Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.
Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (alpha-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the "exposed" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome "footprints."
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000648:ultrastructure*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003852:Deoxyribonucleoproteins / Q000032:analysis; D005557:Formaldehyde / Q000494:pharmacology*; D009707:Nucleosomes / Q000648:ultrastructure; D011402:Pronase; D012097:Repressor Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D009707:Nucleosomes; D012097:Repressor Proteins; D005557:Formaldehyde; D004247:DNA; D011402:Pronase