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-2'-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 5'-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.