Mechanisms Of Transcriptional Regulation By CTCF

CTCF 的转录调控机制

• 批准号: 6521454
• 负责人: VICTOR LOBANENKOV
• 金额: --
• 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/6521454
• 关键词: Wilms' tumor; breast neoplasms; gene mutation; genetic promoter element; genomic imprinting; neoplasm /cancer genetics; prostate neoplasms; protooncogene; tissue /cell culture; transcription factor

In 2000-01, the Molecular Pathology Section (MPS) in the LIP NIAID continued to work on better understanding of the molecular mechanisms that underlie normal function of the transcriptional factor, called CTCF, in development, cell-cycle regulation, and gene imprinting; and of the molecular events causally linked with CTCF malfunction in cancer, and in other human diseases associated with abnormal site-specific DNA methylation at the variety of highly diverged CTCF-target sites (CTSs). The first comprehensive review on CTCF published in "Trends in Genetics" in 2001 by V. Lobanenkov and collaborators, provided a summary of experimental results which show that CTCF is a uniquely versatile transcriptional regulator with diverse functions linked to epigenetics and disease. Our earlier results demonstrated that CTCF is the ubiquitously expressed gene upregulated during the S/G2-stage of the cell cycle. It encodes a nuclear factor containing three major functionally distinct regions with amino acid sequences that were maintained practically identical throughout vertebrate evolution: a DNA-binding domain composed of the 11 Zinc Fingers (ZFs), and two flanking trans-acting transcriptional repressor/activator regions that account for approximately two-thirds of the entire protein Recent review of the literature established CTCF as a true "multivalent multifunctional" protein which utilizes different sets of ZF to form distinct complexes with varying ~50 bp CTCF-target sites (CTS) that mediate distinct functions in regulation of gene expression. Others and we have shown that these functions include context-dependent promoter repression or activation, creation of modular hormone-responsive gene silencers, and formation of diverse vertebrate enhancer-blocking elements (chromatin insulators or boundaries). Functions of varying CTCF/DNA complexes may be regulated by post-translational protein modifications; by physical interactions with other multifunctional nuclear proteins that include, among others, RNA/DNA binding factor YB-1 and the repression-associated mSin3A/HDACs; and by attenuation of the interactions with DNA via specific methylation of CpG pairs involved in recognition of specific CTS by the protein. For example, the latter class of conserved targets which require particular sets of CTCF ZF for formation of the very high-affinity complexes with CTCF, were characterized [see Trends in Genetics 17:520-7 (2001) for review] within the Imprinting Control Region (ICR) between growth-regulating gene IGF2 and a candidate tumor suppressor gene, H19. In collaboration with R. Ohlsson lab in Sweden, we showed that specific CpG methylation eliminates interaction of CTCF with the ICR, allowing the protein to distinguish normally differentially methylated maternal versus paternal IGF2/H19 alleles IN VIVO; and that methylation-regulated formation of CTCF/ICR complexes controls activity and conformation of the chromatin insulator that regulates imprinted IGF2 and H19 expression. In addition to the IGF2/H19 ICR CTSs, critical regulatory regions at the promoters of vertebrate MYC oncogenes have been shown to contain CTS that mediate negative transcriptional control by CTCF modulated by the carboxyterminal phosphorylation. Moreover, a number of novel functional CTS were identified; in respect to cell proliferation control, some of these are neutral while some others are important, for example the CTS in mouse/human PLK and p19ARF genes and mouse/human PIM1 oncogene among others. We have also found that disrupting the spectrum of functional CTCF/DNA complexes either (i) by selective ZF point-mutations observed in some tumors with frequent LOH at CTCF locus mapped on chromosome 16q22 (10) or (ii) by abnormal CpG-methylation of CTS that constitute insulator sites upstream of IGF2 observed in tumors with LOI, is associated with cancer development. We demonstrated recently that CTCF normally inhibits cell cycle progression. In three separate experimental systems, it was shown that the cell cycle profiles of growth arrested cells expressing ectopic CTCF did not noticeably differ from control populations in log phase growth. This ability of CTCF to apparently freeze cells at any stage of cell cycle progression seems to be unprecedented and suggests that CTCF may control expression of genes that arrest cells at each stage in this progression. This model implies function of CTCF as a universal coordinator of an intertwined network of genes in which, if considered separately one from another, each network may perform only strictly specialized tasks in cell cycle control. The same concept may also point to a driving force for exceptional conservation of CTCF, and for evolving of the unusual ability of CTCF ZFs to recognize diverse spectrum of binding sites. On the other hand, it also predicts a very complex multilevel mode for regulation of CTCF itself that may include regulation of CTCF promoter activity during cell cycle, posttranslational modifications including phosphorylation, interactions with multifunctional protein partners, and other mechanisms yet to be discovered. Furthermore, it is conceivable that effects of CTCF on cell cycle progression may depend not only on transcriptional regulation of a variety target genes by CTCF, but also on DNA-independent direct interactions of CTCF with certain components and regulators of the cyclin-CDK complexes, and with some of the proteins directly involved in replication machinery and in cytokinesis. Our preliminary studies of numerous CTCF-interacting functional partners identified by an affinity chromatography of total cell extracts on matrix-immobilized pure recombinant CTCF, by GST-CTCF-mediated protein "pull-down" approach with nuclear extracts, and by the yeast two-hybrid method, have provided evidence that CTCF may indeed be a subject of such interactions. Thus, CTCF functions may go beyond of being just a transcription factor. However, it is clear that better understanding of cell growth regulation by CTCF will require an in depth evaluation of a complete repertoire of CTCF target genes and CTCF-interacting partners. To this end, the program "Mechanisms of Transcriptional Regulation by CTCF" takes advantage of cancer-prone mouse CTCF knock-out models that we developed earlier, and of Drosophila genetics based on identification and cloning of the CTCF homologue in flies. Identification and functional characterization of critically important CTCF target genes and protein partners that are involved in regulation of CTCF function in somatic cells and in germline lead the Section well beyond studies of CTCF per se. By September 2001, some of these proprietary genes have already been characterized and are now being patented by NIAID NIH, and evaluated for diagnostic and therapeutic purposes. Several other genes encoding CTCF-interacting proteins are at the initial stages of deciphering their role(s).

