Brother of the Regulator of Imprinted Sites (BORIS) is a mammalian CTCF paralog with the same central 11Zn fingers (11ZF) that mediate specific interactions with varying f50-bp target sites. Regulated in vivo occupancy of such sites may yield structurally and functionally distinct CTCF/DNA complexes involved in various aspects of gene regulation, including epigenetic control of gene imprinting and X chromosome inactivation. The latter functions are mediated by meCpGsensitive 11ZF binding. Because CTCF is normally present in all somatic cells, whereas BORIS is active only in CTCF-and 5-methylcytosine-deficient adult male germ cells, switching DNA occupancy from CTCF to BORIS was suggested to regulate site specificity and timing of epigenetic reprogramming. In addition to 11ZF-binding paternal imprinting control regions, cancer-testis gene promoters also undergo remethylation during CTCF/BORIS switching in germ cells. Only promoters of cancer testis genes are normally silenced in all somatic cells but activated during spermatogenesis when demethylated in BORIS-positive germ cells and are found aberrantly derepressed in various tumors. We show here that BORIS is also expressed in multiple cancers and is thus itself a cancer-testis gene and that conditional expression of BORIS in normal fibroblasts activates cancer-testis genes selectively. We tested if replacement of CTCF by BORIS on regulatory DNA occurs in vivo on activation of a prototype cancer-testis gene, MAGE-A1. Transition from a hypermethylated/silenced to a hypomethylated/activated status induced in normal cells by 5-aza-2V -deoxycytidine (5-azadC) was mimicked by conditional input of BORIS and is associated with complete switching from CTCF to BORIS occupancy at a single 11ZF target. This site manifested a novel type of CTCF/BORIS 11ZF binding insensitive to CpG methylation. Whereas 5-azadC induction of BORIS takes only few hours, derepression of MAGE-A1 occurred 1 to 2 days later, suggesting that BORIS mediates cancer-testis gene activation by 5-azadC. Indeed, infection of normal fibroblasts with anti-BORIS short hairpin RNA retroviruses before treatment with 5-azadC blocked reactivation of MAGE-A1. We suggest that BORIS is likely tethering epigenetic machinery to a novel class of CTCF/BORIS 11ZF target sequences that mediate induction of cancer-testis genes. (Cancer Res 2005; 65(17): 7751-62)

Regulatory sequences recognized by the unique pair of paralogous factors, CTCF and BORIS, have been implicated in epigenetic regulation of imprinting and X chromosome inactivation. Lung cancers exhibit genome-wide demethylation associated with derepression of a specific class of genes encoding cancer-testis (CT) antigens such as NY-ESO-1. CT genes are normally expressed in BORIS-positive male germ cells deficient in CTCF and meCpG contents, but are strictly silenced in somatic cells. The present study was undertaken to ascertain if aberrant activation of BORIS contributes to derepression of NY-ESO-1 during pulmonary carcinogenesis. Preliminary experiments indicated that NY-ESO-1 expression coincided with derepression of BORIS in cultured lung cancer cells. Quantitative reverse transcription-PCR analysis revealed robust, coincident induction of BORIS and NY-ESO-1 expression in lung cancer cells, but not normal human bronchial epithelial cells following 5-aza-2V -deoxycytidine (5-azadC), Depsipeptide FK228 (DP), or sequential 5-azadC/DP exposure under clinically relevant conditions. Bisulfite sequencing, methylation-specific PCR, and chromatin immunoprecipitation (ChIP) experiments showed that induction of BORIS coincided with direct modulation of chromatin structure within a CpG island in the 5V -flanking noncoding region of this gene. Cotransfection experiments using promoter-reporter constructs confirmed that BORIS modulates NY-ESO-1 expression in lung cancer cells. Gel shift and ChIP experiments revealed a novel CTCF/BORIS-binding site in the NY-ESO-1 promoter, which unlike such sites in the H19-imprinting control region and X chromosome, is insensitive to CpG methylation in vitro. In vivo occupancy of this site by CTCF was associated with silencing of the NY-ESO-1 promoter, whereas switching from CTCF to BORIS occupancy coincided with derepression of NY-ESO-1. Collectively, these data indicate that reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter mediates epigenetic regulation of this CT gene in lung cancer cells, and suggest that induction of BORIS may be a novel strategy to augment immunogenicity of pulmonary carcinomas. (Cancer Res 2005; 65(17): 7763-74)

