Hong Julie A, Kang Yang, Abdullaev Ziedulla, Flanagan Patrick T, Pack Svetlana D, Fischette Maria R, Adnani Mina T, Loukinov Dmitri I, Vatolin Sergei, Risinger John I, Custer Mary, Chen G Aaron, Zhao Ming, Nguyen Dao M, Barrett J Carl, Lobanenkov Victor V, Schrump David S
Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-1201, USA.
Cancer Res. 2005 Sep 1;65(17):7763-74. doi: 10.1158/0008-5472.CAN-05-0823.
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.
由一对独特的旁系同源因子CTCF和BORIS识别的调控序列，已被证明与印记和X染色体失活的表观遗传调控有关。肺癌表现出全基因组去甲基化，这与一类特定的编码癌胚（CT）抗原（如NY-ESO-1）的基因的去抑制相关。CT基因通常在缺乏CTCF和甲基化CpG含量的BORIS阳性雄性生殖细胞中表达，但在体细胞中则严格沉默。本研究旨在确定BORIS的异常激活是否在肺癌发生过程中导致NY-ESO-1的去抑制。初步实验表明，在培养的肺癌细胞中，NY-ESO-1的表达与BORIS的去抑制相一致。定量逆转录PCR分析显示，在临床相关条件下，用5-氮杂-2'-脱氧胞苷（5-azadC）、缩肽FK228（DP）或序贯5-azadC/DP处理后，肺癌细胞中BORIS和NY-ESO-1的表达有强烈的、一致的诱导，但在正常人支气管上皮细胞中则没有。亚硫酸氢盐测序、甲基化特异性PCR和染色质免疫沉淀（ChIP）实验表明，BORIS的诱导与该基因5'侧翼非编码区CpG岛内染色质结构的直接调节相一致。使用启动子-报告基因构建体的共转染实验证实，BORIS在肺癌细胞中调节NY-ESO-1的表达。凝胶迁移和ChIP实验揭示了NY-ESO-1启动子中一个新的CTCF/BORIS结合位点，与H19印记控制区和X染色体中的此类位点不同，该位点在体外对CpG甲基化不敏感。CTCF在体内对该位点的占据与NY-ESO-1启动子的沉默相关，而从CTCF占据转换为BORIS占据则与NY-ESO-1的去抑制相一致。总的来说，这些数据表明CTCF和BORIS与NY-ESO-1启动子的相互结合介导了肺癌细胞中该CT基因的表观遗传调控，并提示BORIS的诱导可能是增强肺癌免疫原性的一种新策略。
