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: 8946422
• 负责人: Victor Lobanenkov
• 金额: $ 68.58万
• 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/8946422
• 关键词: 20q13; Adult; Affect; Alleles; Area; B-Lymphocytes; Binding; Binding Sites; Biological Assay; Breast; Brothers; Cancer cell line; Cell Line; Cells; ChIP-seq; Chromatin; Chromatin Loop; Chromosome Mapping; Code; Complementary DNA; DNA; DNA Binding; DNA Binding Domain; DNA Methylation; DNA Sequence; Development; Diagnostic; Diploidy; Down-Regulation; Epigenetic Process; Exons; Family; Fingers; Gametogenesis; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomics; Germ Cell Cancers; Germ Cells; Goals; H19 gene; Haploidy; Hot Spot; Human; Human Chromosomes; Human Pathology; Integration Host Factors; Lead; Lung; Lymphoid Cell; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of testis; Mediating; Meiosis; Methylation; Mitotic; Modeling; Molecular; Mus; Oncogenes; Ovarian; PAX5 gene; Pathologic; Pattern; Play; Primary Neoplasm; Primates; Property; Protease Gene; Protein Isoforms; Proteins; RNA; Regulation; Regulatory Element; Repression; Role; Site; Somatic Cell; Spermatids; Spermatocytes; Spermiogenesis; Stem cells; Structure; Sum; System; T-Cell Lymphoma; TERT gene; Telomerase; Testing; Testis; Tissues; Transcriptional Activation; Transfection; Translational Research; Tumor Suppressor Genes; Vaccination; Work; Workplace; base; cancer cell; cancer site; cancer testis antigen; cancer therapy; cell immortalization; cell type; clinical application; cofactor; embryonic stem cell; follow-up; genome-wide; human embryonic stem cell; immortalized cell; imprint; lymph nodes; male; mutant; neuronal cell body; novel; osteosarcoma; paired box 5 protein (B-cell lineage specific activator); paralogous gene; prevent; promoter; research study; senescence; tumor; tumorigenesis; vector

(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 mediatedby 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.

(1)BORIS和种系发育。 我们继续研究 BORIS（印记位点调节器的兄弟）——我们发现的 CTCF 旁系同源物。 BORIS 与 CTCF 共享几乎相同的 11 锌指 (11ZF) DNA 结合域 (DBD)，但它们的侧翼 NH2 和 COOH 末端区域不同。 11ZF 区域之前在实验室中被鉴定为多价 DBD，它能够识别并结合延伸的（约 50bp）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，它可能通过利用新的CTCF/BORIS位点，通过特定的环形成，参与在Igf2/H19基因座的特定印记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 靶位点，这已针对几个不相关的印记位点进行了验证。除了在发育中的作用外，BORIS 可能在肿瘤发生中发挥着关键作用。事实上，虽然 BORIS 表达在正常体细胞中被沉默，但它在癌细胞中被激活。即 BORIS 是一种所谓的睾丸癌 (CT) 基因。我们和其他人之前对子宫癌、乳腺癌、肺癌、前列腺癌、骨肉瘤等中的 BORIS 表达进行了表征。 由于BORIS本身是基因表达调控因子，因此推测BORIS介导的启动子调控是负责多个CT基因表达的调控网络。 (2)BORIS与癌症：DNA结合的BORIS与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BORIS直接调节癌症/睾丸特异性蛋白酶基因TSP50，而TSP50又受到p53的负调节。我们之前发现DNA甲基化在人类端粒酶基因hTERT（细胞永生化关键因子之一）的调节中发挥双重作用。甲基化可阻止具有阻遏活性的 CTCF 的结合，但核心启动子的部分低甲基化对于 hTERT 表达是必需的。然而，在淋巴细胞中，端粒酶似乎是通过不依赖甲基化的机制被激活的。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启动子是非甲基化的。 B 细胞特异性转录因子 PAX5 可以超越 CTCF 的抑制功能，并通过甲基化独立机制激活端粒酶阳性 B 细胞中的 hTERT。最近的研究表明，甲基化本身并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，即这些癌细胞中 BORIS 的异常激活会阻止 CTCF 与一些关键位点（包括 hTERT 启动子）的结合。 使用已经记录了 BORIS 异常表达的人类癌细胞系，以及短暂表达 BORIS 编码载体的细胞，我们发现 BORIS 与第一个外显子内的 hTERT 基因结合并促进其转录。在瞬时转染实验中，BORIS 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可能发挥CTCF拮抗剂的作用，使hTERT在癌细胞和永生化细胞中表达，但它不是一个简单的二元系统。通过使用废除 hTERT 外显子 1 内 CTCF 结合的突变体，揭示了 hTERT 调控的复杂性。在这个突变体中，BORIS 能够激活 hTERT 转录。这些结果表明，该基因中存在隐秘的 BORIS 结合位点，该位点与 BORIS 同种型结合，或者该调节是由其他未知因素介导的。 (3) 关于BORIS在干细胞中的作用的研究也得出了一些重要的发现。 BORIS 在小鼠和人 ES 细胞中表达，其表达在分化后关闭。根据 RNA 保护测定，胚胎干细胞中的 BORIS 信息似乎与成人睾丸中的不同。我们已经成功地产生了带有 floxed BORIS 基因座的小鼠 ES 细胞，使我们能够去除小鼠 BORIS 并用人类 cDNA 替代。使用这些细胞，我们将能够使用我们的人 BORIS 单克隆抗体进行 ChIP-seq，这在 ChIP 中效果非常好。