DNA-binding shared by CTCF, BORIS, NATASHA

CTCF、BORIS、NATASHA 共享 DNA 结合

• 批准号: 7196660
• 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/7196660
• 关键词: DNA binding protein; DNA methylation; biotechnology; cell line; chromatin; chromatin immunoprecipitation; developmental genetics; gel mobility shift assay; gene expression; genetic mapping; genetically modified animals; genomic imprinting; germ cells; laboratory mouse; neoplasm /cancer genetics; nuclear proteins; ovary; testis; transcription factor; tumor suppressor genes

From 10-01-04 to 9-01-05, we continued our efforts on a genome wide characterization of various functions mediated in chromatin of normal and cancer cells by the 50-bp-long DNA sequences capable of specific binding to the highly conserved 11 Zinc Finger (11ZF) DNA-binding domain (DBD) shared in two unique nuclear factors, CTCF and BORIS. CTCF is highly conserved, multifunctional, multivalent, nuclear factor with the properties of a tumor suppressor at 16q22. It is ubiquitously expressed in all somatic cells, and involved in promoter activation and repression, hormone-inducible gene silencing, and creation of constitutive or methylation-sensitive chromatin boundaries. An exciting breakthrough in understanding the epigenetic mechanisms was our discovery of a paralogue of CTCF termed BORIS (for Brother Of Regulator of Imprinted Sites). Normally, only CTCF but not BORIS is expressed in all somatic cells. During male germ cell differentiation, the pair displays mutually exclusive expression pattern that correlates with the patterns for (1) genome-wide re-establishing of DNA-methylation marks and (2) certain testis-specific chromatin-remodeling factors, and DNMTs. BORIS is normally expressed only during MALE-germ-cell differentiation, in a mutually exclusive with CTCF pattern that is tightly coupled with epigenetic reprogramming. We showed earlier that CTCF and BORIS share identical exons encoding the 11 ZF DBD to interact with the same spectrum of CTCF/BORIS-binding sites, but diverge at the amino- and carboxy-termini. Both genes have originally been identified and molecularly cloned by V. Lobanenkov with collaborators and co-workers, who first submitted to GenBank the full-length cDNA sequences of CTCF in April 1993 (Acc. No. Z22605) and of BORIS in January 2001 (Acc. No. AF336042). Any later submissions of BORIS cDNA and/or protein sequences under "CTCF-L" name and other accession numbers (for an EST, or a hypothetical "CTCF-like" protein) are incorrect and misleading, because there are ABSOLUTELY NO SIMILARITIES between CTCF and BORIS amino acid sequences in the 11ZF-flanking N-terminal and C-terminal parts that account for the two-thirds of each polypeptide of similar total length, thereby making BORIS to be a factor, which UNLIKE CTCF can direct a totally distinct from CTCF function to a shared set of target DNA-sequences recognized by the 11ZF-domain. By 9-01-04, two reviews on CTCF and BORIS genes, written by V. Lobanenkov and co-workers, are available. Thus, somatic CTCF and testis-specific BORIS could serve as counteracting genes by manifesting and reprogramming PATERNAL epigenetic states, respectively, by DNA-sequences binding to the 11 ZF DBD shared by the two already-published genes CTCF and BORIS. The sites for MATERNAL epigenetic marking