Deregulation of CTCF in Epigenetic Gene Silencing in Human Cancers

描述(由申请人提供)： 项目摘要。通过表观遗传去调控使肿瘤抑制基因沉默是人类恶性肿瘤中常见的现象。我们发现，依赖于CTCF的染色质边界的丢失会导致p16INK4a肿瘤抑制基因的转录失活，p16INK4a肿瘤抑制基因是许多类型人类癌症的表观遗传学沉默的常见靶点，被认为是乳腺癌发生的早期事件。在乳腺癌细胞系中，CTCF结合的丢失也与另外两个抑癌基因RASSF1A和CDH1的高甲基化和沉默有关。CTCF从这些抑癌基因的边界元件上解离是由于CTCF的多聚(ADP-核糖基)缺陷所致，这使其失去了应有的功能。在这项提案中，我们计划使用原代人类乳腺上皮细胞(HMECs)来分析CTCF在启动乳腺肿瘤发生的分子异常中的作用。在这个系统中，正常的人脐静脉内皮细胞克服了无限生长的障碍，出现了不同的细胞亚群。变异的HMECs表现出的第一个障碍与p16基因沉默相吻合，随后是表观遗传可塑性和染色体畸变率的增加，类似于在早期人类乳腺癌中观察到的那样。我们的具体目标是：使用生化方法(目标1)表征来自HMEC和p16沉默的vHMECs的天然CTCF蛋白复合体；使用基于细胞的方法(Aim 2)[和生化研究(Aim 3)][分析CTCF蛋白复合体在p16基因调控中的作用(Aim 4)]；[分析vHMEC中有缺陷的CTCF蛋白复合物的基础(Aim 2)[和生化研究(Aim 3)]。我们推测，由功能失调的CTCF复合体引起的特定染色体边界的不稳定可能是人类乳腺癌发生过程中的一个起始事件，其原因是关键抑癌基因的早期失活和表观遗传调节的正常基因组模式的丧失。

DESCRIPTION (provided by applicant): Project Summary. Silencing of tumor suppressor genes by epigenetic deregulation is a common occurrence in human malignancies. We find that loss of CTCF-dependent chromatin boundaries, which protects genes from adjacent heterochromatin domains, results in transcriptional inactivation of the p16INK4a tumor suppressor gene, which is a frequent target of epigenetic silencing in many types of human cancers and considered to be an early event in breast carcinogenesis. Loss of CTCF binding also correlates with hypermethylation and silencing of two other tumor suppressor genes, RASSF1A and CDH1, in breast cancer cell lines. CTCF dissociation from the boundary elements of these tumor suppressor genes results from defective poly(ADP-ribosyl)ation of CTCF, which abrogates its proper function. In this proposal, we plan to use primary human mammary epithelial cells (HMECs) to analyze the role of CTCF in molecular aberrations that initiate breast tumorigenesis. In this system, distinct subpopulations of cells emerge from normal HMECs that have overcome barriers to indefinite growth. The first barrier exhibited by "variant" HMECs coincides with p16 gene silencing followed by increasing epigenetic plasticity and chromosomal aberrations similar to those observed in early human breast cancers. Our specific aims are to: characterize native CTCF protein complexes from HMECs and p16-silenced vHMECs using biochemical approaches (Aim 1); examine the basis of defective CTCF PARlation in vHMECs using cell- based (Aim 2) [and biochemical studies (Aim 3)]; and [analyze the role of CTCF protein complexes in p16 gene regulation (Aim 4).] We postulate that destabilization of specific chromosomal boundaries by dysfunctional CTCF complexes may be an initiating event in the genesis of human breast cancers by early inactivation of critical tumor suppressor genes and loss of normal genomic patterns of epigenetic regulation.