ROLE OF TRANSCRIPTION FACTOR CTCF IN TUMOR DEVELOPMENT

在人类恶性肿瘤发病机制中发挥作用的突变经常发生在肿瘤抑制基因(TSG)中，TSG通常具有严格控制细胞生长的功能。我们的团队和合作者已经鉴定并克隆了包含11个锌指(ZF)结构域的转录因子CTCF，该结构域在果蝇、青蛙、鸟类、老鼠和人中保守。不同的CTCF靶点(CTS)由CTCFZF的不同组合识别，执行不同的调节功能。根据上下文的不同，不同的CTS在CTCF的转录调控中发挥着不同的作用，包括启动子的抑制、激活和甲状腺激素反应沉默的产生。这些研究发表了30多篇论文，并于1999年发布了“CTCF”专利。脊椎动物染色质绝缘体，包括珠蛋白基因位点的边界，最近被费森菲尔德实验室(NIDDKD，NIH)准确地确定为模型系统中CTCF驱动的增强子阻断活性所必需和充分的不同CTS。此外，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珠蛋白绝缘体CTS相比，具有增强子阻断功能的不同CpG亚集CTS在体内以甲基化敏感的方式结合在Igf2/H19基因座印记控制区的CTCF。这一发现对与IGF2激活相关的基因印记和肿瘤发生的影响将是非常深远的，并可能对癌症的整个分子生物学和遗传学领域产生重大影响，正如刚刚在《自然》、《科学》和《当代生物学》杂志上发表的关于CTCF的评论中所讨论的那样。在CTCF调控的基因中有重要的细胞生长调节因子，包括MYC、PIM-1、Polo样激酶和p19ARF。在后者启动子中，已知的是肿瘤中经常发生高甲基化，CTCF结合依赖于CpG甲基化。在MYC基因座上，CTCF似乎扮演着双重角色，在远离启动子区域的构成核酸酶敏感部位，作为5个质点的染色质边界，以及作为紧接在三个MYC启动子下游的抑制因子。这些发现可能对我们理解正常和经常在癌症中异常表达的MYC和/或p19ARF的分子机制有重要影响。我们对这些基因中重要的CTCF靶点的鉴定表明，CTCF的获得性突变可能与癌症的发生有关。为了支持这一点，我们将CTCF定位在染色体16q22.1上与癌症相关的一个狭窄“热点”内。各种癌症，包括一些乳腺癌、前列腺癌和肾母细胞瘤，都表现出该基因的缺失，并伴有印迹丢失和/或MYC表达紊乱，以及p19ARF异常甲基化。CTCF基因位于16q22.1的最小重叠区域，是乳腺癌、前列腺癌、肾母细胞瘤和其他几种肿瘤中常见的杂合性丢失(LOH)，以及在这些肿瘤中常见的CTCF调节的靶基因(如Igf2和MYC)的去调控，表明CTCF参与了肿瘤的发生，即CTCF=TSG。今年，我们分析了在乳腺、前列腺和肾母细胞瘤中发现的几种不同的CTCF体细胞突变，分析了编码CTCF11-ZF结构域(占整个蛋白质的三分之一)的外显子上的16q22 LOH。突变发生在两个ZF中的任何一个内，并导致ZF形成或DNA碱基识别关键位置的氨基酸替换。每个突变取消或大大减少了CTCF与控制细胞生长的基因(Igf2绝缘子、MYC、PIM-1、Polo-like Kinase和p19ARF的启动子)中不同靶点的结合，但不改变CTCF与生长控制无关的贝珠蛋白绝缘子、溶菌酶沉默因子或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ZF识别不同的位点，有时需要相同的ZF与一个位点结合，但不需要与另一个位点结合。这一发现对于多ZF蛋白质来说是前所未有的。因此，与导致功能丧失的其他TSG中的肿瘤相关突变不同，CTCF中的突变是选择性的功能障碍，允许野生型与某些位点结合，同时完全取消对其他位点的识别。因此，我们得到了CTCF=TSG的直接证据。因此，CTCF突变改变了结合特异性的光谱，可能代表了肿瘤细胞逃避生长控制的一种新机制。这一发现将对与CTCF相关的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.