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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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，该热点在多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 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，但可能具有不同的特性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，它可能通过利用新型 CTCF，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。 /鲍里斯网站。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。我们和其他人对子宫癌、乳腺癌、骨肉瘤、肺癌和前列腺癌中的 BORIS 表达进行了表征。然而，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。使用Boris KO模型，我们证明BORIS直接调节睾丸特异性蛋白酶基因TSP50，而TSP50又受到p53的负调节。我们发现癌症中 BORIS 和 TSP50 基因的异常表达常常一致，表明 BORIS 在作为下游靶点的 CT 基因激活中的作用。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（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，甲基化本身并不是抑制 CTCF 在 hTERT 上结合的主要机制。我们测试了一个假设，即癌细胞中 BORIS 的异常激活会阻止 CTCF 与某些关键位点（包括 hTERT 启动子）的结合。使用记录有 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。