E-GEOD-5650 - Genomewide demarcation of RNA PolII transcription units by physical fractionation of chromatin- ORF-INT enrichment
Submitted on 27 August 2006, released on 30 August 2006, last updated on 10 June 2011
Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states. Keywords: Genomewide mapping of regulatory elements through differential fractionation of crosslinked chromatin based on nucleosome occupancy. ORF-INT enrichment- Whole cells were fixed by addition of 37% formaldehyde/11% methanol to the growth medium to a final concentration of 1% formaldehyde at 30°C for 30 min. Glycine was added to 125mM from a 2.5 M stock and incubated for 5 min. The cells were centrifuged in a Sorvall RT7 at 3,000 rpm for 5 min at 4°C and washed twice with PBS and once with sterile water. Without reversing crosslinks, extracts were prepared by glass-bead disruption, sonication (fragment size 200-2,000 bp, peak at 900 bp), and standard phenol-chloroform extraction.
Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatin. Nagy PL, Cleary ML, Brown PO, Lieb JD.