Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene.
We have used formaldehyde-mediated protein-DNA crosslinking within intact cells to examine the in vivo chromatin structure of the D. melanogaster heat shock protein 70 (hsp70) genes. In agreement with previous in vitro studies, we find that the heat shock-mediated transcriptional induction of the hsp70 genes perturbs their chromatin structure, resulting in fewer protein-DNA contacts crosslinkable in vivo by formaldehyde. However, contrary to earlier in vitro evidence that histones may be absent from actively transcribed genes, we show directly, by immunoprecipitation of in vivo-crosslinked chromatin fragments, that at least histone H4 remains bound to hsp70 DNA in vivo, irrespective of its rate of transcription. The formaldehyde-based in vivo mapping techniques described in this work are generally applicable, and can be used both to probe protein-DNA interactions within specific genes and to determine the genomic location of specific chromosomal proteins.
Solomon
M J
MJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Larsen
P L
PL
Varshavsky
A
A
D000818:Animals; D002500:Centrifugation, Isopycnic; D002843:Chromatin / Q000032:analysis*; D003432:Cross-Linking Reagents; D004247:DNA / Q000032:analysis / Q000235:genetics*; D003902:Detergents; D004331:Drosophila melanogaster; D004587:Electrophoresis, Agar Gel; D005557:Formaldehyde; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000235:genetics*; D006657:Histones / Q000235:genetics*; D007118:Immunoassay; D009693:Nucleic Acid Hybridization; D011402:Pronase / Q000378:metabolism; D012319:RNA Polymerase II / Q000235:genetics; D012521:Sarcosine / Q000031:analogs & derivatives; D014158:Transcription, Genetic
D002843:Chromatin; D003432:Cross-Linking Reagents; D003902:Detergents; D006360:Heat-Shock Proteins; D006657:Histones; D005557:Formaldehyde; C025231:sarkosyl; D004247:DNA; D012319:RNA Polymerase II; D011402:Pronase; D012521:Sarcosine