Two-dimensional electrophoretic mobility shift assay: Identification and mapping of transcription factor CTCF target sequences within an FXYD5–COX7A1 region of human chromosome 19

Ветчинова, А. С.; Akopov, S. B.; Чернов, И. П.; Николаев, Л. Г.

doi:10.1016/j.ab.2006.03.052

Cited by 16 publications

(22 citation statements)

References 37 publications

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“…First, this microarray represented only a relatively small subpopulation of CTCF binding sites, which is approximately 5 to 7% of all potential CTCF target sites (40). This number, however, may be lower (ϳ0.75 to 1%) if the criteria described by Vetchinova et al are used for assessment (61). Second, some of the previously identified and characterized CTSs (e.g., MYC and H19 ICR) could not be assessed in the screening as they were not present on the microarray (40).…”

Section: Discussionmentioning

confidence: 99%

“…Among vertebrate insulators controlled by CTCF are the ␤-globin (3) and the H19 imprinting control region (ICR) (42,44) insulators. In our previous report, the number of CTCF binding sites in the mouse genome was estimated as ϳ4,000 (40) but the real number may be much higher (ϳ30,000 in the human genome), as suggested in a more recent publication (61). Many of these sites are methylation sensitive and map to promoter, inter-and intragenic regions, and introns; some sites contain Alu-like repeated elements (40,61).…”

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confidence: 99%

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CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Chernukhin

Shamsuddin

Kang

et al. 2007

Molecular and Cellular Biology

150

128

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CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Chernukhin

Shamsuddin

Kang

et al. 2007

Molecular and Cellular Biology

150

128

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“…Within this gene-rich human chromosome 19 region, we previously identified insulator-like sequences (24), CTCF transcription-factor binding sites (25), and nuclear matrix attachment regions (S/MARs) (18). These and other available data on human promoters (7) and transcribed sequences (26) make this locus one of the best characterized human genome regions regarding cis -regulatory elements.…”

Section: Discussionmentioning

confidence: 98%

A New Technique for Selective Identification and Mapping of Enhancers Within Long Genomic Sequences

Чернов

Stukacheva²,

Akopov³

et al. 2008

BioTechniques

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We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns.

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“…The number of identified sites was, however, not sufficient to make a reliable conclusion about their distribution in the whole genome. An attempt to map the majority of CTCF binding sites in a 1 megabase human genome region was undertaken [88] using both in vitro and in vivo approaches. Ten binding sites were identified therein that allowed to estimate the total number of CTCF binding sites in the human genome at about 30,000.…”

Section: Ctcf and Genome Functioningmentioning

confidence: 99%

Vertebrate Protein CTCF and its Multiple Roles in a Large-Scale Regulation of Genome Activity

Николаев¹,

Akopov²,

Didych³

2009

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The CTCF transcription factor is an 11 zinc fingers multifunctional protein that uses different zinc finger combinations to recognize and bind different sites within DNA. CTCF is thought to participate in various gene regulatory networks including transcription activation and repression, formation of independently functioning chromatin domains and regulation of imprinting. Sequencing of human and other genomes opened up a possibility to ascertain the genomic distribution of CTCF binding sites and to identify CTCF-dependent cis-regulatory elements, including insulators. In the review, we summarized recent data on genomic distribution of CTCF binding sites in the human and other genomes within a framework of the loop domain hypothesis of large-scale regulation of the genome activity. We also tried to formulate possible lines of studies on a variety of CTCF functions which probably depend on its ability to specifically bind DNA, interact with other proteins and form di- and multimers. These three fundamental properties allow CTCF to serve as a transcription factor, an insulator and a constitutive dispersed genome-wide demarcation tool able to recruit various factors that emerge in response to diverse external and internal signals, and thus to exert its signal-specific function(s).

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Two-dimensional electrophoretic mobility shift assay: Identification and mapping of transcription factor CTCF target sequences within an FXYD5–COX7A1 region of human chromosome 19

Cited by 16 publications

References 37 publications

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

A New Technique for Selective Identification and Mapping of Enhancers Within Long Genomic Sequences

Vertebrate Protein CTCF and its Multiple Roles in a Large-Scale Regulation of Genome Activity

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