In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray.
Chromatin immunoprecipitation (ChrIP or ChIP) has commonly been used to map protein-DNA interaction sites at specific genomic loci through use of formaldehyde-induced crosslinking. However, formaldehyde alone has proved inadequate for crosslinking of certain proteins such as the yeast histone deacetylase Rpd3. We report here a modified crosslinking procedure that includes a protein-protein crosslinking agent in addition to formaldehyde. Using this double crosslinking method, we have successfully mapped Rpd3 binding sites in vivo. We also describe the use of ChrIP in combination with DNA microarrays (ChrIP-array) to determine the pattern of Rpd3 binding genomewide. This approach couples the versatility of ChrIP with that of microarrays to identify binding patterns that would otherwise be hidden in a gene-by-gene survey.
Kurdistani
Siavash K
SK
Department of Biological Chemistry, University of California School of Medicine, Boyer Hall, University of California, Los Angeles, CA 90095, USA.
Grunstein
Michael
M
D001483:Base Sequence; D002843:Chromatin / Q000235:genetics* / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D005557:Formaldehyde; D016678:Genome; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011233:Precipitin Tests / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics
D002843:Chromatin; D003432:Cross-Linking Reagents; D017931:DNA Primers; D005557:Formaldehyde; D004247:DNA