Direct study of DNA-protein interactions in repressed and active chromatin in living cells.
Current methods for analysis of chromatin architecture are invasive, utilizing chemicals or nucleases that damage DNA, making detection of labile constituents and conclusions about true in vivo structure problematic. We describe a sensitive assay of chromatin structure which is performed in intact, living yeast. The approach utilizes expression of SssI DNA methyltransferase (MTase) in Saccharomyces cerevisiae to provide an order-of-magnitude increase in resolution over previously introduced MTases. Combining this resolution increase with the novel application of a PCR-based, positive chemical display of modified cytosines provides a significant advance in the direct study of DNA-protein interactions in growing cells that enables quantitative footprinting. The validity and efficacy of the strategy are demonstrated in mini-chromosomes, where positioned nucleosomes and a labile, operator-bound repressor are detected. Also, using a heterologous system to study gene activation, we show that in vivo hormone occupancy of the estrogen receptor is required for maximal site-specific DNA binding, whereas, at very high receptor-expression levels, hormone-independent partial occupancy of an estrogen-responsive element was observed. Receptor binding to a palindromic estrogen-responsive element leads to a footprint with strand-specific asymmetry, which is explicable by known structural information.
Kladde
M P
MP
Department of Biochemistry and Molecular Biology and The Center for Gene Regulation, The Pennsylvania State University, University Park 16802, USA.
Xu
M
M
Simpson
R T
RT
D002843:Chromatin / Q000737:chemistry*; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics; D009707:Nucleosomes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011960:Receptors, Estrogen / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D015533:Transcriptional Activation
D002843:Chromatin; D004268:DNA-Binding Proteins; D004967:Estrogens; D009707:Nucleosomes; D011960:Receptors, Estrogen; D012097:Repressor Proteins; D003596:Cytosine; D004247:DNA; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases