The 5'-HS4 chicken beta-globin insulator is a CTCF-dependent nuclear matrix-associated element - PubMed (original) (raw)

The 5'-HS4 chicken beta-globin insulator is a CTCF-dependent nuclear matrix-associated element

Timur M Yusufzai et al. Proc Natl Acad Sci U S A. 2004.

Abstract

The protein CTCF plays an essential role in the action of a widely distributed class of vertebrate enhancer-blocking insulators, of which the first example was found in a DNA sequence element, HS4, at the 5' end of the chicken beta-globin locus. HS4 contains a binding site for CTCF that is necessary and sufficient for insulator action. Purification of CTCF has revealed that it interacts with proteins involved in subnuclear architecture, notably nucleophosmin, a 38-kDa nucleolar phosphoprotein that is concentrated in nuclear matrix preparations. In this report we show that both CTCF and the HS4 insulator element are incorporated in the matrix; HS4 incorporation depends on the presence of an intact CTCF-binding site. However the DNA sequence in the neighborhood of HS4 is not like that of canonical matrix attachment regions, and its incorporation into the matrix fraction is not sensitive to ribonuclease, suggesting that the insulator is a distinct matrix-associated element.

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Figures

Fig. 1.

Fig. 1.

Schematic representation and sequence of the HS4 core insulator (underlined) and flanking regions. The binding of CTCF and nucleophosmin are based on previous chromatin immunoprecipitation data (16).

Fig. 2.

Fig. 2.

Extraction of CTCF protein from nuclei reveals soluble/insoluble fractions. Nuclei were digested with DNase I and then extracted with buffer of increasing ionic strength. Release of CTCF protein was monitored by Western analyses and compared to known insoluble lamins and soluble histone H3. The extraction of CTCF was partially improved with detergent.

Fig. 3.

Fig. 3.

The chicken HS4 insulator behaves as a matrix attachment site in vitro. An in vitro MAR assay by using a 32P-labeled 1.2-kb HS4 insulator probe (Ins) or a control λ DNA probe. Prepared matrices (lithium salt and 2 M NaCl) were incubated with the labeled DNA fragments and washed, and bound DNA was precipitated and resolved by agarose gel electrophoresis. The 1.2-kb insulator specifically bound, whereas the control DNA did not.

Fig. 4.

Fig. 4.

Matrix attachment properties of DNA in the neighborhood of the chicken HS4 insulator. (A) An in vivo MAR assay of the endogenous HS4 insulator. Matrices from pre-erythroid 6C2 cells or primary 10-d RBCs were prepared by using the lithium salt extraction procedure to preserve native attachment sites. After extensive DNase I digestion, residual DNA fragments were precipitated and analyzed by quantitative PCR by using primers across the chicken β-globin locus. Only DNA fragments of the HS4 insulator remained. (B) Representation of PCR analyses of matrices from 6C2 cells centered on the HS4 core and surrounding sequences. All primer sets were tested with genomic DNA. Open bars represent amplified regions for the genomic DNA but were absent from the matrix preparation. Filled bars represent amplified products for both genomic and matrix DNA samples.

Fig. 5.

Fig. 5.

The intact CTCF site is responsible for the matrix attachment. Transgenic cell lines containing a fragment of the HS4 insulator and flanking sequences were generated in parallel with lines carrying transgenes with a mutation in the CTCF-binding site. After matrix preparation, only the lines containing the WT CTCF-binding site were found to be associated with the nuclear matrix.

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