apparently only high-affinity CTCF-sites have been detected and such sites are commonly seen in insulators but not in gene-promoters); and 3) ChIPed-probe-amplification methods (because many CTCF-sites are found to be embedded into extremely CT- and CG-rich sequences that are very difficult, if not possible at all, to PCR by using Taq polymerase). We also continued in depth characterization of the role of CTCF in regulation of several genomic loci that we study in the lab. First, we reported a peculiar interplay of CTCF repression of hTERT gene and methylation of hTERT 5 regulatory region. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. Now we used electrophoretic mobility shift assay (EMSA) and ChIP to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression. Second, while studying regulation by CTCF of imprinted loci we performed systematic chromosome conformation capture 4C analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF site within Igf2 DMR1. This systematic 4C analysis of an imprinted gene-cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome. While studying maternally imprinted KvDMR1 locus we characterized two novel CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated allele. Using a number of reporter assays, we showed that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Next, we studied function of another group of CTCF-binding sites mapped in the Wilms tumor 1 gene (WT1) and characterized the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for CTCF. Potentiation of the silencer activity was demonstrated after CTCF protein was knocked-down, thereby suggesting a novel silencer-blocking activity for CTCF. We also assessed the ARR methylation in developmental and in tumor tissues including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumorigenesis. Finally for this FY report, we discovered the presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters. We suggested (based on the analogy to CTCF/BORIS-sites mapped and functionally characterized in MAGE-A1 and NY-ESO-1 gene promoter-regulation, as we published earlier) that their activation in spermatogenesis is mediated by the programmed replacement of the DNA-occupancy in vivo from CTCF to occupancy of the same site by BORIS. Finally, this year we reported cloning of CTCF from Danio rerio - a valuable vertebrate model organism. It shows very high similarity with CTCF from mammals as well as similar developmental expression pattern and promoter regulation. Taken together with our earlier report on identification and cloning of Drosophila CTCF (H. Moon, et.al., EMBO R., 2005, v.6, pp. 165-170), this work put forward our continuing efforts (which begun in early 90s from cloning of CTCF from birds, mice, rats, and humans) to identify and molecularly clone CTCF from all species that have this gene. To this end, we have identified (but not yet published) a cDNA sequence of CTCF from C. elegans.

