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: 8336243
• 负责人: Victor Lobanenkov
• 金额: $ 86.88万
• 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/8336243
• 关键词: 20q13; Affect; B-Lymphocytes; Binding; Cancer cell line; Cell Line; Cells; Chromatin; Chromatin Loop; Code; Collaborations; DNA; DNA Binding Domain; DNA Methylation; DNA Sequence; Development; Distant; Down-Regulation; Enhancers; Epigenetic Process; Exons; Fingers; Functional RNA; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Germ Cells; Goals; H19 gene; Hot Spot; Human; Human Chromosomes; Individual; Knock-in Mouse; Lead; Link; Lymphoid Cell; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Malignant neoplasm of testis; Maps; Mediating; Methylation; Modeling; Molecular; Mus; Oncogenes; Ovarian; PAX5 gene; Pathologic; Pattern; Play; Primary Neoplasm; Property; Protease Gene; Proteins; Publications; RNA Splicing; Regulation; Regulatory Element; Role; Site; Structure; Sum; System; T-Cell Lymphoma; TERT gene; Telomerase; Testing; Testis; Tissues; Transcriptional Activation; Transfection; Transgenic Organisms; Translational Research; Uterine Cancer; Work; base; cancer cell; cell immortalization; clinical application; embryonic stem cell; follow-up; genome-wide; immortalized cell; imprint; in vivo; lymph nodes; male; malignant breast neoplasm; model development; mutant; novel; osteosarcoma; paired box 5 protein (B-cell lineage specific activator); paralogous gene; prevent; promoter; research study; tumor; tumorigenesis; vector

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.

BORIS是具有候选癌基因和/或细胞永生化基因的性质的癌症/睾丸（CT）基因。它映射到人类染色体20 q13上的癌症相关热点，该热点在各种人类肿瘤中显示出拷贝数和/或扩增的频繁增加。与其他CT类似，BORIS在大多数原发性肿瘤和相关癌细胞系中异常表达。作为CTCF的一个副产物，BORIS与CTCF共享一个几乎相同的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，但可能具有不同的特性，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基因座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。我们和其他研究人员描述了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。使用Boris KO模型，我们证明了BORIS直接调节睾丸特异性蛋白酶基因TSP 50，而TSP 50又受p53负调控。我们发现，BORIS和TSP 50基因在癌症中的异常表达往往是一致的，这表明BORIS在激活作为下游靶点的CT基因中的作用。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，甲基化本身不是抑制CTCF与hTERT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。使用记录了BORIS异常表达的人类癌细胞系，以及具有BORIS编码载体的瞬时表达的细胞，我们表明BORIS在第一外显子内结合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在癌症和永生化细胞中表达，但它不是一个简单的二元系统。