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

• 批准号: 10272128
• 负责人: Victor Lobanenkov
• 金额: $ 85.59万
• 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/10272128
• 关键词: 3-Dimensional; 4T1; ATAC-seq; Adenoviruses; Affect; Alphavirus; Animals; Antigens; Attention; Autologous; Binding; Binding Sites; Breast Cancer Model; Brothers; CRISPR/Cas technology; Cancer Patient; Cancer cell line; Cell Maintenance; Cells; Cessation of life; ChIP-seq; Chromatin; Chromatin Loop; Chromatin Remodeling Factor; Clinic; Clinical trial protocol document; Control Animal; Coupled; DNA; DNA Binding; DNA Binding Domain; Data; Defect; Dendritic Cells; Dependence; 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; Peripheral Blood Mononuclear Cell; 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; Ukraine; 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; driving force; 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; stemness; subfertility; therapy resistant; tissue culture; tool; transcriptome sequencing; treatment site; tumor; tumor growth; virtual; working group; zinc finger nuclease

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. The above data suggest that BORIS is likely to be not only a marker, but also a driving force for the cancer stemness phenotype. Notably, BORIS has been included in the list of high priority TAAs generated by the NCI Translational Working Group (NCI TWG) 91. As a proof of principle two animal breast cancer models have been successfully tested. On this basis, we developed UVAX-002, an autologous cellular vaccine comprising peripheral blood mononuclear cell-derived DC transfected with adenovirus-containing BORIS. The clinical trial protocol was already approved in the Ukraine by a Coordinating Center for extra-corporal cell maintenance and returning DCs to real patients

BORIS是全球三维基因组组织者CTCF的一个类似项目。虽然CTCF是普遍表达的，但BORIS的表达仅限于生殖细胞和癌细胞，使BORIS成为癌睾丸基因。我们对CTCF和BORIS的研究重点是开发合适的工具来研究这两个因素的功能。首先，我们建立了这两个因子的敲除动物，发现CTCF对胚胎的存活至关重要，而BORIS则参与正常的精子发生。BORIS-/-小鼠表现出低生育表型和多种精子发生缺陷。BORIS-/-小鼠的基因表达谱揭示了BORIS在精子发生过程中许多睾丸特异性基因的转录激活中的作用。特别是，通过BORIS结合内含子CTCF位点，可以诱导Gal3st1和FerT睾丸特异性亚型的表达。有趣的是，这两种转录本都是体细胞中表达的男性生殖细胞特异性同源基因的产物，两种转录本在癌细胞中都异常激活，与BORIS表达一致。为了将这两个例子扩展到全基因组范围，我们最近进行了染色质免疫沉淀结合下一代测序（ChIP-seq）来绘制几种癌细胞系的CTCF和BORIS结膜位点。结合我们的ChIP-Seq、RNA-seq和deep-CAGE-seq数据和ENCODE数据，我们发现BORIS在癌细胞中的异常表达导致CTCF内含子结合位点的多个睾丸特异性基因被激活。BORIS位点的种间保守性分析显示，人类和小鼠之间所有位点的高度保守性超过80%，这使我们确认了BORIS在人类癌细胞系和小鼠圆形精子中相似的全基因组占用。此外，我们的数据表明，尽管单个CTCF位点总是仅被CTCF结合（在所有细胞类型中），但在含有2xcts的reg的空间邻近驱动下，BORIS通过重新形成BORIS和CTCF异源二聚体而被cta基因以及许多内部肿瘤抑制基因和抗死亡促癌基因启动子募集时，正常的CTCF功能会丢失。DNA元件被两个相邻的11 ZF DNA结合域同时结合。