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: 8156922
• 负责人: Victor Lobanenkov
• 金额: $ 73.5万
• 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/8156922
• 关键词: Pathologic

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. CTCF is an 11-zinc-finger (ZF) DNA-binding protein that coordinates the spatial organization of chromatin with the regulation of gene expression. As we discovered, this control acts through two major mechanisms: either direct regulation of a gene downstream of CTSes or indirect regulation, via the formation of chromatin loops stabilized by CTCF dimerization that affects relationships between the promoter, enhancer and/or imprinted control region (ICR). 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 core biological functions. Furthermore, the loop-forming activity of CTCF can be naturally extended to formation of localized somatic inter-chromosome pairing sites that therefore acquire potential for epigenetic co-regulation such as transcription factories, DNA replication factories, 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 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. We also identified a novel CTCF activity that directly links CTCF to transcriptional machinery binding of CTCF to Pol II. This novel pathway provides a mechanism for opening loop-independent transcription start sites for either coding or non-coding transcripts throughout the genome. Mechanistically, the 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. While CTCF is mostly known as a regulator of gene expression, our data on its potential functions in heterochromatin and centrosomes, as well as its roles in mitosis and meiosis, suggested a significant housekeeping role of CTCF in genome organization and chromosome segregation. CTCF was previously shown to undergo a variety of posttranslational modifications, and we expanded these studies to characterize novel modifications. Another pathological aspect of the deregulated CTCF occupancy of promoter targets sites is aberrant DNA methylation in cancers. Both of these novel biological roles of CTCF are subjects of ongoing studies in the MPS. 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. 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 very early embryonic stages). 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 loci in order to understand contributions of CTCF CTSes to their regulation. They included genes important for immune responses, as well as genes with a potential for the development of approaches for cancer treatment. As a general rule, we have found that if a CTCF binding site is located upstream of the transcriptional start site, it tends to play an activator role, while CTSes located downstream of (+1) usually behave as repressors. The gene for catalytic subunit of human telomerase (hTERT) is one of the most prominent genes in this study. It was also recently revealed that up to 22% of genes in a typical human being are regulated in the allele-specific manner, so that same genes on homologous chromosomes are expressed differentially. SNPs are potentially one of the major factors directly underlying allelic variations in gene expression. Upon completion of mapping for sites that bind CTCF in individuals and families, it was established that CTCF binding to SNPs is the potent facilitator of allele-specific expression. This is a major breakthrough in our understanding of the role played by noncoding polymorphisms in the human genome. Moreover, because SNPs are frequently associated with a variety of human syndromes, including rare and neglected diseases, our long-tem goals include GWAS on CTCF binding with respect to SNPs in those syndromes. Finally, we found the correlation between DNAse I-hypersensitive sites and CTCF binding in the HIV genome. Moreover, CpG methylation regions linked to epigenetic regulation of HIV-1 latency also correlated with CTCF binding sites. Thus, these important findings form a strong foundation for systematic research on the CTCF involvement in regulation of viral infections.

CTCF是一种高度保守的多功能核因子，既参与全局基因组结构，又在基因调节的许多方面，后者从直接基因抑制/激活到增强子阻断和激素促成沉默。 CTCF是一种11-锌指（ZF）DNA结合蛋白，可将染色质的空间组织与基因表达的调节进行协调。正如我们发现的那样，该控制通过两种主要机制起作用：通过形成CTCF二聚体稳定的染色质环的形成，可以直接调节CTSE的下游基因或间接调节，从而影响启动子，增强子，或/或压印控制区域（ICR）之间的关系。在大多数细胞类型中，与DNA结合的CTCF的二聚化活性可能是其活性的核心，这是其核心生物学功能的基础。此外，CTCF的循环形成活性可以自然扩展到形成局部的体细胞间染色体配对位点，从而获得表观遗传共同调节的潜力，例如转录工厂，DNA复制工厂和DNA修复灶。 CTCF调节了许多其他染色质锚定功能，例如建立印迹痕迹及其读数，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。我们还确定了一种新型的CTCF活性，该活性将CTCF与CTCF与POL II的转录机械结合联系起来。这种新型途径为整个基因组中的编码或非编码转录本开放循环转录启动位点提供了一种机制。从机械上讲，受调节的募集和随后从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。 尽管CTCF主要被称为基因表达的调节剂，但我们关于其在异染色质和中心体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在基因组组织和染色体分离中具有重要的家政作用。先前显示CTCF进行了多种翻译后修饰，我们扩大了这些研究以表征新颖的修饰。启动子靶点的放松管制CTCF占用位点的另一个病理方面是癌症中异常的DNA甲基化。 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。 我们先前首次分析了全基因组CTCF靶标（Cell 2007，第128卷，PP1231-1245），并且通过CTCF靶位点（CTS）与人类基因组中的基因位置有很强的相关性，CTCF在细胞功能中的基本作用得到了验证。由于具有如此多的重要功能，因此CTCF成为脊椎动物中的重要基因，因为CTCF-KNOCKOUT小鼠是不可行的（在非常早期的胚胎阶段的致死性）。 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座，以了解CTCF CTSE对其法规的贡献。它们包括对免疫反应重要的基因以及具有开发癌症治疗方法的基因。通常，我们发现，如果CTCF结合位点位于转录起始位点的上游，则它倾向于扮演激活剂的角色，而位于（+1）下游的CTSE通常会作为阻遏物。人端粒酶（HTERT）催化亚基的基因是本研究中最突出的基因之一。 最近还揭示了典型人中多达22％的基因受等位基因特异性的调节，因此同源染色体上的相同基因差异表达。 SNP可能是基因表达中等位基因变化的主要因素之一。在完成在个人和家庭中结合CTCF的位点的映射后，已经确定CTCF与SNP结合是等位基因特异性表达的有效促进者。这是我们的重大突破 了解非编码多态性在人类基因组中所起的作用。此外，由于SNP经常与各种人类综合症有关，包括罕见和被忽视的疾病，因此我们的长TEM目标包括与这些综合症中SNP的CTCF结合的GWA。 最后，我们发现了HIV基因组中的DNase I-甲状化位点与CTCF结合之间的相关性。此外，与HIV-1潜伏期相关的CPG甲基化区域也与CTCF结合位点有关。因此，这些重要的发现为CTCF参与病毒感染调节的系统研究构成了强大的基础。