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Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

Kotova ES, Akopov SB, Didych DA, Petrova NV, Iarovaia OV, Razin SV, Nikolaev LG - Acta Naturae (2016 Jan-Mar)

Bottom Line: So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus.Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro.During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.

View Article: PubMed Central - PubMed

Affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St., Moscow 117997, Russia.

ABSTRACT
A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.

No MeSH data available.


Preparation and characterization of the library of CTCF-binding fragments. (A)Selection of CTCF-binding fragments by means of the two-dimensionalelectrophoretic mobility shift assay (2D-EMSA). The results of two-dimensionalelectrophoresis for the second selection round are shown. Region containingselected CTCF-binding fragments is outlined by the oval. For detail, see text.(B) Estimation of the degree of enrichment with the CTCF-binding fragments forthe library obtained. Initial DNA and DNA after first and second 2D-EMSAselection rounds were used as a template for PCR with primers targeted toCTCF-binding sequences from the chicken alpha-globin locus: CDS (CTCF-dependentsilencer) and M9 sequence. Sequence from HBAD gene exon whichdoes not bind CTCF was used as a negative control. (C) Rarefaction curveobtained during sequencing of the CTCF-binding fragments library
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Figure 1: Preparation and characterization of the library of CTCF-binding fragments. (A)Selection of CTCF-binding fragments by means of the two-dimensionalelectrophoretic mobility shift assay (2D-EMSA). The results of two-dimensionalelectrophoresis for the second selection round are shown. Region containingselected CTCF-binding fragments is outlined by the oval. For detail, see text.(B) Estimation of the degree of enrichment with the CTCF-binding fragments forthe library obtained. Initial DNA and DNA after first and second 2D-EMSAselection rounds were used as a template for PCR with primers targeted toCTCF-binding sequences from the chicken alpha-globin locus: CDS (CTCF-dependentsilencer) and M9 sequence. Sequence from HBAD gene exon whichdoes not bind CTCF was used as a negative control. (C) Rarefaction curveobtained during sequencing of the CTCF-binding fragments library

Mentions: To obtain libraries of CTCF-binding sequences by two-dimensional EMSA (2D-EMSA,[12]), the artificial bacterialchromosome (BAC) containing a 227366 bp insert, which overlaps the chickenalpha-globin locus and includes extensive flanking regions, was digested tocompletion with either the Sau3AI or Csp6I restriction enzyme. Syntheticadapters were attached to the resulting sticky ends, amplified by PCR, and bothhydrolysates were mixed in equal proportions. The resulting library of shortfragments (approximately 1,000 fragments with an average length of ca. 500 bp)was 32P-labeled and mixed with a protein fraction enriched infull-length CTCF, expressed in COS-1 cells [17].The reaction mixture was then electrophoreticallyseparated by non-denaturing polyacrylamide gel (first dimension). The regionwith the sample was cut out, incubated in SDS-containing buffer to disrupt theDNA-protein complexes, and the DNA fragments were separated in SDS-containinggel (second dimension). The region containing the most fragments originallybound to CTCF (outlined by the ovalin Fig. 1A) was cut outfrom the gel and the DNA fragments were eluted and amplified.The procedure was repeated to improve the efficiency of selection.


Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

Kotova ES, Akopov SB, Didych DA, Petrova NV, Iarovaia OV, Razin SV, Nikolaev LG - Acta Naturae (2016 Jan-Mar)

Preparation and characterization of the library of CTCF-binding fragments. (A)Selection of CTCF-binding fragments by means of the two-dimensionalelectrophoretic mobility shift assay (2D-EMSA). The results of two-dimensionalelectrophoresis for the second selection round are shown. Region containingselected CTCF-binding fragments is outlined by the oval. For detail, see text.(B) Estimation of the degree of enrichment with the CTCF-binding fragments forthe library obtained. Initial DNA and DNA after first and second 2D-EMSAselection rounds were used as a template for PCR with primers targeted toCTCF-binding sequences from the chicken alpha-globin locus: CDS (CTCF-dependentsilencer) and M9 sequence. Sequence from HBAD gene exon whichdoes not bind CTCF was used as a negative control. (C) Rarefaction curveobtained during sequencing of the CTCF-binding fragments library
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837575&req=5

Figure 1: Preparation and characterization of the library of CTCF-binding fragments. (A)Selection of CTCF-binding fragments by means of the two-dimensionalelectrophoretic mobility shift assay (2D-EMSA). The results of two-dimensionalelectrophoresis for the second selection round are shown. Region containingselected CTCF-binding fragments is outlined by the oval. For detail, see text.(B) Estimation of the degree of enrichment with the CTCF-binding fragments forthe library obtained. Initial DNA and DNA after first and second 2D-EMSAselection rounds were used as a template for PCR with primers targeted toCTCF-binding sequences from the chicken alpha-globin locus: CDS (CTCF-dependentsilencer) and M9 sequence. Sequence from HBAD gene exon whichdoes not bind CTCF was used as a negative control. (C) Rarefaction curveobtained during sequencing of the CTCF-binding fragments library
Mentions: To obtain libraries of CTCF-binding sequences by two-dimensional EMSA (2D-EMSA,[12]), the artificial bacterialchromosome (BAC) containing a 227366 bp insert, which overlaps the chickenalpha-globin locus and includes extensive flanking regions, was digested tocompletion with either the Sau3AI or Csp6I restriction enzyme. Syntheticadapters were attached to the resulting sticky ends, amplified by PCR, and bothhydrolysates were mixed in equal proportions. The resulting library of shortfragments (approximately 1,000 fragments with an average length of ca. 500 bp)was 32P-labeled and mixed with a protein fraction enriched infull-length CTCF, expressed in COS-1 cells [17].The reaction mixture was then electrophoreticallyseparated by non-denaturing polyacrylamide gel (first dimension). The regionwith the sample was cut out, incubated in SDS-containing buffer to disrupt theDNA-protein complexes, and the DNA fragments were separated in SDS-containinggel (second dimension). The region containing the most fragments originallybound to CTCF (outlined by the ovalin Fig. 1A) was cut outfrom the gel and the DNA fragments were eluted and amplified.The procedure was repeated to improve the efficiency of selection.

Bottom Line: So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus.Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro.During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.

View Article: PubMed Central - PubMed

Affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya St., Moscow 117997, Russia.

ABSTRACT
A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.

No MeSH data available.