Allele-Specific Binding of CTCF to the Multipartite Imprinting Control Region KvDMR1

Abstract

Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of theKcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.

Functional interplay between MyoD and CTCF in regulating long-range chromatin interactions during differentiation

“…As shown in Fig. 2A, and in agreement with a previous report (Fitzpatrick et al, 2007), we observed that CTCF binds to both the F1 and F2 subregions (corresponding to the CTS1 and CTS2 fragments described by Fitzpatrick and co-workers). In addition, as shown in the same figure, we found that CTCF also binds to the F3 subregion in all three cell types analyzed, implying the existence of a further and previously unidentified CTCF-binding site within KvDMR1.…”

“…The inspection of ChIP-seq data from ENCODE/Caltech for CTCF and MyoD in mouse C2C12 muscle cells (a cell line related to C2.7) showed weak but detectable signals for both factors along KvDMR1, further supporting our ChIP results. Interestingly, whereas CTCF binding to F1 is specific for the paternal allele (Fitzpatrick et al, 2007), we observed that CTCF binds biallelically to F3 (Fig. 2B), just as we had previously found for MyoD (Busanello et al, 2012).…”

“…1). Two of these sites, previously demonstrated to be bound by CTCF in mouse fibroblasts (Fitzpatrick et al, 2007), are located in the F1 and F2 subregions, whereas the third, predicted by MatInspector tool, is located in the F3 subregion, which we had previously demonstrated to be functionally relevant for MyoD-induced rearrangement (Busanello et al, 2012). We also noticed the presence of at least one putative CTCF-binding site within the p57 promoter (Fig.…”

Genome-wide and parental allele-specific analysis of CTCF and cohesin DNA binding in mouse brain reveals a tissue-specific binding pattern and an association with imprinted differentially methylated regions

“…CTCF has long been associated with genomic imprinting due to its selective binding of the unmethylated maternal allele of the Igf2/H19 ICR resulting in parent-of-origin-specific expression of Igf2 and H19 in mouse and human (Bell and Felsenfeld 2000;Hark et al 2000;Kanduri et al 2000;Fedoriw et al 2004;Szabo et al 2004). CTCF has been studied at several other imprinted loci, and it binds the unmethylated allele at the gDMRs of Rasgrf1, Peg13, Kcnq1ot1 (Yoon et al 2005;Fitzpatrick et al 2007;Singh et al 2011), and Grb10 (Hikichi et al 2003;Mukhopadhyay et al 2004). CTCF-mediated regulation is postulated to be one of two major control mechanisms operating at ICRs (Lewis and Reik 2006;Kim et al 2009).…”

A cross-talk between DNA methylation and H3 lysine 9 dimethylation at the KvDMR1 region controls the induction of Cdkn1c in muscle cells

“…The first one would involve the enhancer-blocking activity of paternal KvDMR1, mediated by the insulator CCCTC-binding factor (CTCF). 15 The second one would involve chromatin silencing by paternally expressed Kcnq1ot1, a macro noncoding RNA, the promoter of which is comprised within the KvDMR1 CpG island, 16 and which has been suggested to regulate in cis the silencing of adjacent genes by recruiting epigenetic modifiers. 17 As found for many imprinted genes, especially those involved in growth control, alterations in the dosage of Cdkn1c expression cause important functional consequences.…”