Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I.
Most methods for assessment of chromatin structure involve chemical or nuclease damage to DNA followed by analysis of distribution and susceptibility of cutting sites. The agents used generally do not permeate cells, making nuclear isolation mandatory. In vivo mapping strategies might allow detection of labile constituents and/or structures that are lost when chromatin is swollen in isolated nuclei at low ionic strengths. DNase I has been the most widely used enzyme to detect chromatin sites where DNA is active in transcription, replication or recombination. We have introduced the bovine DNase I gene into yeast under control of a galactose-responsive promoter. Expression of the nuclease leads to DNA degradation and cell death. Shorter exposure to the active enzyme allows mapping of chromatin structure in whole cells without isolation of nuclei. The validity and efficacy of the strategy are demonstrated by footprinting a labile repressor bound to its operator. Investigation of the inter-nucleosome linker regions in several types of repressed domains has revealed different degrees of protection in cells, relative to isolated nuclei.
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Department of Biochemistry and Molecular Biology, 308 Althouse Laboratory, The Pennsylvania State University, University Park, PA 16802, USA.
Simpson
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002843:Chromatin / Q000737:chemistry*; D018983:DNA Footprinting; D004271:DNA, Fungal / Q000032:analysis / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D009707:Nucleosomes / Q000737:chemistry; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
D002843:Chromatin; D004271:DNA, Fungal; D009707:Nucleosomes; D012097:Repressor Proteins; D003850:Deoxyribonuclease I