Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

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From 10-01-06 to 9-01-07, we continued our studies of BORIS (an acronym for Brother of the Regulator of Imprinted Sites) - a unique CTCF-paralogous gene that we discovered. In humans and in mice, CTCF and BORIS genes encode polypeptides of similar size that share a centrally positioned near identical DNA binding domain (DBD) composed of 11 Zn-fingers (11ZF). In the mid 90s, this remarkable 11ZF region was defined as a multivalent DBD with respect to the diversity of unusually extended (average 50bp) target sequences recognized in double-stranded DNA. This DBD region shared in BORIS and CTCF is encoded by genomic sequences displaying an accurate duplication of a region containing all ZF-coding exons of CTCF gene from an early mammal. Therefore, while sharing with CTCF a duplicate of the 11ZF DBD allows BORIS to bind specifically to the same DNA-target sequences that interact with CTCF, in vivo binding of BORIS to a given CTCF-site would result to a different functional outcome compared to CTCF binding to the same site. Normally, CTCF and BORIS siblings are not expressed in the same cell to avoid functional interference caused by competition for binding to the same spectrum of 11ZF DNA-targets. Although numerous CTCF-target sequences do not always contain sites of mammalian methylation (meCpG), a subset of sites sensitive to this DNA modification have been associated with regulation and maintenance of normal mono-allelic DNA methylation and gene expression, histone-modifications patterns, presence of non-coding RNAs, and some additional features collectively known as epigenetic marks. Several methylation-sensitive CTCF-binding sites have been strongly implicated in the processes of epigenetic regulation in somatic cells, in ES cells, and in germline cells. Such sites have been universally mapped in differentially methylated domains of imprinting control regions (ICR) that regulate a parent-of-origin-dependent mono-allelic expression within clusters of imprinted genes. In 2000, three laboratories have simultaneously and independently reported mapping and functional characterization of a novel set of CTCF sites regulated by CpG-methylation in the imprinting control region (ICR) of the Igf2/H19 locus. Moreover, in our early papers on CTCF and imprinting, we predicted that in spite of the lack of any obvious homology between various ICRs mapped in different imprinted loci, most (if not all) of ICR sequences shall contain meCpG-sensitive CTCF-sites. This prediction turned out to be correct, since our collaborative efforts with several labs resulted in three more recent publications (see references in this and previous FY reports) describing CTCF-driven methylation-sensitive insulators in ICRs of Rasgrf-1, AWT1/WT1-AS, and KvDMR imprinted gene-loci. Using a biochemical system of Xenopus oocyte injection for expression of BORIS and its epigenetic co-factors Jelinic et. al. showed that BORIS recruits a complex containing PRMT7 and DNMTs to target CpG to CTCF-sites of the H19 ICR in a pre-assembled chromatin context. BORIS is normally strictly silenced in somatic cells, but activated together with a particular family of genes, called cancer-testis antigens (CTAs). Since promoter regions of genes encoding X-linked CTAs are activated during development of male germ cells undergoing genome-wide demethylation (and having decreased levels of CTCF), we asked if BORIS involved in coordinated regulation of CTA gene activation. In 2005, we discovered and published a novel class of CpG-containing CTCF-target sequences that manifested methylation-insensitive rather than methylation-sensitive interactions with CTCF. This type of CTCF/BORIS-target sites was at first found and characterized in MAGE-A1 and NY-ISO-1 - two best-studied examples of the CTA genes. Characterization of this new class of CTCF/BORIS-target-sequences allowed us to explain how CTCF can occupy in vivo a DNA region that is methylated/silenced as a part of inactivated chromatin, and how BORIS can bind to such DNA sequences to recruit demethylation to a hypermethylated CTCF-site, which results in demethylation and activation of CTA gene-promoters. Over a dozen of additional CTA and other germ/stem-specific genes, normally co-expressed in testis with BORIS in male germ cells, can also be activated by ectopic BORIS expression in normal primary human fibroblasts. Next, we showed that BORIS itself belongs to the CTA family, aberrantly activated in many types of human cancers. While CTCF is located at 16q22 locus at smallest region of overlap for LOH in wide variety of cancers, BORIS is located at 20q13 locus that is commonly copy-gained/amplified in these cancers. Therefore, we continued our studies directed to understanding the role of BORIS as an interfering mutation for tumor-suppressor activity of CTCF. Our ongoing studies suggest that competition of BORIS with CTCF for binding to the same common set of target sites can deregulate proliferation/apoptosis-related CTCF-target genes including c-myc, p27, p21, p53, and p19/ARF, PAX6, Wsb1/Nf1, BRCA1, Rb and hTERT, thus having an oncogenic effect. Highly restricted expression and unique function of BORIS provides a great opportunity for diagnostics, immunotherapeutic, or other targeted therapies. BORIS appears to be aberrantly expressed with a much higher then other CT-genes incidence in many tumors of different histological origin including, but not limited to breast, prostate, ovary, lung, gastric, liver, endometrial, glia, colon, esophagus but also is capable of up-regulating expression of other CT genes. Weve shown previously that BORIS when abnormally expressed in tumors induces antibody response in cancer patients (patent pending). More recently, we also showed (in collaboration with Dr. E. Klenova of the Essex University, UK) that at least breast cancer patients have BORIS expression in polymorphonuclear granulocytes. This tumor-related occurrence is a phenomenon not observed in donors with injuries and immune and inflammatory diseases. Detection of BORIS in a high proportion of patients with various types of breast tumors indicates that BORIS can be a valuable early blood marker of breast cancer. As a therapeutic approach weve tried vaccine approach. First, to obtain a solid proof of feasibility, Loukinov et al., tested BORIS as a vaccine in a very aggressive, highly metastatic, and poorly immunogenic murine model of mammary carcinoma. Immunizations with a DNA encoding the mutant form of murine BORIS antigen (excluding its 11ZF region) significantly prolonged survival, and inhibited tumor growth in BALB/c mice inoculated with 4T1 cells. Boosting with adenoviral vector expressing mBORIS was generally more effective, suggesting that the vaccination protocol could be further optimized. Then in collaboration with M. Agadjanyan we generated DNA- and protein-based mouse BORIS anti-tumor vaccines using again non-DNA-binding version of the BORIS molecule. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4+ T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production. Importantly, potent anticancer CD8+-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. Moreover, these cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma.