Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

BORIS 对正常和病理 CTCF 功能的表观遗传调控

• 批准号: 7964638
• 负责人: Victor Lobanenkov
• 金额: $ 59.08万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964638
• 关键词: 20q13; Antigens; Binding; Brothers; Cancer cell line; Cell Line; Cells; Chromatin; CpG Islands; DNA; DNA Binding Domain; DNA Methylation; DNA Sequence; Data; Databases; Development; Employee; Epigenetic Process; Fingers; GPR17 gene; Gene Expression; Gene Silencing; Gene Targeting; Genes; Germ Cells; Goals; Growth; Head and Neck Squamous Cell Carcinoma; Hot Spot; Hour; Human; Human Chromosomes; Lead; Legal patent; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Malignant neoplasm of testis; Maps; Mediating; Modeling; Molecular; Mucous Membrane; National Institute of Allergy and Infectious Disease; Non-Small-Cell Lung Carcinoma; Normal tissue morphology; Oncogenes; Oncogenic; Pathologic; Pathway interactions; Pattern; Play; Primary Neoplasm; Property; Proteins; Proto-Oncogenes; Regulation; Relaxation; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Site; Somatic Cell; Structure; TP53 gene; Testing; Testis; Tetracyclines; Tissues; Transcriptional Activation; Transfection; Translational Research; Tumor Suppressor Genes; Up-Regulation; Uterine Cancer; Work; base; cancer cell; cancer site; clinical application; cofactor; comparative; demethylation; derepression; functional loss; immortalized cell; imprint; mRNA Expression; male; malignant breast neoplasm; novel; osteosarcoma; overexpression; paralogous gene; promoter; research study; response; senescence; therapeutic target; tumor; tumorigenesis; tumorigenic

(1) BORIS and cancer. During the last year, we advanced our studies of BORIS (Brother Of the Regulator of Imprinted Sites) - a CTCF-paralog, which we discovered. BORIS shares with CTCF a nearly identical 11 Zn-finger (11ZF) DNA binding domain (DBD), but their flanking NH2- and COOH-terminal regions are divergent. The 11ZF region was previously identified in the lab as a multivalent DBD, which is able to recognize and bind extended (around 50bp) target sequences. By virue of sharing the identical DBD, CTCF and BORIS can recignize the same DNA sequences, but likely have distinct regulation and form different associations with protein cofactors. Furthermore, due to the tissue-specific expression of BORIS in male germ cells, it is likely involved in re-establishment of paternal-specific DNA methylation patterns at particular imprinted sites of the Igf2/H19 locus through specific loop formation, by utilizing novel CTCF/BORIS sites. Based on our studies we predicted that most ICR sequences would contain meCpG-sensitive CTCF/BORIS target sites, which was validated for several unrelated imprinted loci. In addition to its role in development, BORIS likely plays a key role in oncogenesis. Indeed, while BORIS expression is silenced in normal somatic cells, it is activated in cancer cells; i.e. BORIS is a so-called