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 CTCF function is lost on some tumor-suppressor and oncogene promoters via BORIS recruitment and/or through BORIS and CTCF heterodimer formation at 2xCTSes binding sites. 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 repetitive 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. 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 stringent 4T1 mouse model of human breast cancer that was used for pre-clinical experiments with the HER2-based immunotherapy approved eventually 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 immunogen inhibited tumor growth and reduced metastass number 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 was capable of cancer elimination as we were able to cure 50% of animals compared to a 100% lethal outcome with untreated control animals. Based on the above data we believe that on ongoing attempt of translating CTCFL/BORIS targeting immunotherapeutic strategies to the clinic will provide new avenues for improving survival of cancer patients with advanced metastatic CSC-driven disease.

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Lobanenkov, Victor V; Zentner, Gabriel E (2018) Discovering a binary CTCF code with a little help from BORIS. Nucleus 9:33-41
Teplyakov, Evgeny; Wu, Qiongfang; Liu, Jian et al. (2017) The downregulation of putative anticancer target BORIS/CTCFL in an addicted myeloid cancer cell line modulates the expression of multiple protein coding and ncRNA genes. Oncotarget 8:73448-73468
Rivero-Hinojosa, Samuel; Kang, Sungyun; Lobanenkov, Victor V et al. (2017) Corrigendum: Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells. Sci Rep 7:46891
Rivero-Hinojosa, Samuel; Kang, Sungyun; Lobanenkov, Victor V et al. (2017) Testis-specific transcriptional regulators selectively occupy BORIS-bound CTCF target regions in mouse male germ cells. Sci Rep 7:41279
Pugacheva, Elena M; Rivero-Hinojosa, Samuel; Espinoza, Celso A et al. (2015) Comparative analyses of CTCF and BORIS occupancies uncover two distinct classes of CTCF binding genomic regions. Genome Biol 16:161
Schwarzenbach, Heidi; Eichelser, Corinna; Steinbach, Bettina et al. (2014) Differential regulation of MAGE-A1 promoter activity by BORIS and Sp1, both interacting with the TATA binding protein. BMC Cancer 14:796
Kemp, Christopher J; Moore, James M; Moser, Russell et al. (2014) CTCF haploinsufficiency destabilizes DNA methylation and predisposes to cancer. Cell Rep 7:1020-9
Mendez-Catala, Claudia Fabiola; Gretton, Svetlana; Vostrov, Alexander et al. (2013) A Novel Mechanism for CTCF in the Epigenetic Regulation of Bax in Breast Cancer Cells. Neoplasia 15:898-912
Shen, Yin; Yue, Feng; McCleary, David F et al. (2012) A map of the cis-regulatory sequences in the mouse genome. Nature 488:116-20
Moore, James M; Rabaia, Natalia A; Smith, Leslie E et al. (2012) Loss of maternal CTCF is associated with peri-implantation lethality of Ctcf null embryos. PLoS One 7:e34915

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