Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

(1)BORIS和癌症在过去的一年里，我们推进了我们对BORIS（印记网站监管者的兄弟）的研究-我们发现的CTCF-paradox。BORIS与CTCF共享几乎相同的11锌指（11 ZF）DNA结合结构域（DBD），但它们的侧翼NH 2-和COOH-末端区域是不同的。11 ZF区域先前在实验室中被鉴定为多价DBD，其能够识别并结合延伸的（约50 bp）靶序列。CTCF和BORIS由于具有相同的DBD，可以识别相同的DNA序列，但可能具有不同的调节作用，并与蛋白质辅因子形成不同的结合。此外，由于BORIS在雄性生殖细胞中的组织特异性表达，它可能通过利用新的CTCF/BORIS位点，通过特异性环形成，参与在Igf 2/H19基因座的特定印迹位点处重建父系特异性DNA甲基化模式。基于我们的研究，我们预测大多数ICR序列将包含meCpG敏感的CTCF/BORIS靶位点，这在几个不相关的印迹基因座中得到了验证。除了在发育中的作用外，BORIS可能在肿瘤发生中起关键作用。事实上，虽然BORIS表达在正常体细胞中沉默，但它在癌细胞中被激活;即BORIS是所谓的癌症睾丸（CT）基因。我们和其他人先前表征了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本身是一种基因表达调节剂，因此假设BORIS介导的启动子控制是负责多个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）以及药理学脱甲基细胞系中BORIS表达进行了全面的功能分析，以鉴定HNSCC中异常脱甲基和表达的候选原癌基因和癌症睾丸抗原。这项研究的结果表明，BORIS通过协调启动子去甲基化，重新激活了人类癌症中表观遗传学沉默的基因。BORIS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激活诱导协调的原癌基因特异性启动子去甲基化和非致瘤细胞中的表达，以及转化的NIH 3 T3细胞。这些结果阐明了功能性p53丧失后BORIS调节的启动子沉默的松弛机制。作为第一步，我们采用了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（Affytron U133 A mRNA表达微阵列，16，383个探针组）。使用统计分析，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个基因含有启动子相关的CpG岛。在一个平行实验中，我们分析了32，500个基因（U133 plus 2微阵列）对未包括在原发性肿瘤表达分析中的标准化细胞系的5-aza/TSA处理的响应上调。在所鉴定的46个靶基因中，30个被证实具有CpG岛。然后通过定量RT-PCR和功能性地通过相应基因的瞬时转染证实5-aza/TSA处理后9个靶基因样品的转录上调。这些基因包括MAGEA 2、MAGEA 3/6、MAGEA 11、TKTL 1、MAGEA 4、C19 ORF 28和GRIN 1。由于几个法师基因靶点的存在表明BORIS或CTCF可能参与这种表观遗传调控，我们评估了36例原发性HNSCC的单独样本中BORIS的表达。BORIS过表达与6种原癌基因的过表达显著相关，包括：MAGEA 3/6、MAGEA 4、MAGEA 11、GPR 17和C19 ORF 28。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位表达，90%的肿瘤的BORIS表达水平至少超过100%。所有基因的平均表达量。这些数据证实了异常的BORIS 表达是人类癌症的普遍特征。最后，我们使用四环素诱导的pBIG 2 i-BORIS构建体瞬时转染到NIH-3 T3和OKF 6-Tert 1 R细胞系中，以模拟BORIS对候选靶基因的作用。在表达BORIS的OKF 6-Tert 1 R细胞中，9个靶基因中有7个的表达显著增加，在BORIS诱导后48小时，9个靶基因中有6个的去甲基化启动子增加超过100%。因此，BORIS是参与促进生长的原癌基因候选者的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，对多种致癌途径产生直接影响。 (2)关于BORIS在非癌细胞中的作用的研究也导致了几个重要的发现。因此，提交了一份雇员发明报告（E-165-2009/0-EIR-00），列出了Svetlana Pack，Ziyedulla N。Abdullayev和维克托V. Lobanenkov作为发明人。NIAID已批准提交专利申请，计划于2009年10月提交。

(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.