Analysis of in vivo nucleosome positions by determination of nucleosome-linker boundaries in crosslinked chromatin.
We describe a procedure for the determination of nucleosome boundaries that utilizes single-stranded mononucleosomal DNA obtained from fixed cells as the template in a primer extension assay. The procedure entails treatment of cells with formaldehyde, a reversible protein-DNA crosslinking agent used with the object of fixing the histone octamers to DNA in vivo, followed by preparation of nucleosomal DNA with micrococcal nuclease, reversal of the crosslinks, and isolation of the mononucleosomal material. Full-length single-stranded mononucleosomal DNA is then prepared and used as a template in a linear amplification primer extension assay. The use of single-stranded DNA templates eliminates interference from nicked DNA present in double-stranded preparations. Because of its reversibility, the use of formaldehyde permits the preparation of DNA suitable as a template in DNA synthesis. We present evidence demonstrating the efficiency of histone-DNA crosslinking and the reversibility of the crosslinking reaction as applied to the regeneration of native DNA, active in DNA synthesis. Use of this methodology removes the impact that mobility of the histone octamer and the presence of nicks on nucleosomal DNA have on the determination of nucleosome boundaries.
Fragoso
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Laboratory of Molecular Virology, National Cancer Institute, NIH, Bethesda, Maryland 20892-5055, USA.
Hager
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GL
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002843:Chromatin / Q000648:ultrastructure*; D003432:Cross-Linking Reagents; D004247:DNA / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D007202:Indicators and Reagents; D008836:Micrococcal Nuclease; D009707:Nucleosomes / Q000648:ultrastructure*; D016133:Polymerase Chain Reaction / Q000379:methods; D013698:Templates, Genetic
D002843:Chromatin; D003432:Cross-Linking Reagents; D017931:DNA Primers; D006657:Histones; D007202:Indicators and Reagents; D009707:Nucleosomes; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; D008836:Micrococcal Nuclease