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PMID: 8947052 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Direct study of DNA-protein interactions in repressed and active chromatin in living cells.

The EMBO journal ·Vol. 15 ·No. 22 ·1996-11-15 ·Pages 6290-300

Kladde MP, Xu M, Simpson RT

Abstract

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.

MeSH Terms
Chromatin/chemistry Cytosine/metabolism DNA/metabolism DNA Footprinting DNA Methylation DNA-Binding Proteins/metabolism DNA-Cytosine Methylases/metabolism Estrogens/pharmacology Gene Expression Regulation/genetics Nucleosomes/metabolism Polymerase Chain Reaction Promoter Regions, Genetic/genetics Receptors, Estrogen/metabolism Repressor Proteins/genetics Saccharomyces cerevisiae/metabolism Transcriptional Activation
Chemicals
Chromatin DNA-Binding Proteins Estrogens Nucleosomes Receptors, Estrogen Repressor Proteins Cytosine DNA DNA modification methylase SssI DNA-Cytosine Methylases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kladde M P
Department of Biochemistry and Molecular Biology and The Center for Gene Regulation, The Pennsylvania State University, University Park 16802, USA.
Xu M
Simpson R T
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1996-11-15
Pages
6290-300
Language
English
Region
England
NLM ID
8208664
PMCID
PMC452452
Subset
IM
Grants
NIGMS NIH HHS · R01 GM052908 · United States
NIGMS NIH HHS · GM52908 · United States
External Links
PubMed source PMC Full Text View journal details
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