The INK4B-ARF-INK4A (INK/ARF) locus is composed of three tumor suppressor genes, which are kept silenced by DNA methylation in different cancer types. In addition. a non-coding RNA (ANRIL) is transcribed in the anti-sense orientation upstream of the ARF gene. The resulting divergent promoter region is bound by the chromatin insulator protein CTCF in association with histone H3 tri-methylated on lysine 4, irrespective of transcription of ANRIL and ARF. Methylation of the overlapping CpG island abolishes CTCF binding and the associated modification, which can be restored by 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment. shRNA knock down of CTCF expression dramatically reduces the induction of ANRIL and ARF, but also that of INK4A and INK4B expression by 5-Aza-dC. We propose that CTCF is an essential factor for transcription of the INK/ARF locus and that abrogation of its binding by DNA methylation contributes to the permanent silencing of several genes of the locus in tumors. (C) 2010 Elsevier Inc. All rights reserved.