权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Regulation of CTCF Functions and Target Sites by Cancer/Testis-specific CTCF Like BORIS Factor

癌症/睾丸特异性 CTCF 样 BORIS 因子对 CTCF 功能和靶位点的调节

基本信息

批准号：
10014136
负责人：
Victor Lobanenkov
金额：
$ 81.94万
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/10014136
关键词：
3-Dimensional 4T1 ATAC-seq Affect Alphavirus Animals Antigens Attention Binding Binding Sites Breast Cancer Model Brothers CRISPR/Cas technology Cancer Patient Cancer cell line Cells Cessation of life ChIP-seq Chromatin Chromatin Loop Chromatin Remodeling Factor Clinic Control Animal Coupled DNA DNA Binding DNA Binding Domain Data Defect Dendritic Cells Dependence Dimensions Disease Distant ERBB2 gene Elements Embryo Endogenous Retroviruses Epigenetic Process FDA approved Family Gametogenesis Gene Expression Gene Expression Profiling Gene Expression Regulation Generations Genes Genetic Genetic Transcription Genome Genomic Segment Germ Cells Goals Housekeeping Human Immune Immunize Immunological Diagnosis Immunotherapeutic agent Immunotherapy In Vitro Journals K-562 K562 Cells Knock-out Knockout Mice Lead Light Link MCF7 cell MYCN gene Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of testis Maps Mediating Meiosis Methods Mitotic Modeling Molecular Mus Mutate NIH 3T3 Cells NIH Mouse Names Nature Neoplasm Metastasis Neuroblastoma Oncogenic Organ Outcome Pathologic Patients Phenotype Play Population Primates Protein Isoforms Proteins Protocols documentation Rattus Recombinants Recurrence Regulation Regulatory Element Reporting Research Resistance Retrotransposon Role Side Site Somatic Cell Specificity Spermatids Spermatogenesis Structure Tandem Repeat Sequences Testing Testis Tissues Transcript Transcription Initiation Site Transcriptional Activation Translating Translational Research Tumor Suppressor Genes Undifferentiated Up-Regulation Ursidae Family Vaccines base cancer cell cancer immunotherapy cancer site cancer stem cell cancer survival cancer therapy cell type chemotherapy chromatin immunoprecipitation chromatin remodeling clinical application design disease diagnosis experimental study gene product genome-wide human model humanized mouse immunogenic imprint improved in vivo inhibitor/antagonist knock-down knockout animal male malignant breast neoplasm mouse model next generation sequencing novel overexpression paralogous gene pre-clinical programs promoter recruit senescence stem-like cell subfertility therapy resistant tissue culture tool transcriptome sequencing treatment site tumor tumor growth virtual zinc finger nuclease

