ROLE OF TRANSCRIPTION FACTOR CTCF IN TUMOR DEVELOPMENT

在人类恶性肿瘤的发病机制中发挥作用的突变通常发生在肿瘤抑制基因（TSGs）中，TSGs通常起着严格控制细胞生长的作用。我们的团队和合作者已经鉴定并克隆了包含11锌指（ZF）结构域的转录因子CTCF，该转录因子在果蝇、青蛙、鸟类、小鼠和人类中都是保守的。不同的CTCF-target sites （CTSs）被不同的CTCF- ZFs组合识别，执行不同的调控功能。根据不同的背景，不同的CTSs在CTCF的转录调控中发挥不同的作用，包括启动子抑制、激活和甲状腺激素应答沉默子的产生。这些研究成果发表了30多篇论文，并于1999年获得了“CTCF”专利。包括珠蛋白基因位点边界在内的脊椎动物染色质绝缘体最近被Felsenfeld实验室（NIDDKD， NIH）确定为模型系统中ctcf驱动的增强子阻断活性所必需和充分的不同CTSs。此外，在与Ohlsson实验室（瑞典乌普萨拉大学）的合作中，我们发现并发表了与球蛋白绝缘体CTSs相比，具有增强子阻断功能的含有cpg的CTSs亚群以甲基化敏感的方式在体内Igf2/H19位点的印迹控制区结合CTCF。这一发现对于与IGF2激活相关的基因印迹和肿瘤发生的意义将是非常深远的，并可能对整个癌症分子生物学和遗传学领域产生重大影响，正如刚刚发表在《Nature》、《Science》和《Current Biology》杂志上的关于CTCF的综述所讨论的那样。通过CTCF调控的基因包括重要的细胞生长调节因子，包括MYC、PIM-1、polo样激酶和p19ARF。在后一个启动子中，已知在肿瘤中经常发生高甲基化，CTCF结合依赖于cpg甲基化。在MYC基因座中，CTCF似乎扮演着双重角色：在远离启动子区域的组成型核酸酶超敏感位点充当5- '染色质边界，并在三个MYC启动子的下游充当阻遏物。这些发现可能对我们理解癌症、MYC和/或p19ARF表达正常和经常失调的分子机制有重要影响。我们对这些基因中功能重要的CTCF靶点的表征表明，CTCF的获得性突变可能参与了癌症的发展。为了支持这一点，我们将CTCF定位在染色体16q22.1上一个狭窄的癌症相关“热点”上。包括一些乳腺癌、前列腺癌和Wilms肿瘤在内的多种癌症在该位点上显示缺失，并伴有印记缺失和/或MYC表达失调，以及p19ARF甲基化异常。CTCF基因位于16q22.1，位于乳腺、前列腺、Wilms等肿瘤中常见的杂合性缺失（LOH）的最小重叠区域内，并且在这些肿瘤中经常发现octcf调控的靶基因（如Igf2和MYC）的失调，提示CTCF参与肿瘤形成，即CTCF=TSG。今年，我们通过分析编码CTCF 11- zf结构域（整个蛋白的三分之一）的外显子的16q22 LOH，在乳腺癌、前列腺癌和Wilms肿瘤病例中发现了几种不同的CTCF体细胞突变。突变发生在两个ZF中的任何一个，并导致对ZF形成或DNA碱基识别至关重要的氨基酸位置的取代。每个突变都消除或大大减少了CTCF与控制细胞生长的基因（Igf2绝缘子、MYC启动子、PIM-1启动子、polo样激酶启动子和p19ARF）中不同靶点的结合，但没有改变CTCF与生长控制无关的β -珠蛋白绝缘子、溶菌酶沉默子或APP启动子中的靶点的相互作用。我们的结果还表明，CTCF通常通过组合使用相同的单个zf来识别不同的位点，有时需要与一个位点结合而不需要与另一个位点结合。这一发现对于多zf蛋白来说是前所未有的。因此，与其他tsg中导致功能丧失的肿瘤相关突变不同，CTCF中的突变是选择性功能失调的，允许野生型结合到某些位点，同时完全取消对其他位点的识别。因此，我们获得了CTCF=TSG的直接证据。因此，改变结合特异性谱的CTCF突变可能代表了肿瘤细胞逃避生长控制的新机制。这一发现将对CTCF相关癌症的分子诊断和治疗产生可预见的影响。

Mutations that play a role in the pathogenesis of human malignancies often occur in tumor suppressor genes (TSGs), which normally function to keep cell growth under strict control. Our team and collaborators have identified and cloned the transcription factor CTCF containing an 11 Zn finger (ZF) domain conserved from Drosophila to frogs to birds to mice to men. Different CTCF-target sites (CTSs), recognized by different combinations of CTCF ZFs, perform distinct regulatory functions. Depending on the context, different CTSs play distinct roles in transcriptional regulation by CTCF including promoter repression, activation, and creation of the thyroid hormone-responsive silencers. These studies resulted in more then 30 publications, and the "CTCF" Patent issued in 1999.Vertebrate chromatin insulators including the boundaries of the globin gene locus have been recently pinpointed by the Felsenfeld lab (NIDDKD, NIH) to be the different CTSs that are necessary and sufficient for CTCF-driven enhancer-blocking activity in a model system. Furthermore, in collaboration with the Ohlsson lab (Uppsala Univ., Sweden) we found and published that compared with the globin insulator CTSs, different CpG-containing subset of CTSs with an enhancer-blocking functions bind CTCF in the imprinting control region of the Igf2/H19 locus in vivo in a methylation-sensitive manner. Implications of this finding for gene imprinting and tumorigenesis associated with IGF2 activation will are very far-reaching and may have a