ChIC and ChEC; genomic mapping of chromatin proteins.
To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.
Departments of Biochemistry and Molecular Biology, NCCR Frontiers in Genetics, University of Geneva, 30, Quai Ernest-Ansermet, CH1211, Geneva 4, Switzerland.
Manfred
M
Schmid
Thérèse
T
Durussel
Ulrich K
UK
Laemmli
D000906:Antibodies; D000918:Antibody Specificity; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D008836:Micrococcal Nuclease; D025941:Protein Interaction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013205:Staphylococcal Protein A; D035181:TATA-Box Binding Protein / Q000378:metabolism; D016615:Telomere / Q000378:metabolism
D000906:Antibodies; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D013205:Staphylococcal Protein A; D035181:TATA-Box Binding Protein; D008836:Micrococcal Nuclease