Analysis of chromatin structure by in vivo formaldehyde cross-linking.
Recent advances leave no doubt that higher order chromatin structures play a fundamental role in many developmentally important mechanisms of gene regulation. In particular analyses in genetic model systems like yeast and Drosophila uncovered novel proteins that are involved in the regulation of chromatin structures. Many of these proteins do not bind directly to DNA but interact in large multimeric complexes. To identify the DNA elements regulated by these multiprotein complexes, alternative approaches to the standard methods of DNA-protein analysis had to be devised. Here we present a method that preserves the architecture of the higher order chromatin structures by cross-linking cells in vivo with formaldehyde. An immunoprecipitation strategy is then used to identify the DNA targets of chromosomal proteins of interest. This method can be applied to study the distribution of proteins at high resolution over extended chromosomal regions.
Orlando
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V
ZMBH, Center for Molecular Biology, University of Heidelberg, Germany.
Strutt
H
H
Paro
R
R
D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern / Q000379:methods; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D002843:Chromatin / Q000648:ultrastructure*; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D004331:Drosophila melanogaster; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004625:Embryo, Nonmammalian; D005557:Formaldehyde; D008961:Models, Structural; D016133:Polymerase Chain Reaction / Q000379:methods; D014465:Ultrasonics
D002843:Chromatin; D003432:Cross-Linking Reagents; D005557:Formaldehyde; D004247:DNA