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) BORIS与种系发育。我们继续研究BORIS（印迹位点监管者的兄弟）-我们发现的ctcf类似物。BORIS与CTCF具有几乎相同的11 Zn-finger (11ZF) DNA结合域（DBD），但它们两侧的NH2-和cooh末端区域是不同的。11ZF区域先前在实验室中被鉴定为多价DBD，能够识别和结合延伸（约50bp）的靶序列。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控，与蛋白质辅因子形成不同的关联。此外，由于BORIS在男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位点形成特异性环，参与在Igf2/H19位点的特定印迹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靶点，这在几个不相关的印迹位点上得到了验证。除了在发育中发挥作用外，BORIS可能在肿瘤发生中发挥关键作用。事实上，BORIS的表达在正常体细胞中是沉默的，但在癌细胞中却被激活；即BORIS是一种所谓的癌睾丸（CT）基因。我们和其他研究人员先前研究了BORIS在子宫癌、乳腺癌、肺癌、前列腺癌、骨肉瘤等肿瘤中的表达。由于BORIS本身是一个基因表达调控因子，因此我们假设BORIS介导的启动子调控是多个CT基因表达的调控网络。