项目摘要

BORIS is a paralog of CTCF, the global three-dimensional genome organizer. While CTCF is ubiquitously expressed, BORIS expression is restricted to germ and cancer cells, making BORIS as a cancer-testis gene. Our research on CTCF and BORIS is focused on developing appropriate tools to study the functions of both factors. First, we generated knockout animals for the two factors and found that CTCF is essential for survival of the embryo, while BORIS is involved in normal spermatogenesis. BORIS-/- mice show a sub-fertility phenotype and multiple defects in spermatogenesis. Gene expression profiling of BORIS-/- mice uncovered a role for BORIS in transcriptional activation of many testis-specific genes during spermatogenesis. In particular, expression of Gal3st1 and FerT testes-specific isoforms is induced by BORIS binding to intronic CTCF sites. Interestingly, both transcripts are products of genes that are male germ cell-specific homologs of genes expressed in somatic cells and both transcripts are aberrantly activated in cancer cells, coinciding with BORIS expression. To extend these two examples to a genome-wide scale, we recently performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) to map both CTCF and BORIS vinding sites in several cancer cell lines. Combining our ChIP-Seq, RNA-seq, and deep-CAGE-seq data with ENCODE data, we found that an aberrant expression of BORIS in cancer cells results in activation of multiple testis-specific genes from intronic CTCF binding sites. The interspecies conservation analysis of BORIS sites revealed the high conservation of over 80% of all sites between human and mice, allowing us to confirm a similar BORIS genome-wide occupancy in both human cancer cell lines and mouse round spermatids. Moreover, our data suggest that although single CTCF sites are always bound by CTCF-only (in all cells types), normal CTCF function gets lost upon recruitment of BORIS to CTA-genes as well as to many house-keeping tumor suppressor genes and anti-death pro-oncogenic gene' promoters through de novo formation of BORIS and CTCF heterodimers driven by spatial proximity at 2xCTS-containing reg. DNA elements bound simultaneously by two adjacent paralogous 11 ZF DNA-bindig domains. Remarkably, this and our other major conclusions on aberrant chromatin regulatory interactions in BORIS-positive cancer cell lines have been convincingly confirmed in actual human neuroblastoma tumors with ALK-mutated & MYCN-amplified cells that gained resistance against chemotherapy with TAE684 ALK inhibitor through the initial loss of MYCN expression followed by subsequent activation and increasing over-expression of BORIS leading to the concomitant switch in proliferation dependence from MYCN to BORIS as described recently in a prestigious journal (Nature, Aug 2019) available at https://doi.org/10.1038/s41586-019-1472-0 online. Notably, aberrantly activated BORIS in the treatment-resistant cancer cells of real human neuroblastoma patients has been found to be enriched at 3D DNA-looping anchoring-points mapped with BORIS- vs. CTCF- specific ChIP_Seq and Hi-C methods within de-regulated genomic regions defined, as expected, by the same dual 2xCTS rather that single 1xCTS elements that we have previously mapped in a number of human cancer lines with high BORIS levels, including undifferentiated K562 and OvCa cells. We also recently confirmed that intronic CTCF sites could be reprogrammed into alternative transcription start sites via BORIS occupancy through epigenetic remodeling of chromatin. To identify the molecular mechanisms by which BORIS activates alternative transcription from intronic promoters, we plan to analyze what protein-partners are recruited by BORIS to initiate transcription and what kind of chromatin remodeling factors are involved in the activation of intronic class of Transcription Start Sites (TSSs) and whether any CTCF-assisted chromatin looping could be also involved by such TSS as well. RNA-seq following BORIS knockdown revealed a widespread upregulation of SVA repeat expression, suggesting that BORIS acts as a repressor of SVA transcription. Given that SVA repeats are primate-specific, these observations suggest that germline-restricted BORIS continued to evolve after the divergence of the primate lineage, acquiring a specific function in germline defense. A deeper analysis of CTCF and BORIS binding to a vast majority of currently known mouse and human repetitive DNA elements also revealed a striking distinction between repeat-contained CTCF-only, CTCF & BORIS, and BORIS-only sites. CTCF-only sites were enriched in evolutionarily ancient and inactive types of repeats, while CTCF & BORIS sites were mainly located in many other tandem repeats. In contrast, BORIS-only sites were found primarily within the evolutionarily young SVA class of repeats. SVA elements are primate specific, active retrotransposons, and so their uncontrolled activity presents a threat to the stability of the germline. Generation of BORIS humanized mice would provide an ideal model to test direct link between normal BORIS expression transposition-prone repeats including human-specific HERV and SVA families. Furthermore, CTCFL/BORIS has recently received additional attention as a very attractive immunotherapy target because it was found expressed in cancer stem cells (CSC) in vivo and in CSC-like side populations of cancer cell lines in tissue culture. Since SCS are believed to drive tumor growth recurrence, metastasis, and treatment resistance, while CTCFL/BORIS silencing lead to senescence and death of CSC, it appears therefore that our immunotherapeutic strategy that targets CTCFL/BORIS may lead to the selective destruction of CSC and potential eradication of metastatic disease. Indeed, the high immunotherapeutic potential of CTCFL/BORIS was shown in the same FDA-approved stringent 4T1 mouse model of human breast cancer that was used for pre-clinical experiments designed to develop the HER2-based immunotherapy that has been eventually approved by the FDA. Using these highly metastatic, poorly immunogenic carcinoma cells inoculated into Th2 prone mice we showed that Dendritic Cells (DC) fed with recombinant CTCFL/BORIS as immune antigen inhibited tumor growth and reduced metastases numbers in distant organs. While about 20% of CTCFL/BORIS immunized animals become tumor-free, approximately 50% (i.e., every second one) of the BORIS-immunized animals remained metastasis-free. Hence, at least the same widely used rat model of breast cancer showed that alphavirus-based CTCFL/BORIS immunotherapy (reviewed recently by D. Loukinov) was capable of cancer elimination as we were able literally cure 50% of animals compared to a 100% lethal outcome with untreated control animals. Based on the above data we believe that our most recently advanced and currently ongoing attempt of translating CTCFL/BORIS targeting immunotherapeutic strategies to the clinic will succeed to provide new avenues for improving survival of cancer patients with advanced metastatic disease driven by a therapy-resultant cell population of BORIS-expressing CSCs also known as Cancer Stem Cells.