E-GEOD-5662 - Genomewide demarcation of RNA PolII transcription units by physical fractionation of chromatin- ALL Arrays
Submitted on 28 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 demarcation of RNA PolII transcription units by physical fractionation of chromatin- Intergenic enrichment This series contains all the arrays associated with Nagy et al (PNAS 2003 May 27;100(11):6364-9). The arrays can be divided into four logical sets: Intergenic enrichment (GSE5648), ORF enrichment (GSE5649), ORF-Intergenic enrichment (GSE5650), and Neutral enrichment (GSE5651). Shown with each logical set is a series accession number containing the arrays associated with that set. Below is an explanation for the experimental procedure used to derived the samples for this manuscript. Standard DNA Preparation- (Experiments 1–8) All DNA was prepared by glass-bead disruption of cells and a standard phenol-chloroform extraction. The extract was centrifuged for 5 s at 14,000 x g, and the supernatant was sonicated and subsequently phenol-chloroform extracted. Intergenic Enrichment Procedure- (Experiments 9–27) 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 125 mM 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. ORF Enrichment Procedure- (Experiments 31–32) Cells were crosslinked as above, and isolated nuclei were used to prepare solubilized chromatin. Crosslinks were then reversed by incubation at 65°C, and DNA was prepared. In initial experiments (nos. 28–30), immunoprecipitation using antimethyl-lysine histone H3 antibody was performed before the crosslinks were reversed. However, the IPs were not required for ORF enrichment .
Jason D Lieb <email@example.com>, Michael L Cleary, Patrick O Brown, Peter L Nagy
Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatin. Nagy PL, Cleary ML, Brown PO, Lieb JD.