A new fractionation assay, based on the size of formaldehyde-crosslinked, mildly sheared chromatin, delineates the chromatin structure at promoter regions.
To explore the higher order structure of transcribable chromatin in vivo, its local configuration was assessed through the accessibility of the chromatin to crosslinking with formaldehyde. The application of crosslinked and mildly sheared chromatin to sedimentation velocity centrifugation followed by size-fractionation of the DNA enabled us to biochemically distinguish between chromatin with heavily versus sparsely crosslinkable structures. The separated fractions showed a good correlation with gene expression profiles. Genes with poor crosslinking around the promoter region were actively transcribed, while transcripts were hardly detected from genes with extensive crosslinking in their promoter regions. For the inducible gene, Il2, the distribution of the promoter shifted in the gradient following T-cell receptor stimulation, consistent with a change in structure at this locus during activation. The kinetics of this switch preceded the chromatin change observed in a DNase I accessibility assay. Thus, this new chromatin fractionation technique has revealed a change in chromatin structure that has not been previously characterized.
Ishihara
Satoru
S
Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. satorui@fujita-hu.ac.jp
Varma
Rajat
R
Schwartz
Ronald H
RH
D000818:Animals; D002843:Chromatin / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004247:DNA / Q000302:isolation & purification; D005260:Female; D005557:Formaldehyde / Q000737:chemistry*; D020869:Gene Expression Profiling; D007376:Interleukin-2 / Q000235:genetics; D008213:Lymphocyte Activation; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000276:immunology; D014461:Ultracentrifugation / Q000379:methods*
D002843:Chromatin; D003432:Cross-Linking Reagents; D007376:Interleukin-2; D011506:Proteins; D005557:Formaldehyde; D004247:DNA