2000- 2001年，LIP NIAID的分子病理学组（MPS）继续致力于更好地理解转录因子（CTCF）在发育、细胞周期调控和基因印记中的正常功能的分子机制；以及在癌症中与CTCF功能障碍因果相关的分子事件，以及在各种高度分化的CTCF靶位点（CTSs）上与异常位点特异性DNA甲基化相关的其他人类疾病中。2001年，V. Lobanenkov及其合作者在《遗传学趋势》（Trends in Genetics）上发表了关于CTCF的第一篇综合综述，总结了实验结果，表明CTCF是一种独特的多功能转录调节剂，具有与表观遗传学和疾病相关的多种功能。我们早期的研究结果表明，CTCF是细胞周期S/ g2期上调的普遍表达基因。它编码一个核因子，包含三个主要的功能不同的区域，氨基酸序列在脊椎动物进化过程中几乎保持一致：最近的文献综述表明，CTCF是一种真正的“多价多功能”蛋白，它利用不同的ZF集形成不同的复合物，具有不同的~50 bp的CTCF靶位点（CTS），介导不同的基因表达调节功能。其他人和我们已经证明，这些功能包括上下文依赖的启动子抑制或激活，模块化激素响应基因沉默子的产生，以及各种脊椎动物增强子阻断元件（染色质绝缘体或边界）的形成。不同CTCF/DNA复合物的功能可能受到翻译后蛋白修饰的调节；通过与其他多功能核蛋白的物理相互作用，包括RNA/DNA结合因子YB-1和与抑制相关的mSin3A/ hdac；通过CpG对的特定甲基化来减弱与DNA的相互作用，CpG对参与了蛋白质对特定CTS的识别。例如，在生长调节基因IGF2和候选肿瘤抑制基因H19之间的印迹控制区（ICR）中，后一类保守靶标需要特定的CTCF ZF集才能与CTCF形成非常高亲和力的复合物[见《遗传学趋势》17:20 20-7（2001）回顾]。与瑞典的R. Ohlsson实验室合作，我们发现特异性CpG甲基化消除了CTCF与ICR的相互作用，使该蛋白能够在体内区分正常甲基化的母体与父亲的IGF2/H19等位基因；甲基化调节的CTCF/ICR复合物的形成控制了染色质绝缘体的活性和构象，而染色质绝缘体调节印迹IGF2和H19的表达。除了IGF2/H19 ICR CTS外，脊椎动物MYC癌基因启动子的关键调控区域已被证明含有CTS，这些CTS通过羧基端磷酸化调节CTCF介导负转录控制。此外，还发现了一些新的功能性CTS；在细胞增殖控制方面，其中一些是中性的，而另一些是重要的，例如小鼠/人类PLK和p19ARF基因中的CTS以及小鼠/人类PIM1致癌基因等。我们还发现，破坏功能性CTCF/DNA复合物的光谱(i)通过选择性ZF点突变观察到的位于16q22染色体上的CTCF位点频繁LOH的肿瘤（10）或（ii）通过在LOI肿瘤中观察到的构成IGF2上游绝缘体位点的CTS的异常cpg甲基化，与癌症的发展有关。我们最近证明CTCF通常会抑制细胞周期进程。在三个独立的实验系统中，表明表达异位CTCF的生长阻滞细胞的细胞周期谱在对数期生长中与对照群体没有显着差异。CTCF在细胞周期进程的任何阶段明显冻结细胞的能力似乎是前所未有的，这表明CTCF可能控制在细胞周期进程的每个阶段阻止细胞的基因表达。该模型暗示CTCF作为一个相互交织的基因网络的普遍协调者的功能，如果单独考虑，每个网络可能只在细胞周期控制中执行严格的专门任务。同样的概念也可以指出CTCF异常保护的驱动力，以及CTCF ZFs识别不同结合位点的异常能力的进化。另一方面，它还预测了CTCF本身的一个非常复杂的多层调控模式，可能包括细胞周期中CTCF启动子活性的调控、磷酸化等翻译后修饰、与多功能蛋白伴侣的相互作用以及其他尚未发现的机制。此外，可以想象，CTCF对细胞周期进程的影响可能不仅取决于CTCF对多种靶基因的转录调控，还取决于CTCF与周期蛋白- cdk复合物的某些成分和调节因子，以及与一些直接参与复制机制和细胞分裂的蛋白质的dna独立的直接相互作用。