从2006年10月1日到2007年9月1日，我们继续研究CTCF -一种具有多种功能的转录因子，从基因激活和抑制到染色质绝缘子功能和基因印迹的调节。尽管许多这些功能依赖于CTCF- dna相互作用，但人们逐渐认识到CTCF依赖的分子过程涉及CTCF与其他蛋白质的相互作用。我们证明了染色质中的CTCF亚群直接与RNA聚合酶II （Pol II）复合物相互作用。我们发现Pol II的最大亚基（LS Pol II）是一种在细胞核中与CTCF显著共定位的蛋白质，并在体内和体外与CTCF特异性相互作用。通过观察到LS Pol II与体内CTCF靶点的关联依赖于完整的CTCF结合序列，CTCF作为DNA和LS Pol II之间的纽带的作用得到了加强。“双”ChIP分析显示，CTCF和LS Pol II均存在于增殖的HD3细胞的β -珠蛋白绝缘体中，但不存在于分化的珠蛋白合成细胞中。此外，在稳定转染的细胞中，单个野生型CTCF靶位点（N-Myc-CTCF），而不是缺乏CTCF结合的突变位点，足以激活无启动子报告基因的转录。最后，利用CTCF靶点文库的微阵列进行ChIP-on-Chip杂交分析显示，许多基因间CTCF靶点序列与CTCF和LS Pol II相互作用。接下来，我们对人类基因组中的CTCF位点进行了全基因组表征。我们能够在人类基因组的潜在绝缘体中描述13,804个新的ctcf结合位点，这些位点是在人类原代成纤维细胞中实验发现的。这些序列大多位于远离转录起始位点的位置，其分布与基因密切相关。它们中的大多数符合高度保守的共识基序，适用于预测其他脊椎动物基因组中CTCF驱动的可能绝缘体。此外，CTCF的定位在不同的细胞类型中基本上是不变的。这些结果为研究绝缘子功能以及特定CTCF位点的其他可能的一般和进化保守活性提供了资源。此外，这些数据提出了一个重要的问题，即芯片对芯片CTCF方法的整体有效性，因为使用它肯定会错过许多CTCF结合位点，特别是基因启动子中的位点。显然，主要问题与：1)抗ctcf抗体的类型有关（因为染色质中不同类型的ctcf - dna复合物被不同的辅助因子相互作用不同地掩盖）；2)在IP步骤之前将CTCF与DNA交联的方法（因为显然只检测到高亲和力的CTCF位点，并且这种位点常见于绝缘子而未见于基因启动子）；3) chiped探针扩增方法（因为许多ctcf位点被发现嵌入到极其丰富的CT和cg序列中，使用Taq聚合酶进行PCR是非常困难的，如果根本不可能的话）。我们还继续深入表征CTCF在实验室研究的几个基因组位点调控中的作用。首先，我们报道了CTCF抑制hTERT基因和htert5调控区甲基化的特殊相互作用。我们之前的研究表明，hTERT启动子的甲基化是其转录所必需的，CTCF可以通过结合第一个外显子抑制hTERT的转录。现在，我们使用电泳迁移率转移试验（EMSA）和ChIP表明CTCF不结合甲基化的hTERT第一外显子。用5- azadc处理端粒酶阳性细胞导致hTERT 5'-调控区域的强烈去甲基化，CTCF结合的重新激活和hTERT的下调。尽管完全的hTERT启动子甲基化与完全的转录抑制有关，但详细的图谱显示，在端粒酶阳性细胞中，并非所有的CpG位点都被甲基化，尤其是在启动子区域。通过甲基化盒实验，核心启动子内110bp的选择性去甲基化显著增加了hTERT的转录活性。在我们的模型中，hTERT甲基化阻止CTCF抑制因子的结合，但核心启动子的部分低甲基化对于hTERT表达是必要的。其次，在研究CTCF对印迹位点的调控时，我们在Igf2/H19区域进行了系统的染色体构象捕获4C分析，分析了超过160 kb的区域，确定了与远端增强子和ICR物理相互作用的序列。我们发现，在父系染色体上，增强子与Igf2启动子相互作用，但在母系等位基因上，这被H19 ICR内的CTCF结合所阻止。母体ICR中的CTCF结合调节其与Igf2位点的基质附着区（MAR）3和DMR1的相互作用，从而在母体Igf2位点周围形成一个紧密的环，这可能是其沉默的原因之一。H19 ICR中CTCF位点的突变导致Igf2 DMR1中CTCF位点的重新甲基化和CTCF结合的丧失。对印迹基因簇的系统性4C分析表明，CTCF在基因组中高阶染色质结构和基因沉默的表观遗传调控中起着关键作用。在研究母体印迹的KvDMR1位点时，我们在KvDMR1中发现了两个新的CTCF结合位点，这些位点在体内仅位于未甲基化的等位基因上。通过一系列报告分析，我们发现KvDMR1 ICR由多个独立的顺式作用模块组成。接下来，我们研究了Wilms肿瘤1基因（WT1）中另一组ctcf结合位点的功能，并表征了WT1 ARR差异甲基化区域，并表明它包含一个转录沉默子，作用于AWT1和WT1- as启动子。沉默子的DNA甲基化导致转录抑制增加，并且沉默子也被证明是CTCF的体外和体内靶点。CTCF蛋白被敲除后，沉默子活性增强，从而提示CTCF具有新的沉默子阻断活性。我们还评估了发育组织和肿瘤组织中的ARR甲基化，包括对Wilms肿瘤前体病变、肾源性病变的首次分析。值得注意的是，CTCF靶位点内CpG残基的甲基化状态似乎区分了单等位基因和双等位基因的表达状态。我们的数据表明，在肾脏发育早期，WT1 ARR的甲基化扩散失败，随后印记消除，发生在Wilms的肿瘤发生过程中。最后，在本年度报告中，我们在SPANX启动子中发现了CTCF及其睾丸特异性对话物BORIS的结合位点。我们提出（基于在MAGE-A1和NY-ESO-1基因启动子调控中定位和功能表征的CTCF/BORIS位点的类比，如我们之前发表的），它们在精子发生中的激活是由体内dna占用从CTCF到BORIS占用相同位置的编程替代介导的。最后，今年我们报道了从达尼奥-一种有价值的脊椎动物模式生物中克隆CTCF。它与哺乳动物CTCF具有很高的相似性，并且具有相似的发育表达模式和启动子调控。结合我们早期关于果蝇CTCF的鉴定和克隆的报告（H. Moon, et al.）。， EMBO R., 2005, v.6, pp. 165-170)，这项工作推动了我们继续努力（从90年代初开始从鸟类、小鼠、大鼠和人类克隆CTCF），从所有具有该基因的物种中识别和分子克隆CTCF。为此，我们鉴定了秀丽隐杆线虫CTCF的cDNA序列（但尚未发表）。