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）鲍里斯和癌症。在过去的一年中，我们对Boris（印记网站的监管者的兄弟）进行了研究，这是我们发现的CTCF -PARALOG。 Boris与CTCF共享几乎相同的11 Zn手指（11ZF）DNA结合结构域（DBD），但它们的侧翼NH2-和COOH末端区域是不同的。先前在实验室中确定了11ZF区域为多价DBD，该DBD能够识别并结合扩展（约50bp）的目标序列。通过共享相同DBD的病毒，CTCF和Boris可以重新签署相同的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，由于鲍里斯在男性生殖细胞中的组织特异性表达，因此可能通过利用新型的CTCF/Boris遗址，通过特定的循环形成在IGF2/H19基因座的特定印迹位点重新建立。基于我们的研究，我们预测大多数ICR序列将包含对MECPG敏感的CTCF/BORIS目标位点，该目标位点已在几个无关的印迹基因座进行验证。除了其在发育中的作用外，鲍里斯还可能在肿瘤发生中起关键作用。实际上，虽然在正常的体细胞细胞中保持鲍里斯的表达是在癌细胞中激活的。即鲍里斯是一种所谓的癌症 - ct）基因。我们和其他人以前以前表征了子宫癌，乳腺癌，骨肉瘤，肺癌和前列腺癌中的鲍里斯表达。但是，由于鲍里斯本身是一种基因表达调节剂，因此假设鲍里斯介导的启动子的控制是负责多个CT基因表达的调节网络。最近，我们对原代头和颈部鳞状细胞癌（HNSCC）以及药理学脱甲基化的细胞系进行了全面的功能分析，以鉴定HNSCC中异常脱甲基化和表达的候选原始癌和癌症抗原。这项研究的结果暗示了鲍里斯通过协调的启动子脱甲基化在人类癌中表观遗传沉默的基因的重新激活。异常的鲍里斯表达与多种人类恶性肿瘤（包括原发性非小细胞肺癌和HNSCC）中的候选原癌基因的上调特别相关。此外，鲍里斯激活诱导了非氨基苯基细胞中的原始癌基因特异性启动子脱甲基化和表达，并转化了NIH3T3细胞。这些结果阐明了功能性p53丧失后，骨调节启动子沉默的放松机理。作为第一步，我们采用了一种利用癌症分析分析（COPA）的比较表观遗传学方法来测试49个原发性HNSCC和19个正常粘膜组织，以实现mRNA表达水平（Affymetrix U133A mRNA表达微阵列，16,383个探针集）。使用统计分析，我们确定了在癌细胞中显着上调的106个基因。选择了最高的26个基因进行进一步分析，其中17个被发现包含与启动子相关的CPG岛。在一个平行的实验中，我们分析了32,500个基因（U133PLUS2微阵列）相对于它们的上调，以响应5-aza/TSA治疗标准化细胞系，而这些细胞系未包括在原发性肿瘤表达分析中。在确定的46个靶基因中，有30个已确认有CPG岛。然后通过定量RT-PCR确认了9个目标基因样品的转录上调，并通过相应基因的瞬时转染确认。在此类基因中，有magea2，magea3/6，magea11，tktl1，magea4，c19orf28和grin1。由于几个法师基因的存在表明鲍里斯或CTCF可能参与了这种表观遗传调节，因此我们在36个原发性HNSCC的单独样本中评估了鲍里斯的表达。鲍里斯的过表达与6种原始基因的过表达显着相关，其中包括：MageA3/6，MageA4，MageA11，GPR17和C19orf28。此外，对公共癌症数据库的分析表明，所有肿瘤中有59％的骨水平超过了所有基因的中位表达，而90％的肿瘤至少具有超过boris的表达水平。所有基因的中位表达。这些数据证实了异常的鲍里斯 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，以模拟鲍里斯对候选靶基因的影响。在表达鲍里斯的OKF6-TERT1R细胞中，九个靶基因中有7个显着增加，而九个靶标中的六个显示出诱导鲍里斯后48小时的脱甲基化启动子的增长大于100％。因此，鲍里斯是参与促进生长促进原始癌基因候选物A的协调压缩的关键转录调节剂。因此，鲍里斯可能对多种致癌途径的定向作用提出了有希望的治疗靶标。 （2）关于鲍里斯在非癌细胞中作用的研究也导致了一些重要的发现。结果，提交了一份员工发明报告（E-165-2009/0-EIR-00），列出了Svetlana Pack，Ziyedulla N. Abdullayev和Victor V. Lobanenkov作为发明者。 NIAID已批准提交专利申请，该申请计划于2009年10月。