MECHANISMS OF TRANSCRIPTIONAL REGULATION BY CTCF

我们的团队和合作者已经鉴定并克隆了转录因子CTCF，其包含从果蝇到青蛙到鸟类到小鼠到人类的11个锌指（ZF）结构域。由CTCF ZF的不同组合识别的不同CTCF靶位点（CTS）执行不同的调节功能。根据上下文，不同的CTS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。这些研究发表了30多篇论文，并于1999年获得了“CTCF”专利。最近，脊椎动物染色质绝缘体（包括珠蛋白基因位点的边界）被确定为模型系统中CTCF驱动的增强子阻断活性所必需和充分的不同CTS。此外，我们发现并发表了与珠蛋白绝缘子CTS相比，具有增强子阻断功能的不同含CpG的CTS子集在体内以甲基化敏感的方式结合Igf 2/H19位点的印迹控制区中的CTCF。这一发现对IGF 2激活相关的基因印迹和肿瘤发生的意义将是非常深远的，可能对整个癌症分子生物学和遗传学领域产生重大影响。在通过CTCF调控的基因中，重要的细胞生长调节因子包括MYC、PIM-1、POLO样激酶和p19 ARF。 在后一种启动子中，已知其在肿瘤中经常变得高度甲基化，CTCF结合是CpG甲基化依赖性的。 在MYC基因座，CTCF似乎发挥双重作用，作为一个5-总理染色质边界的组成性核酸酶超敏感位点远离启动子区域，并作为一个阻遏物的下游立即每三个MYC启动子。这些发现可能对我们理解正常的分子机制产生重要影响，并且在癌症中经常失调，MYC和/或p19 ARF表达。我们对这些基因中功能重要的CTCF靶点的表征表明，CTCF中的获得性突变可能参与癌症的发展。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”内。多种肿瘤在该位点显示缺失，伴随着印记丢失和/或MYC表达失调以及p19 ARF异常甲基化。今年，我们在乳腺癌、前列腺癌和肾母细胞瘤病例中鉴定了几种不同的CTCF体细胞突变，并在编码CTCF 11-ZF结构域（整个蛋白质的三分之一）的外显子处分析了16 q22洛。突变发生在两个ZF中的任一个内，并导致ZF形成或DNA碱基识别的关键位置处的氨基酸取代。我们的研究结果还表明，CTCF通常识别不同的网站，通过组合使用的ZF与相同的个人ZF有时需要结合到一个网站，但不是另一个。这一发现对于多ZF蛋白是前所未有的。因此，与导致功能丧失的其他TSG中的肿瘤相关突变不同，CTCF中的突变是选择性功能障碍的，允许野生型结合到一些位点，同时完全废除对其他位点的识别。因此，我们获得了CTCF=TSG的直接证据。CTCF突变改变了结合特异性的范围，因此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重大影响。在肿瘤中CTCF中发现的突变可以帮助定义CTCF蛋白伴侣和靶基因，其与CTCF的相互作用或由CTCF的调节受癌症相关突变的影响，预期将揭示参与控制正常和恶性生长的新的调节途径，从而允许鉴定参与癌症发展的新基因。

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 to be the different CTSs that are necessary and sufficient for CTCF-driven enhancer-blocking activity in a model system. Furthermore, 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.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 tumors display deletions at this locus accompanied by loss of imprinting, and/or deregulated MYC expression, and p19ARF aberrant methylation. 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. 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 great impact on molecular diagnostics and therapy of cancers associated with CTCF. Mutations found in CTCF in tumors maay help to define CTCF protein partners and target genes whose interaction with, or regulation by, CTCF is affected by cancer-associated mutations are expected to uncover new regulatory pathways involved in control of normal and malignant growth thus allowing identification of new genes involved in cancer development.