DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity

Dmitry A Didych,Elena S Kotova,Segey B Akopov,Eugene D Sverdlov,Lev G Nikolaev

doi:10.1186/1756-0500-5-178

Abstract

BackgroundEarlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome.ResultsTen CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk) gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations.ConclusionsWe demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

Highlights

Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region
We studied the relationship between CTCF binding and enhancer-blocking activity of 10 CTCF-binding genomic fragments identified earlier in our laboratory [22]
Using a functional test described above, we demonstrated that all fragments which bind CTCF both in vitro and in vivo were capable of blocking activation of the CMV minimal promoter by the CMV enhancer in stably transfected CHO cells

Summary

Introduction

Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. One approach is based on the ChIP-on-chip or ChIP-seq techniques with antibodies to known insulator-binding proteins, like CTCF or CP190 [8,9,10] This approach can be used for the whole-genome analysis, but has a drawback that binding of a certain protein may be insufficient for insulator activity, which may result in many false positives. Another approach is based on a functional enhancer-blocking test in stably transfected cells [11,12] but is applicable to only relatively short (several megabases) genomic sequences

Methods

Results

Discussion

Conclusion