Epigenetic Regulation of the Human Retinoblastoma Tumor Suppressor Gene Promoter by CTCF

Abstract

Epigenetic misregulation is a more common feature in human cancer than previously anticipated. In the present investigation, we identified CCCTC-binding factor (CTCF), the multivalent 11-zinc-finger nuclear factor, as a regulator that favors a particular local chromatin conformation of the human retinoblastoma gene promoter. We show that its binding contributes to Rb gene promoter epigenetic stability. Ablation of the CTCF binding site from the human Rb gene promoter induced a rapid epigenetic silencing of reporter gene expression in an integrated genome context. CTCF DNA binding is methylation sensitive, and the methylated Rb-CTCF site is recognized by the Kaiso methyl-CpG-binding protein. This is the first evidence suggesting that CTCF protects the Rb gene promoter, a classic CpG island, against DNA methylation, and when such control region is abnormally methylated Kaiso, and probably its associated repressor complex, induce epigenetic silencing of the promoter. Our results identify CTCF as a novel epigenetic regulator of the human retinoblastoma gene promoter. [Cancer Res 2007;67(6):2577-85]

Epigenetic regulation of the human p53 gene promoter by the CTCF transcription factor in transformed cell lines

“…We next carried out a chromatin immunoprecipitation (ChIP) assay using an antibody against human CTCF and by using primers that flank the novel CTCF-binding sequence in several cell lines. As a negative amplification control we used the exon 27 of the Rb gene, as reported previously (De La Rosa-Vela´zquez et al, 2007). Interestingly, we observed enrichment of the CTCF immunoprecipitated fraction in SW480 and HeLa cell lines, but not in the U87MG and T98G glioma cells lines in which p53 gene expression is silenced (Figure 3c).…”

“…We found a putative CTCFbinding motif in a DNA fragment located at B700 bp upstream of the p53 transcription start site (Figure 3a) (De La Rosa-Vela´zquez et al, 2007;Kim et al, 2007). We performed a gel shift analysis using total extracts from HeLa cells to determine whether CTCF associates with this sequence in vitro (Figure 3b).…”

“…HeLa cells were chosen because they show an intermediate level of expression, between SW480 cells and primary normal fibroblasts (Figure 1a). In addition, HeLa cells were used in our previous analysis to show the epigenetic contribution of CTCF to human retinoblastoma gene regulation (De La Rosa-Vela´zquez et al, 2007). Thus, CTCF knockdown will allow us to perform a comparative analysis between p53 and Rb gene expression profiles.…”

“…(c) ChIP assays were carried out on different cell lines, including the two glioma cell lines. As a negative amplification control we used primers from exon 27 of the human retinoblastoma gene (De La Rosa-Vela´zquez et al, 2007). (d) ChIP assays were performed on T98G glioma cells, untreated (T98G mock) and treated with 5-aza-2'-deoxycytidine (5-azadC).…”

“…We hypothesize that different classes of CpG islands might be regulated differently, not only at the level of DNA methylation and/or Polycomb family members but also in terms of selective and differential use of transcription factors. In accordance, in this study we address whether different kinds of CpG island promoters are epigenetically regulated by alternative mechanisms (De La Rosa-Vela´zquez et al, 2007). We found that the multifunctional CCCTC-binding site factor (CTCF) is involved in p53 epigenetic regulation by harboring an open chromatin configuration.…”