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.

鲍里斯和癌症。在过去的一年里，我们进一步研究了BORIS（印迹位点监管机构的兄弟），这是我们发现的ctcf类似物。BORIS与CTCF具有几乎相同的11 Zn-finger (11ZF) DNA结合域（DBD），但它们两侧的NH2-和cooh末端区域是不同的。11ZF区域先前在实验室中被鉴定为多价DBD，能够识别和结合延伸（约50bp）的靶序列。由于共享相同的DBD， CTCF和BORIS可以识别相同的DNA序列，但可能具有不同的调控，与蛋白质辅因子形成不同的关联。此外，由于BORIS在男性生殖细胞中的组织特异性表达，它可能通过利用新的CTCF/BORIS位点形成特异性环，参与在Igf2/H19位点的特定印迹位点重建父本特异性DNA甲基化模式。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靶点，这在几个不相关的印迹位点上得到了验证。除了在发育中发挥作用外，BORIS可能在肿瘤发生中发挥关键作用。事实上，BORIS的表达在正常体细胞中是沉默的，但在癌细胞中却被激活；即BORIS是一种所谓的癌睾丸（CT）基因。我们和其他人先前研究了BORIS在子宫癌、乳腺癌、骨肉瘤、肺癌和前列腺癌中的表达。然而，由于BORIS本身是一个基因表达调控因子，因此我们假设BORIS介导的启动子调控是多个CT基因表达的调控网络。最近，我们对原发性头颈部鳞状细胞癌（HNSCC）和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通过协同启动子去甲基化参与了人类癌症中表观遗传沉默基因的再激活。BORIS的异常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异性相关。此外，BORIS激活在非致瘤性细胞和转化的NIH3T3细胞中诱导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缺失后，boris调控启动子沉默的放松机制。作为第一步，我们采用比较表观遗传学方法，利用癌症异常分析（COPA）检测49例原发性HNSCC和19例正常粘膜组织的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个在癌细胞中显著上调的基因。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5-aza/TSA处理正常化细胞系时的上调反应，这些细胞系未包括在原发肿瘤表达分析中。在所鉴定的46个靶基因中，有30个被证实存在CpG岛。5-aza/TSA处理后，9个目标基因样本的转录上调，然后通过定量RT-PCR和相应基因的瞬时转染功能证实。这些基因包括MAGEA2、MAGEA3/6、MAGEA11、TKTL1、MAGEA4、C19ORF28和GRIN1。由于多个MAGE基因靶点的存在表明BORIS或CTCF可能参与了这种表观遗传调控，我们在36例原发性HNSCC的单独样本中评估了BORIS的表达。BORIS过表达与MAGEA3/6、MAGEA4、MAGEA11、GPR17、C19ORF28等6个原癌基因过表达显著相关。此外，对公共癌症数据库的分析显示，59%的肿瘤BORIS表达水平超过所有基因的中位数，90%的肿瘤BORIS表达水平至少超过中位数。所有基因的中位数表达。这些数据证实了异常的BORIS