require a third factor with the same DBD to be expressed in embryonic ovaries. Indeed, we discovered a FEMALE-germ-cell-specific counterpart of BORIS, named "Natasha" to highlight the difference both with the MALE-germ-cell-specific BORIS and with somatic CTCF. This year, we have proven the stunning presence in the ORF of the partial Natasha cDNA clones of the same 11ZF DBD that was earlier described in the unique CTCF & BORIS PAIR. Identification of Natasha has completed our search of all three factors required for the re-establishing of parental epigenetic marks at chromatin regions with DNA-sequences recognized by the same 11ZF DBD shared by these three unique factors. We called such sequences CTCF/BORIS/NATASHASHA-target sites, or "CBN-sites". Our working hypothesis suggests that regulation of the in vivo access to CBN-sites, and functionally different outcomes from the in vivo occupancy of the same genomic sites by competing against each other distinct factors equipped with the same 11ZF-DBD, may serve to target, to maintain, or to alter local status of epigenetic modifications genome wide in immortalized/transformed cells. Indeed, unlike normal somatic cells with CTCF, the vast majority of cancer cells are found to abnormally co-express CTCF and BORIS together. In accord with this hypothesis, several studies of the IGF2/H19 LOI by altering mono-allelic CTCF-binding in tumors with aberrant bi-allelic hypo- and/or hyper- methylation of at least one of seven CTCF-sites in the H19 ICR, suggested that CTCF-binding sequences, or CBN sites, are taking the central stage in human cancer epigenetics. In some human families, Mother Nature herself performed some of the most revealing experiments on the role of CBN-sites, for example, by showing that a narrow deletion of only two CTCF-binding ICR sites in two different families (or abnormal bi-allelic methylation of the same sites in several other families) results in the Beckwith-Wiedemann syndrome (BWS) including prenatal death on maternal transmission with the signs of BWS. Various CBN 11ZF-DBD-targets can incorporate additional binding sites for other (lineage-specific) transcriptional factors, because formation of various 11ZF/DNA-complexes generates 50-60-bp-long footprints with different contexts of ZF-contacting nucleotides on both DNA strands of a CBN site, which explains how CTCF and BORIS are capable of discriminating functionally distinct targets, form so many distinct geometry complexes with DNA, and result in a SELECTIVE interactions of the complexes one with another. The ability of CTCF and BORIS to discriminate in vivo functionally distinct targets suggested that the functional outcomes from changing a particular mode of the in vivo occupancy at the 11ZF-DBD-binding sites can differ and be cell-type-specific. Indeed, for example, an ectopic tet- expression of BORIS (but not the 11ZF-DBD alone) in primary normal human fibroblasts results in the selective demethylation and re-activation of the CBN target promoters normally regulated by the CTCF-BORIS-switching in germ cells. Such targets include promoters of so called "cancer-testis" genes, such as MAGEs, LAGEs, NY-ISO-1 and other downstream BORIS-targets like the Oct-4 promoter, for which the change of the in vivo occupancy at CTCF/BORIS-sites, initially mapped