The murine Igh locus has a 3 regulatory region (3 RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3 RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3 RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro-and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. The immunoglobulin heavy chain locus (Igh) (see Fig. 1) is arguably the most complex mammalian locus. B-cell development is marked by the progressive expression of Igh genes (via VDJ recombination, somatic hypermutation, and class switch recombination [CSR]). These regulated processes, some involving substantial DNA rearrangements and deletions, are required for the generation of antibody diversity (reviewed in references 47 and 57). Spanning ϳ3 Mb, the murine Igh locus contains a limited number of enhancers, including the intronic enhancer (E), which has been implicated in V to DJ joining and chain expression (reviewed in reference 47), and a complex 3Ј regulatory region (3Ј RR) located immediately downstream of C␣, the last constant region gene. The human Igh locus contains two 3Ј RRs, each located downstream of one of the duplicated C␣ genes (9, 49, 62).The murine 3Ј RR spans ϳ28 kb downstream of C␣ and comprises four enhancers, each associated with DNase I hypersensitivity (hs3A, hs1,2, hs3B, and hs4) (reviewed in reference 33). hs3A and hs3B are located at the termini of a 25-kb inverted repeat flanking hs1,2 (7, 66), and these three enhancers acquire DNase I hypersensitivity at later stages of B-cell development (66). The hs4 enhancer is the only 3Ј enhancer with activity and DNase I hypersensitivity throughout B-cell development (19, 48), although no role for hs4 or any of the 3Ј enhancers in early B-cell development has been shown.Targeted deletions of the 3Ј RR enhancers have shown that hs3A and hs1,2 are individually dispensable for B-cell development and Igh expression (46), while the combination of hs3B and hs4 is critical for the process of CSR (63). The 3Ј RR has also been proposed to regulate Igh expression in plasma cells (23,40,48,63,70). In accord with earlier suggestions that it functions as a locus control region (43), the 3Ј RR shows synergistic activity and position-independent regulation when tested in transgenic model systems (8,33,59). However, copynumber-dependent expression has not been observed (8). In addition to a function within the Igh locus, the 3Ј RR is likely to be responsible for dysregulation of c-myc expression resulting from certain Igh::c-myc translocations in mouse plasmacytoma cells as well as in human Burkitt's lymphoma and multiple myeloma cells (30,36,43).Th...

The choice mechanisms that determine the future inactive X chromosome in somatic cells of female mammals involve the regulated expression of the XIST gene. A familial C(-43)G mutation in the XIST promoter results in skewing of X chromosome inactivation (XCI) towards the inactive X chromosome of heterozygous females, whereas a C(-43)A mutation found primarily in the active X chromosome results in the opposite skewing pattern. Both mutations point to the existence of a factor that might be responsible for the skewed patterns. Here we identify this factor as CTCF, a conserved protein with a 11 Zn-finger (ZF) domain that can mediate multiple sequence-specificity and interactions between DNA-bound CTCF molecules. We show that mouse and human Xist/XIST promoters contain one homologous CTCF-binding sequence with the matching dG-contacts, which in the human XIST include the -43 position within the DNase I footprint of CTCF. While the C(-43)A mutation abrogates CTCF binding, the C(-43)G mutation results in a dramatic increase in CTCF-binding efficiency by altering ZF-usage mode required for recognition of the altered dG-contacts of the mutant site. Thus, the skewing effect of the two -43C mutations correlates with their effects on CTCF binding. Finally, CTCF interacts with the XIST/Xist promoter only in female human and mouse cells. The interpretation that this reflected a preferential interaction with the promoter of the active Xist allele was confirmed in mouse fetal placenta. These observations are in keeping with the possibility that the choice of X chromosome inactivation reflects stabilization of a higher order chromatin conformation impinging on the CTCF-XIST promoter complex.