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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.


CTCF binding to DNA regions in vivo as revealed by chromatinimmunoprecipitation and a real-time PCR analysis. The results for HD3 cells,HD3 induced to erythroid differentiation, and for B-lymphoid DT40 cells arepresented. Primers were targeted to the DNA fragments selected in this work(1-13) and to six fragments identified in [9] (5d1-5d3, 10d1- 10d3). F1, MYC – positive controls;Enh, HBAD – negative controls. The data are normalized to binding CTCFwith the F1 fragment. Error bars indicate the standard errors of the mean.
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Figure 4: CTCF binding to DNA regions in vivo as revealed by chromatinimmunoprecipitation and a real-time PCR analysis. The results for HD3 cells,HD3 induced to erythroid differentiation, and for B-lymphoid DT40 cells arepresented. Primers were targeted to the DNA fragments selected in this work(1-13) and to six fragments identified in [9] (5d1-5d3, 10d1- 10d3). F1, MYC – positive controls;Enh, HBAD – negative controls. The data are normalized to binding CTCFwith the F1 fragment. Error bars indicate the standard errors of the mean.

Mentions: To compare the CTCF binding to DNA in a living cell and detected by EMSA, weperformed chromatin immunoprecipitation for 13 DNA fragments from the globinregion, as well as for the 5d1–5d3 and 10d1–10d3fragments [9] in three cell types: HD3 cells, HD3 inducedto erythroid differentiation, and B-lymphoid DT40. The positions of DNAfragments amplified during chromatin immunoprecipitation are shown inFig. 3 (ChIP panel),and the results of immunoprecipitation are presentedin Fig. 4.


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)

CTCF binding to DNA regions in vivo as revealed by chromatinimmunoprecipitation and a real-time PCR analysis. The results for HD3 cells,HD3 induced to erythroid differentiation, and for B-lymphoid DT40 cells arepresented. Primers were targeted to the DNA fragments selected in this work(1-13) and to six fragments identified in [9] (5d1-5d3, 10d1- 10d3). F1, MYC – positive controls;Enh, HBAD – negative controls. The data are normalized to binding CTCFwith the F1 fragment. Error bars indicate the standard errors of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: CTCF binding to DNA regions in vivo as revealed by chromatinimmunoprecipitation and a real-time PCR analysis. The results for HD3 cells,HD3 induced to erythroid differentiation, and for B-lymphoid DT40 cells arepresented. Primers were targeted to the DNA fragments selected in this work(1-13) and to six fragments identified in [9] (5d1-5d3, 10d1- 10d3). F1, MYC – positive controls;Enh, HBAD – negative controls. The data are normalized to binding CTCFwith the F1 fragment. Error bars indicate the standard errors of the mean.
Mentions: To compare the CTCF binding to DNA in a living cell and detected by EMSA, weperformed chromatin immunoprecipitation for 13 DNA fragments from the globinregion, as well as for the 5d1–5d3 and 10d1–10d3fragments [9] in three cell types: HD3 cells, HD3 inducedto erythroid differentiation, and B-lymphoid DT40. The positions of DNAfragments amplified during chromatin immunoprecipitation are shown inFig. 3 (ChIP panel),and the results of immunoprecipitation are presentedin Fig. 4.

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.