Targeted Deletion of Multiple CTCF-Binding Elements in the Human C-MYC Gene Reveals a Requirement for CTCF in C-MYC Expression

Wendy M Gombert,Anton Krumm,Mikhail V Blagosklonny

doi:10.1371/journal.pone.0006109

Abstract

BackgroundInsulators and domain boundaries both shield genes from adjacent enhancers and inhibit intrusion of heterochromatin into transgenes. Previous studies examined the functional mechanism of the MYC insulator element MINE and its CTCF binding sites in the context of transgenes that were randomly inserted into the genome by transfection. However, the contribution of CTCF binding sites to both gene regulation and maintenance of chromatin has not been tested at the endogenous MYC gene.Methodology/Principal FindingsTo determine the impact of CTCF binding on MYC expression, a series of mutant human chromosomal alleles was prepared in homologous recombination-efficient DT40 cells and individually transferred by microcell fusion into murine cells. Functional tests reported here reveal that deletion of CTCF binding elements within the MINE does not impact the capacity of this locus to correctly organize an ‘accessible’ open chromatin domain, suggesting that these sites are not essential for the formation of a competent, transcriptionally active locus. Moreover, deletion of the CTCF site at the MYC P2 promoter reduces transcription but does not affect promoter acetylation or serum-inducible transcription. Importantly, removal of either CTCF site leads to DNA methylation of flanking sequences, thereby contributing to progressive loss of transcriptional activity.ConclusionsThese findings collectively demonstrate that CTCF-binding at the human MYC locus does not repress transcriptional activity but is required for protection from DNA methylation.

Highlights

Insulator and boundary elements shield genomic loci from unscheduled transcriptional activation or repression by regulatory elements
The CTCF-binding site within the MINE is coincident with the boundary of hyperacetylation of the MYC promoter. This arrangement suggested that CTCF functions as a barrier for the MYC locus by the formation and/or maintenance of chromatin structure required for normal MYC gene expression
Deletion of CTCF binding sequences at the MYC gene by homologous recombination The MYC insulator element MINE serves as an efficient boundary and insulator element for both transfected and randomly integrated reporter-gene constructs [7]

Summary

Introduction

Insulator and boundary elements shield genomic loci from unscheduled transcriptional activation or repression by regulatory elements. Recent genome-wide surveys have demonstrated that CTCF is uniquely distributed throughout the genome in a pattern distinct from general transcription factors [3,4] These studies have revealed that the majority of CTCF binding sites are located far from gene promoters with only one fifth of CTCF binding sites mapping within 2 kb of transcription initiation sites. Promoter-proximal CTCF-binding sites generally correlate with low gene activity [4], consistent with previous reports that CTCF can repress transcription [5,6]. The CTCF-binding site within the MINE (site N, Figure 1) is coincident with the boundary of hyperacetylation of the MYC promoter This arrangement suggested that CTCF functions as a barrier for the MYC locus by the formation and/or maintenance of chromatin structure required for normal MYC gene expression. The contribution of CTCF binding sites to both gene regulation and maintenance of chromatin has not been tested at the endogenous MYC gene

Methods

Results

Conclusion