cancer-testis (CT) gene. We and others previously characterized BORIS expression in uterine cancers, breast cancers, osteosarcomas, lung cancers, and prostate cancers. However, as BORIS is itself a gene expression regulator, it was hypothesized that BORIS-mediated control of promoters is the regulatory network responsible for the expression of multiple CT genes. Recently, we conducted a comprehensive functional analysis of BORIS expression in primary head and neck squamous cell carcinoma (HNSCC) as well as pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. The results of this study implicate BORIS in the reactivation of epigenetically silenced genes in human cancers via coordinated promoter demethylation. Aberrant BORIS expression specifically correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC. Furthermore, BORIS activation induced coordinated proto-oncogene-specific promoter demethylation and expression in non-tumorigenic cells, as well as transformed NIH3T3 cells. These results elucidate the mechanism of relaxation of silencing of BORIS-regulated promoters following loss of functional p53. As the first step, we employed a comparative epigenetic approach utilizing Cancer Outlier Profiling Analysis (COPA) to test 49 primary HNSCC and 19 normal mucosal tissues for mRNA expression levels (the Affymetrix U133A mRNA expression microarray, 16,383 probe sets). Using statistical analysis we identified 106 genes that were significantly upregulated in cancer cells. The top scoring 26 genes were selected for further analyses, and 17 of them were discovered to contain promoter-associated CpG islands. In a parallel experiment we analyzed 32,500 genes (U133plus2 microarray) with respect to their upregulation in response to 5-aza/TSA treatment of normalized cell lines that were not included in primary tumor expression analysis. Among 46 target genes identified, 30 were confirmed to have CpG islands. Transcriptional upregulation of a 9 target gene sample after 5-aza/TSA treatment was then confirmed by quantitative RT-PCR and functionally - by transient transfections of the corresponding genes. Among such genes were MAGEA2, MAGEA3/6, MAGEA11, TKTL1, MAGEA4, C19ORF28, and GRIN1. As the presence of several MAGE genes targets indicated that BORIS or CTCF may be involved in such an epigenetic regulation, we assessed BORIS expression in a separate sample of 36 primary HNSCC. BORIS overexpression was significantly correlated with overexpression of 6 proto-oncogenes including: MAGEA3/6, MAGEA4, MAGEA11, GPR17, and C19ORF28. Furthermore, analysis of public cancer databases revealed that 59% of all tumors have BORIS levels exceeding the median expression of all genes, and 90% of tumors have BORIS expression level exceedind at least . of median expression for all genes. These data confirm that aberrant BORIS expression is a universal feature of human cancers. Finally, we used tetracycline-inducible pBIG2i-BORIS constructs transiently