significant impact on the whole field of Molecular Biology and Genetics of cancer as discussed in the reviews on CTCF just published in "Nature", "Science" and "Current Biology" journals. Among genes regulated through CTCF are important cell growth regulators including MYC, PIM-1, POLO-like kinase, and p19ARF. In the latter promoter, that is known to become frequently hypermethylated im tumors, CTCF binding is CpG-methylation dependent. In the MYC locus, CTCF appears to play a dual role by acting as a 5-prime chromatin boundary at the constitutive nuclease-hypersensitive site distant to the promoter region, and as a repressor immediately downstream of each of three MYC promoters. These findings may have an important impact on our understanding of molecular mechanisms of normal, and frequently dysregulated in cancer, MYC and/or p19ARF expression. Our characterization of functionally important CTCF targets in these genes suggested that acquired mutations in CTCF might be involved in cancer development. Supporting this, we mapped CTCF within a narrow cancer-associated "hot spot" on chromosome 16q22.1. A variety of cancers including some breast, prostate and Wilms' tumors display deletions at this locus accompanied by loss of imprinting, and/or deregulated MYC expression, and p19ARF aberrant methylation. The localization of the CTCF gene at 16q22.1 within the smallest region of overlap for losses of heterozygosity (LOH) often observed in breast, prostate, Wilms' and several other tumors and deregulation oCTCF-regulated target genes, such as Igf2 and MYC, frequently noticed in these tumors suggested an involvement of CTCF in neoplasia, i.e. that CTCF=TSG. This year we characterized several distinct somatic mutations of CTCF identified in breast, prostate and Wilms tumor cases with 16q22 LOH analyzed at exons coding for the CTCF 11-ZF-domain (one third of the entire protein). The mutations occurred within either of two ZFs and resulted in substitutions of amino acids at position critical for ZF formation or DNA base recognition. Each mutation abrogated or greatly diminished CTCF binding to different target sites in the genes governing cell growth (Igf2 insulator, and promoters of MYC, PIM-1, Polo-like kinase, and p19ARF) but did not change interaction of CTCF with targets found in growth-control-unrelated betta-globin insulator, lysozyme silencer or APP promoter. Our results also show that CTCF normally recognizes different sites by the combinatorial use of ZFs with the same individual ZFs sometimes being required for binding to one site but not another. This finding is unprecedented for multi-ZF proteins. Thus, unlike tumor-related mutations in other TSGs that lead to loss of function, mutations in CTCF are selectively dysfunctional, permitting wild-type binding to some sites while completely abrogating recognition of others. Thus, we obtained direct evidence that CTCF=TSG. CTCF mutations that shift the spectrum of binding specificities may thus represent a novel mechanism for tumor cell escape from growth control. This finding will have a predictable impact on molecular diagnostics and therapy of cancers associated with CTCF.