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

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.

Keywords: erythroid differentiation; globin genes; transcription factor CTCF.

Fig. 1

Preparation and characterization of the library of CTCF-binding fragments. (A) Selection of CTCF-binding fragments by means of the two-dimensional electrophoretic mobility shift assay (2D-EMSA). The results of two-dimensional electrophoresis for the second selection round are shown. Region containing selected CTCF-binding fragments is outlined by the oval. For detail, see text. (B) Estimation of the degree of enrichment with the CTCF-binding fragments for the library obtained. Initial DNA and DNA after first and second 2D-EMSA selection rounds were used as a template for PCR with primers targeted to CTCF-binding sequences from the chicken alpha-globin locus: CDS (CTCF-dependent silencer) and M9 sequence. Sequence from HBAD gene exon which does not bind CTCF was used as a negative control. (C) Rarefaction curve obtained during sequencing of the CTCF-binding fragments library

Fig. 2

CTCF binding to selected DNA fragments 10 and 13. Anti-CTCF (A) and anti-polyHistidine (B) antibodies were used for the supershift assay. AB – antibodies, NE – nuclear extract

Fig. 3

Distribution of CTCF binding sites and some regulatory elements in the region overlapping the chicken genome alpha-globin domain. Upper map shows the positions of all selected DNA fragments. The arrows indicate DNA regions with high affinity to CTCF. Lower part shows the enlarged map of the immediate surroundings of the globin genes. In the “CTCF sites” panel the identified previously CTCF binding sites M9, C10-C14 [7], CDS [8] and 5d1-5d3, 10d1-10d3 [9] are shown, the “Regulatory elements” panel demonstrates the positions of the MRE [2] and the enhancer and silencer [25]. In the ChIP region the positions of DNA fragments amplified in the chromatin immunoprecipitation experiment are shown (see text)

Fig. 4

CTCF binding to DNA regions in vivo as revealed by chromatin immunoprecipitation and a real-time PCR analysis. The results for HD3 cells, HD3 induced to erythroid differentiation, and for B-lymphoid DT40 cells are presented. 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 CTCF with the F1 fragment. Error bars indicate the standard errors of the mean.