(1) BORIS and germline development. We continued our studies of BORIS (Brother Of the Regulator of Imprinted Sites) - a CTCF-paralog, which we discovered. 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 regulation and form different associations with protein cofactors. Furthermore, due to the tissue-specific expression of BORIS in male germ cells, it is likely involved in the 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. Indeed, while BORIS expression is silenced in normal somatic cells, it is activated in cancer cells;i.e. BORIS is a so-called cancer-testis (CT) gene. We and others previously characterized BORIS expression in uterine cancers, breast, lung, prostate cancers, osteosarcomas and others. 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. (2) BORIS and cancer: antagonism of BORIS and CTCF. 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 previously discovered that DNA methylation plays a 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 methylation per se is not the chief mechanism inhibiting CTCF binding at hTERT in germ cell cancers. We tested a hypothesis that abnormal activation of BORIS in those cancer cells prevents CTCF binding to some key sites, including hTERT promoter. Using human cancer cell lines where abnormal expression of BORIS was already 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. Down-regulation of BORIS led to a decrease of hTERT transcription in transient transfection experiments. However, in testicular and ovarian cell lines BORIS down-regulation did not affect endogenous hTERT transcription. Thus, BORIS might play the role of CTCF antagonist, enabling the expression of hTERT in cancer and immortalized cells, but it is not a simple binary system. The complexity of hTERT regulation was revealed by using a mutant which abolished CTCF binding within the exon1 of hTERT. In this mutant, BORIS was able to activate hTERT transcription. These results suggest that either cryptic BORIS-binding sites exist in this gene, the site(s) are bound by BORIS isoform(s), or that the regulation is mediated by other, yet unknown factors. (3) Studies on the BORIS role in stem cells have also led to several significant findings. BORIS is expressed in both mouse and human ES cells and its expression is shut down after differentiation. Based on RNA protection assays, the BORIS message in embryonic stem cells seems to be different from the one in adult testis. We have successfully produced mouse ES cells with floxed BORIS loci enabling us to remove murine BORIS and substitute with the human cDNA. Using these cells we will be able to perform ChIP-seq using our monoclonals to human BORIS that worked very well in ChIP.

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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
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
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
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
Mkrtichyan, Mikayel; Ghochikyan, Anahit; Davtyan, Hayk et al. (2011) Cancer-testis antigen, BORIS based vaccine delivered by dendritic cells is extremely effective against a very aggressive and highly metastatic mouse mammary carcinoma. Cell Immunol 270:188-97
Lobanenkov, Victor; Loukinov, Dmitry; Pugacheva, Elena (2011) Environmental epigenomics and disease susceptibility. Keystone symposia on molecular and cellular biology. The Grove Park Hotel & Spa, Ashville, NC, USA, 27 March–1 April 2011. Epigenomics 3:261-6

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