CTCF- and LR1-binding sites in the Ccnd1 gene. (A) Schematic representation of putative CTCF- and LR1-binding sites within and around the Ccnd1 gene. (B) Gel retardation assay using a labeled oligonucleotide corresponding to the CTCF2 sequence. dI/dC, unlabeled nonspecific competitor; FII, canonical CTCF site34 ; NPE, nuclear protein extract prepared from HeLa cell nuclei. CTCF2 and CTCF26 indicate unlabeled oligonucleotides tested for their capacity to compete for labeled CTCF2. The arrow points to the position of the CTCF2-specific band. (C) CTCF binding in vivo. ChIP was performed on Granta-519 cells and normal lymphocytes using anti-CTCF dark and light gray bars, CTCF (Ab) or a nonspecific antibody (black bars, ctrl IgG). The precipitated DNA fragments were analyzed by quantitative PCR using primers for amplification of sequences located ∼70 kb centromeric to the Ccnd1 gene or within its exon 4, neither being recognized by CTCF (immunoprecipitation negative controls, “ctrl” and “Ex4”); for CTCF26 and CTCF2, the 2 potential CTCF binding sites identified in Ccnd1. Error bars represent the standard deviation between duplicates within the same experiment. (D) LR1-binding sites in the Ccnd1 gene as transcriptional enhancers. The transcriptional activity of the 3 potential LR1-binding sites identified in the Ccnd1 gene was tested in HeLa cells 48 hours after transfection. The enhancer strength was quantified relative to the luciferase activity generated with the reference SV40 promoter.