transfected into NIH-3T3 and OKF6-Tert1R cell lines to model the effect of BORIS on candidate target genes. Expression of seven of nine target genes was significantly increased in OKF6-Tert1R cell expressing BORIS, and six out of nine targets showed a greater than 100% increase in demethylated promoters 48 hours after induction of BORIS. Thus, BORIS is the key transcriptional regulator involved in the coordinated derepression of a of growth-promoting proto-oncogene candidates. Therefore, BORIS may present a promising therapeutic target for a directed effect on multiple oncogenic pathways. (2) Studies on BORIS role in non-cancer cells have also led to several significant findings. As a result, an Employee Invention Report (E-165-2009/0-EIR-00) was filed listing Svetlana Pack, Ziyedulla N. Abdullayev, and Victor V. Lobanenkov as inventors. NIAID has approved filing a patent application, which is planned for October, 2009.

(1)鲍里斯与癌症。去年，我们推进了我们发现的 CTCF 旁系同源物 BORIS（印记位点调节器兄弟）的研究。 BORIS 与 CTCF 共享几乎相同的 11 锌指 (11ZF) DNA 结合域 (DBD)，但它们的侧翼 NH2 和 COOH 末端区域不同。 11ZF 区域之前在实验室中被鉴定为多价 DBD，它能够识别并结合延伸的（约 50bp）靶序列。通过共享相同的 DBD，CTCF 和 BORIS 可以识别相同的 DNA 序列，但可能具有不同的调节并与蛋白质辅助因子形成不同的关联。此外，由于 BORIS 在雄性生殖细胞中的组织特异性表达，它可能通过利用新型 CTCF，通过特定的环形成，参与在 Igf2/H19 基因座的特定印记位点重建父本特异性 DNA 甲基化模式。 /鲍里斯网站。根据我们的研究，我们预测大多数 ICR 序列将包含 meCpG 敏感的 CTCF/BORIS 靶位点，这已针对几个不相关的印记位点进行了验证。除了在发育中的作用外，BORIS 可能在肿瘤发生中发挥着关键作用。事实上，虽然 BORIS 表达在正常体细胞中被沉默，但它在癌细胞中被激活。即 BORIS 是一种所谓的睾丸癌 (CT) 基因。我们和其他人之前对子宫癌、乳腺癌、骨肉瘤、肺癌和前列腺癌中的 BORIS 表达进行了表征。然而，由于BORIS本身是基因表达调节因子，因此推测BORIS介导的启动子控制是负责多个CT基因表达的调节网络。最近，我们对原发性头颈鳞状细胞癌（HNSCC）以及药理学去甲基化细胞系中的 BORIS 表达进行了全面的功能分析，以鉴定 HNSCC 中异常去甲基化和表达的候选原癌基因和癌睾丸抗原。这项研究的结果表明，BORIS 通过协调启动子去甲基化，重新激活人类癌症中表观遗传沉默的基因。异常的 BORIS 表达与多种人类恶性肿瘤（包括原发性非小细胞肺癌和 HNSCC）中候选原癌基因的上调特别相关。此外，BORIS 激活诱导非致瘤细胞以及转化的 NIH3T3 细胞中协调的原癌基因特异性启动子去甲基化和表达。这些结果阐明了功能性 p53 丧失后 BORIS 调节的启动子沉默松弛的机制。第一步，我们采用比较表观遗传学方法，利用癌症异常值分析 (COPA) 来测试 49 个原发性 HNSCC 和 19 个正常粘膜组织的 mRNA 表达水平（Affymetrix U133A mRNA 表达微阵列，16,383 个探针组）。通过统计分析，我们确定了 106 个在癌细胞中显着上调的基因。选择得分最高的 26 个基因进行进一步分析，发现其中 17 个含有启动子相关的 CpG 岛。在一项平行实验中，我们分析了 32,500 个基因（U133plus2 微阵列）在对未包括在原发性肿瘤表达分析中的标准化细胞系进行 5-aza/TSA 处理时的上调情况。在鉴定的 46 个靶基因中，有 30 个被确认具有 CpG 岛。 5-aza/TSA 处理后 9 个靶基因样品的转录上调通过定量 RT-PCR 得到证实，并通过相应基因的瞬时转染在功能上得到证实。这些基因包括 MAGEA2、MAGEA3/6、MAGEA11、TKTL1、MAGEA4、C19ORF28 和 GRIN1。由于多个 MAGE 基因靶标的存在表明 BORIS 或 CTCF 可能参与这种表观遗传调控，因此我们评估了 36 个原发性 HNSCC 的单独样本中的 BORIS 表达。 BORIS 过表达与 6 个原癌基因的过表达显着相关，包括：MAGEA3/6、MAGEA4、MAGEA11、GPR17 和 C19ORF28。此外，对公共癌症数据库的分析显示，59%的肿瘤的BORIS水平超过所有基因的中值表达，并且90%的肿瘤的BORIS表达水平至少超过。所有基因的中值表达。这些数据证实异常的鲍里斯 表达是人类癌症的普遍特征。最后，我们使用四环素诱导的 pBIG2i-BORIS 构建体瞬时转染到 NIH-3T3 和 OKF6-Tert1R 细胞系中，以模拟 BORIS 对候选靶基因的影响。在表达 BORIS 的 OKF6-Tert1R 细胞中，九个靶基因中的七个的表达显着增加，并且在诱导 BORIS 后 48 小时，九个靶基因中的六个显示去甲基化启动子增加超过 100%。因此，BORIS 是参与一系列促生长原癌基因候选物协调去抑制的关键转录调节因子。因此，BORIS 可能是一个有前途的治疗靶点，可以直接作用于多种致癌途径。 (2) 关于 BORIS 在非癌细胞中作用的研究也得出了一些重要的发现。因此，提交了一份员工发明报告 (E-165-2009/0-EIR-00)，其中将 Svetlana Pack、Ziyedulla N. Abdullayev 和 Victor V. Lobanenkov 列为发明人。 NIAID 已批准提交专利申请，计划于 2009 年 10 月提交。