Structure and Function of CTCF: Mouse Model Studies

CTCF的结构和功能：小鼠模型研究

• 批准号: 7118531
• 负责人: GALINA N FILIPPOVA
• 金额: $ 39.7万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-05 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118531
• 关键词: chromosomes; embryogenesis; flow cytometry; gene expression; gene targeting; genetic promoter element; genetic regulation; genetically modified animals; immunocytochemistry; laboratory mouse; methylation; neoplasm /cancer genetics; northern blottings; polymerase chain reaction; protein structure function; southern blotting; tissue /cell culture; transcription factor; tumor suppressor proteins

DESCRIPTION (provided by applicant): CTCF is a highly conserved 11 Zn finger (ZF) transcription factor and insulator protein. CTCF is involved in multiple aspects of gene regulation including promoter activation and repression, hormone-responsive gene silencing, methylation-dependent chromatin insulation, and genomic imprinting. Moreover, our recent discovery of a methylationsensitive CTCF insulator at the DM1 locus that might be responsible for the severe phenotype of Congenital Myotonic Dystrophy (Filippova et al.,2001), and our preliminary data on the role of CTCF insulators in X chromosome inactivation strongly suggest a link between CTCF and epigenetic regulation in development. Several findings also point to a role for CTCF as a tumor suppressor gene in both cancer epigenetics and genetics. Aberrant methylation of certain CTCF target sites, that is predicted to prevent CTCF-binding, has been demonstrated in a number of tumors. CTCF maps to human chromosome 16q22, within a region that displays frequent cancer-associated deletions (Filippova et al.,1998). We have observed somatic missense mutations within the CTCF 11ZF DNA-binding domain in breast, prostate and Wilms' tumors (Filippova et al.,2002). CTCF +/- mice exhibit enhanced tumor development in multiple tissues. Homozygous deletion of the CTCF results in early embryonic lethality, whereas the CTCF heterozygous mice exhibit decreased embryonic survival. Our broad and longterm hypothesis is that certain functions of CTCF are essential in early development and maintaining cell viability, whereas its other functions, when lost, result in the malignant phenotype. To test this hypothesis our specific aims will: Extend our studies on the role of CTCF in tumorigenesis (Aim 1) and early embryonic development (Aim 2). We will determine what genetic and epigenetic mechanisms are involved in CTCF haploinsufficiency. And finally (Aim 3), we will use the Cre-loxP system to generate a conditional CTCF mutant allele, that in combination with various tissue-specific Cre transgenic mice will allow us to test the hypothesis that complete loss of CTCF function leads to cell death in both normal and/or malignant cells, whereas loss of one CTCF allele in somatic tissue predisposes to tumor development. We will also generate knock-in mice harboring CTCF Zn finger point mutations that we have observed in human tumors to determine the functional significance of such mutated alleles in tumor predisposition. The significance of this proposal is that identifying the mechanisms of the CTCF haploinsufficiency will provide new insight into the broad spectrum of clinical human cancer phenotypes associated with LOH at 16q22.1 where CTCF maps. The health-relatedness is that the identified mechanisms can be targeted for both cancer prevention and therapeutic intervention.

描述(申请人提供)：CTCF是一个高度保守的11锌指(ZF)转录因子和绝缘蛋白。CTCF参与基因调控的多个方面，包括启动子的激活和抑制、激素反应的基因沉默、甲基化依赖的染色质分离和基因组印迹。此外，我们最近在DM1基因座上发现了一个甲基化敏感的CTCF绝缘子，这可能是导致先天性强直性肌营养不良症严重表型的原因(Filippova等人，2001)，以及我们关于CTCF绝缘子在X染色体失活中的作用的初步数据强烈表明CTCF与发育中的表观遗传调节之间存在联系。一些发现也指出了CTCF作为肿瘤抑制基因在癌症表观遗传学和遗传学中的作用。在一些肿瘤中已经证实了某些CTCF靶点的异常甲基化，这被预测为阻止CTCF结合。CTCF定位于人类染色体16q22，该区域显示与癌症相关的频繁缺失(Filippova等人，1998年)。我们在乳腺、前列腺和肾母细胞瘤中观察到CTCF11ZF DNA结合域中的体细胞错义突变(Filippova等人，2002年)。CTCF+/-小鼠在多个组织中表现出增强的肿瘤发展。CTCF纯合子缺失会导致早期胚胎死亡，而CTCF杂合子小鼠的胚胎存活率降低。我们广泛和长期的假设是，CTCF的某些功能在早期发育和维持细胞活力方面是必不可少的，而当它的其他功能丢失时，会导致恶性表型。为了验证这一假设，我们的具体目标是：扩展我们关于CTCF在肿瘤发生(目标1)和早期胚胎发育(目标2)中作用的研究。我们将确定哪些遗传和表观遗传机制参与了CTCF单倍体功能不全。最后(目标3)，我们将使用Cre-loxP系统产生一个条件性CTCF突变等位基因，它与各种组织特异性Cre转基因小鼠相结合，将使我们能够检验这样的假设，即CTCF功能完全丧失会导致正常和/或恶性细胞中的细胞死亡，而在躯体组织中丢失一个CTCF等位基因会导致肿瘤的发生。我们还将产生带有我们在人类肿瘤中观察到的CTCF锌指点突变的敲入小鼠，以确定这些突变的等位基因在肿瘤易感性中的功能意义。这一建议的意义在于，识别CTCF单倍性不足的机制将为临床上与16q22.1的CTCF图谱上的LOH相关的人类癌症表型提供新的见解。与健康相关的是，已识别的机制可以作为癌症预防和治疗干预的目标。