Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

Boris是一种癌症/睾丸(CT)基因，具有候选癌基因和/或细胞永生化基因的特性。它映射到人类染色体20q13上与癌症相关的热点，该热点显示在各种人类肿瘤中拷贝数和/或扩增频繁增加。与其他CT相似，Boris在大多数原发肿瘤和相关癌细胞系中异常表达。作为CTCF-Paralog，Boris与CTCF共享几乎相同的11锌指(11ZF)DNA结合域(DBD)，但他们侧翼的NH2-和COOH-末端区域是不同的。11ZF区先前在实验室中被鉴定为多价DBD，它能够识别和结合延长的(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，但可能具有不同的性质，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，它可能参与了通过利用新的CTCF/Boris位点在Igf2/H19基因座的特定印迹位置通过特定的环形成重建父系特有的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靶点，这在几个不相关的印迹基因座上得到了验证。除了在发育中的作用外，Boris还可能在肿瘤发生中发挥关键作用。我们和其他人研究了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，因此假设Boris介导的启动子调控是负责多个CT基因表达的调控网络。利用Boris KO模型，我们证明了Boris直接调控睾丸特异的蛋白酶基因TSP50，而TSP50又受到P53的负调控。我们发现，Boris和TSP50基因在癌症中的异常表达经常是一致的，这表明Boris作为下游靶点在CT基因的激活中发挥了作用。在另一系列实验中，我们发现DNA甲基化在人类端粒酶基因hTERT的调控中起着双重作用，hTERT是细胞永生化的关键因素之一。甲基化阻止了具有抑制活性的CTCF的结合，但核心启动子的部分低甲基化是hTERT表达所必需的。然而，在淋巴样细胞中，端粒酶似乎是通过甲基化独立的机制激活的。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启动子没有甲基化。在端粒酶阳性的B细胞中，B细胞特异性转录因子PAX5可以通过甲基化非依赖性的机制，推翻CTCF的抑制功能，激活hTERT。综上所述，最近的研究表明，甲基化本身并不是抑制hTERT中CTCF结合的主要机制。我们测试了一个假设，即癌细胞中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。使用发现Boris基因异常表达的人癌细胞以及瞬时表达Boris编码载体的细胞，我们发现Boris结合了hTERT基因的第一个外显子，并促进了其转录。在瞬时转染实验中，Boris基因的下调导致hTERT转录减少。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可能扮演CTCF拮抗剂的角色，使hTERT在癌细胞和永生化细胞中得以表达，但这不是一个简单的二元系统。

BORIS is a cancer/testis (CT) gene with the properties of a candidate oncogene and/or a cell-immortalizing gene. It maps to a cancer-associated hot spot on human chromosome 20q13 that shows frequent gains in copy number and/or amplification in a wide variety of human tumors. Similar to other CT, BORIS is aberrantly expressed in the majority of primary tumors and related cancer cell lines. As a CTCF- paralog, 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 properties and form different associations with protein co-factors. 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. We and others 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 regulation of promoters is the regulatory network responsible for the expression of multiple CT genes. Using the Boris KO model, we demonstrated that BORIS directly regulates the testis-specific protease gene TSP50, which is in turn negatively regulated by p53. We found that aberrant expression of both BORIS and TSP50 genes in cancers often coincide, suggesting the role of BORIS in activation of CT genes as downstream targets. In another line of experiments, we discovered that DNA methylation plays 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 that methylation per se is not the chief mechanism inhibiting CTCF binding at hTERT. We tested a hypothesis that abnormal activation of BORIS in cancer cells prevents CTCF binding to some key sites, including the hTERT promoter. Using human cancer cell lines where abnormal expression of BORIS was 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. Downregulation of BORIS led to a decrease of hTERT transcription in transient transfection experiments. However, in testicular and ovarian cell lines BORIS downregulation did not affect endogenous hTERT transcription. Thus, BORIS may play the role of CTCF antagonist, enabling the expression of hTERT in cancer and immortalized cells, but it is not a simple binary system.