by gel-shifts, was directly shown by the ChIP approach. On the other hand, an ongoing mapping and verifying each novel site for methylation-sensitive binding in vivo, strongly supports an idea that misdirected epigenetic regulation in tumors is mediated through a misuse of varying sequences recognized by the CBN 1ZF DBD, because no exceptions have been yet found for the presence of such sequences in promoters reported in the literature for aberrant hypermethylation, with near 100 of such regions tested, and CBN-sites arrayed for ChIP-to-chip assays. Finally, we showed that Natasha-BORIS-genes are located within the region on 20q13 commonly involved in copy-gain in breast, ovarian, brain and many other cancers, and in fact are abnormally activated in those tumors, thereby making these factors an exceptionally attractive new anti-cancer target and a super-sensitive early diagnostic tool.

从04年10月1日到05年9月1日，我们继续研究了正常细胞和癌细胞染色质中各种功能的全基因组特征，这些功能是通过50 bp长的DNA序列特异性结合到两个独特的核因子CTCF和BORIS共享的高度保守的11锌指（11ZF） DNA结合域（DBD）来实现的。CTCF是一种高度保守、多功能、多价的核因子，在16q22位点具有肿瘤抑制因子的特性。它在所有体细胞中普遍表达，参与启动子的激活和抑制，激素诱导的基因沉默，以及构成或甲基化敏感染色质边界的创造。在理解表观遗传机制方面的一个令人兴奋的突破是我们发现了一个被称为BORIS（印迹位点调控因子的兄弟）的CTCF对偶。正常情况下，所有体细胞只表达CTCF，不表达BORIS。在男性生殖细胞分化过程中，这对基因表现出互斥的表达模式，这种表达模式与(1)dna甲基化标记的全基因组重建和(2)某些睾丸特异性染色质重塑因子和dnmt的模式相关。BORIS通常仅在男性生殖细胞分化过程中表达，与CTCF模式相互排斥，并与表观遗传重编程紧密结合。我们之前发现CTCF和BORIS具有相同的外显子编码11 ZF DBD，与CTCF/BORIS结合位点的相同谱相互作用，但在氨基端和羧基端存在差异。这两个基因最初是由V. Lobanenkov及其同事鉴定和分子克隆的，他们于1993年4月首次向GenBank提交了CTCF的全长cDNA序列(Acc。不。Z22605)和BORIS的2001年1月(Acc。不。AF336042)。任何后来以“CTCF-l”名称和其他加入号提交的BORIS cDNA和/或蛋白质序列（用于EST或假设的“CTCF-like”蛋白质）都是不正确和误导性的，因为CTCF和BORIS在11zf侧n端和c端部分的氨基酸序列之间绝对没有相似性，这些部分占每个相似总长度多肽的三分之二，从而使BORIS成为一个因子。与CTCF不同的是，它可以将完全不同于CTCF的功能引导到11zf结构域识别的一组共享的目标dna序列。截止到04年9月1日，由V. Lobanenkov及其同事撰写的两篇关于CTCF和BORIS基因的综述已经发表。因此，体细胞CTCF和睾丸特异性BORIS可以作为对抗基因，通过dna序列结合到两个已发表的基因CTCF和BORIS共有的11 ZF DBD上，分别表现和重编程PATERNAL表观遗传状态。母体表观遗传标记位点需要具有相同DBD的第三个因子在胚胎卵巢中表达。事实上，我们发现了BORIS的女性生殖细胞特异性对应物，命名为“Natasha”，以突出与男性生殖细胞特异性BORIS和体细胞CTCF的差异。今年，我们已经证明了在ORF中存在与先前在独特的CTCF和BORIS配对中描述的相同的11ZF DBD的部分Natasha cDNA克隆。娜塔莎的鉴定完成了我们在染色质区域重建亲本表观遗传标记所需的所有三个因素的搜索，这些dna序列被这三个独特因素所共享的相同11ZF DBD所识别。我们称这些序列为CTCF/BORIS/ natasha -target sites，或CBN-sites。我们的工作假设表明，体内进入cbn位点的调控，以及通过与具有相同11ZF-DBD的不同因子相互竞争而在体内占用相同基因组位点的功能不同结果，可能有助于靶向、维持或改变永生化/转化细胞中全基因组表观遗传修饰的局部状态。事实上，与具有CTCF的正常体细胞不同，绝大多数癌细胞被发现异常地同时表达CTCF和BORIS。根据这一假设，在H19 ICR中七个ctcf位点中至少有一个的双等位基因低甲基化和/或高甲基化的肿瘤中，通过改变单等位基因ctcf结合的IGF2/H19 LOI的几项研究表明，ctcf结合序列或CBN位点在人类癌症表观遗传学中处于中心地位。在一些人类家庭中，大自然母亲亲自进行了一些关于cbn位点作用的最具揭示性的实验，例如，通过显示在两个不同家庭中仅两个ctcf结合ICR位点的狭窄缺失（或在其他几个家庭中相同位点的异常双等位基因甲基化）导致贝克威斯-维德曼综合征（BWS），包括孕妇传播的产前死亡，并伴有BWS迹象。各种CBN 11ZF- dbd靶标可以结合其他（特异性）转录因子的额外结合位点，因为各种11ZF/DNA复合物的形成在CBN位点的两条DNA链上产生50-60 bp长的足迹，具有不同背景的zf -接触核苷酸，这解释了CTCF和BORIS如何能够区分功能不同的靶标，与DNA形成如此多不同的几何复合物。并导致配合物之间的选择性相互作用。CTCF和BORIS在体内区分不同功能靶点的能力表明，改变11zf - dbd结合位点在体内占用的特定模式可能会产生不同的功能结果，并且具有细胞类型特异性。事实上，例如，BORIS的异位tet-表达（而不是11ZF-DBD）在原代正常人类成纤维细胞中导致CBN靶启动子的选择性去甲基化和再激活，通常由生殖细胞中ctcf -BORIS开关调节。这些靶标包括所谓的“癌睾丸”基因的启动子，如mage、LAGEs、NY-ISO-1和其他下游boris靶标，如Oct-4启动子，这些靶标的体内CTCF/ boris位点的占据变化，最初是通过凝胶位移绘制的，通过ChIP方法直接显示出来。另一方面,一个正在进行的映射和验证每一个小说网站methylation-sensitive绑定体内,强烈支持的想法误导表观遗传调控在肿瘤介导通过滥用不同序列被CBN 1 zf DBD,因为没有发现异常的存在这样的序列在启动子异常甲基化在文献中报道,与测试100点附近的区域,CBN-sites排列到化验。最后，我们发现natasha - boris基因位于20q13上通常参与乳腺癌、卵巢癌、脑癌和许多其他癌症的复制获得的区域内，并且实际上在这些肿瘤中异常激活，从而使这些因子成为一个非常有吸引力的新的抗癌靶点和超敏感的早期诊断工具。