Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

(1)BORIS和生殖系发育。 我们继续我们的研究BORIS（兄弟的印记网站的监管机构）-一个CTCF-paradox，我们发现。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。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节并与蛋白质辅因子形成不同的缔合。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位点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。我们和其他人先前描述了BORIS在子宫癌、乳腺癌、肺癌、前列腺癌、骨肉瘤和其他肿瘤中的表达。 由于BORIS本身是一种基因表达调节剂，因此假设BORIS介导的启动子调节是负责多个CT基因表达的调节网络。 (2)BORIS和癌症：DNA结合的BORIS和CTCF结合位点的正常功能之间的拮抗作用。 使用Boris KO模型，我们证明了BORIS直接调节癌症/睾丸特异性蛋白酶基因TSP 50，而TSP 50又受到p53的负调控。我们先前发现DNA甲基化在人类端粒酶基因hTERT的调控中起双重作用，hTERT是细胞永生化的关键因子之一。甲基化阻止了CTCF的结合，CTCF具有阻遏活性，但是核心启动子的部分低甲基化对于hTERT表达是必需的。然而，在淋巴样细胞中，端粒酶似乎是通过甲基化非依赖性机制激活的。在我们的后续工作中，我们发现在B细胞、一些T细胞淋巴瘤和非肿瘤性淋巴结中，hTERT启动子未甲基化。B细胞特异性转录因子PAX 5可通过甲基化非依赖性机制克服CTCF的抑制功能并激活端粒酶阳性B细胞中的hTERT。最近的研究表明，甲基化本身不是生殖细胞癌中抑制CTCF与hTERT结合的主要机制。 我们测试了一个假设，即在这些癌细胞中BORIS的异常激活阻止了CTCF与一些关键位点的结合，包括hTERT启动子。 使用人癌细胞系，其中BORIS的异常表达已经被记录，以及与BORIS编码载体的瞬时表达的细胞，我们表明，BORIS结合的第一外显子内的hTERT基因，并促进其转录。在瞬时转染实验中，BORIS的下调导致hTERT转录的减少。然而，在睾丸和卵巢细胞系中，BORIS下调并不影响内源性hTERT转录。因此，BORIS可能发挥CTCF拮抗剂的作用，使hTERT在肿瘤和永生化细胞中表达，但它不是一个简单的二元系统。hTERT调控的复杂性是通过使用一个突变体来揭示的，该突变体消除了hTERT外显子1内的CTCF结合。在该突变体中，BORIS能够激活hTERT转录。这些结果表明，在该基因中存在隐蔽的BORIS结合位点，该位点被BORIS同种型结合，或者该调节由其他未知因子介导。 (3)关于BORIS在干细胞中的作用的研究也导致了几个重要的发现。BORIS在小鼠和人ES细胞中都有表达，其表达在分化后被关闭。基于RNA保护分析，胚胎干细胞中的BORIS信息似乎与成人睾丸中的BORIS信息不同。我们已经成功地产生了具有floxed BORIS基因座的小鼠ES细胞，使我们能够去除鼠BORIS并用人cDNA替代。使用这些细胞，我们将能够使用我们的单克隆抗体对人BORIS进行ChIP-seq，该单克隆抗体在ChIP中工作得非常好。

(1) BORIS and germline development. We continued 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 virtue of sharing the identical DBD, CTCF and BORIS can recognize 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 the 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, breast, lung, prostate cancers, osteosarcomas and others. As BORIS is itself a gene expression regulator, it was hypothesized that BORIS-mediated regulation of promoters is the regulatory network responsible for the expression of multiple CT genes. (2) BORIS and cancer: antagonism between DNA-bound BORIS and normal functions of CTCF-binding sites. Using the Boris KO model we demonstrated that BORIS directly regulates the cancer/testis-specific protease gene TSP50, which is in turn negatively regulated by p53. We previously discovered that DNA methylation plays a dual role in the regulation of human telomerase gene, hTERT, one of the key cell immortalization factors. Methylation prevents binding of CTCF, which has repressor activity, but partial hypomethylation of the core promoter is necessary for hTERT expression. In lymphoid cells, however, telomerase appears to be activated through a methylation-independent mechanism. In our follow up work we found that in B cells, some T cell lymphomas, and in non-neoplastic lymph nodes, the hTERT promoter is unmethylated. The B cell-specific transcription factor PAX5 can override the repressive function of CTCF and activate hTERT in telomerase-positive B cells by a methylation-independent mechanism. The sum of recent studies suggests that methylation per se is not the chief mechanism inhibiting CTCF binding at hTERT in germ cell cancers. We tested a hypothesis that abnormal activation of BORIS in those cancer cells prevents CTCF binding to some key sites, including hTERT promoter. Using human cancer cell lines where abnormal expression of BORIS was already documented, as well as cells with transient expression of BORIS-coding vectors, we showed that BORIS binds the hTERT gene within the first exon and facilitates its transcription. Down-regulation of BORIS led to a decrease of hTERT transcription in transient transfection experiments. However, in testicular and ovarian cell lines BORIS down-regulation did not affect endogenous hTERT transcription. Thus, BORIS might play the role of CTCF antagonist, enabling the expression of hTERT in cancer and immortalized cells, but it is not a simple binary system. The complexity of hTERT regulation was revealed by using a mutant which abolished CTCF binding within the exon1 of hTERT. In this mutant, BORIS was able to activate hTERT transcription. These results suggest that either cryptic BORIS-binding sites exist in this gene, the site(s) are bound by BORIS isoform(s), or that the regulation is mediated by other, yet unknown factors. (3) Studies on the BORIS role in stem cells have also led to several significant findings. BORIS is expressed in both mouse and human ES cells and its expression is shut down after differentiation. Based on RNA protection assays, the BORIS message in embryonic stem cells seems to be different from the one in adult testis. We have successfully produced mouse ES cells with floxed BORIS loci enabling us to remove murine BORIS and substitute with the human cDNA. Using these cells we will be able to perform ChIP-seq using our monoclonals to human BORIS that worked very well in ChIP.