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: 8745467
• 负责人: Victor Lobanenkov
• 金额: $ 76.58万
• 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/8745467
• 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; Code; Complementary DNA; DNA; DNA Binding; DNA Binding Domain; DNA Methylation; DNA Sequence; Development; Diagnostic; Down-Regulation; Epigenetic Process; Exons; Family; Fingers; Gametogenesis; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomics; Germ Cell Cancers; Germ Cells; Goals; H19 gene; Hot Spot; Human; Human Chromosomes; Human Pathology; Integration Host Factors; Lead; Lung; Lymphoid Cell; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of testis; Maps; 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; 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 therapy; cell immortalization; 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 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.

（1）鲍里斯和种系的发展。 我们继续研究鲍里斯（印刷地点的监管者的兄弟） - 我们发现的CTCF -PARALOG。 Boris与CTCF共享几乎相同的11 Zn手指（11ZF）DNA结合结构域（DBD），但它们的侧翼NH2-和COOH末端区域是不同的。先前在实验室中确定了11ZF区域为多价DBD，该DBD能够识别并结合扩展（约50bp）的目标序列。通过共享相同的DBD，CTCF和Boris可以识别相同的DNA序列，但可能具有不同的调节并与蛋白质辅助因子形成不同的关联。此外，由于鲍里斯在雄性生殖细胞中的组织特异性表达，它很可能参与了通过利用新型CTCF/Boris遗址的特定环路形成IGF2/H19基因座的特定印迹位点的父亲特异性DNA甲基化模式。基于我们的研究，我们预测大多数ICR序列将包含对MECPG敏感的CTCF/BORIS目标位点，该目标位点已在几个无关的印迹基因座进行验证。除了其在发育中的作用外，鲍里斯还可能在肿瘤发生中起关键作用。实际上，虽然在正常的体细胞细胞中保持鲍里斯的表达是在癌细胞中激活的。即鲍里斯是一种所谓的癌症 - ct）基因。我们和其他人以前曾在子宫，乳房，肺，前列腺癌，骨肉瘤等中表达鲍里斯的表达。 由于鲍里斯本身是一种基因表达调节剂，因此假设鲍里斯介导的启动子的调节是负责多个CT基因表达的调节网络。 （2）鲍里斯和癌症：结合DNA结合的鲍里斯与CTCF结合位点的正常功能之间的拮抗作用。 使用Boris KO模型，我们证明了鲍里斯直接调节癌症/睾丸特异性蛋白酶基因TSP50，而这反过来又受p53负调节。我们先前发现，DNA甲基化在人类端粒酶基因HTERT的调节中起双重作用，HTERT是关键细胞永生因子之一。甲基化阻止了CTCF的结合，CTCF具有抑制剂活性，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细胞淋巴瘤和非塑性淋巴结中，HTERT启动子是未甲基化的。 B细胞特异性转录因子PAX5可以通过非甲基化机制覆盖CTCF的抑制作用，并激活端粒酶阳性B细胞中的HTERT。最近的研究总和表明，甲基化本身并不是抑制生殖细胞癌中HTERT结合CTCF结合的主要机制。 我们检验了一个假设，即这些癌细胞中鲍里斯的异常激活阻止CTCF与某些关键部位（包括HTERT启动子）结合。 使用已经记录了鲍里斯异常表达的人类癌细胞系，以及具有鲍里斯编码载体的瞬时表达的细胞，我们表明鲍里斯在第一个外显子内结合了HTERT基因并促进其转录。鲍里斯的下调导致瞬时转染实验中HTERT转录的减少。但是，在睾丸和卵巢细胞系中，骨下调不影响内源性HTERT转录。因此，鲍里斯（Boris）可能会扮演CTCF拮抗剂的作用，从而使HTERT在癌症和永生细胞中的表达表达，但这不是一个简单的二元系统。通过使用一种消除HTERT外显子内CTCF结合的突变体来揭示HTERT调节的复杂性。在这个突变体中，鲍里斯能够激活HTERT转录。这些结果表明，在该基因中存在隐性骨结合位点，该位点由鲍里斯同工型绑定，或者该调节是由其他但未知的因素介导的。 （3）关于鲍里斯在干细胞中作用的研究也导致了一些重要的发现。鲍里斯在小鼠和人ES细胞中均表达，其表达在分化后被关闭。基于RNA保护测定，胚胎干细胞中的鲍里斯消息似乎与成人睾丸中的消息不同。我们已经成功地用Floxed Boris loci产生了小鼠ES细胞，使我们能够去除鼠鲍里斯并用人cDNA替代。使用这些细胞，我们将能够使用我们的单克隆人对芯片效果很好的人类鲍里斯（Boris）进行CHIP-SEQ。