Genomewide demarcation of RNA PolII transcription units by physical fractionation of chromatin- ORF enrichment

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 enrichment- Cells were crosslinked and nuclei isolated to prepare solubilized chromatin. Crosslinks were then reversed by incubation at 65°C, and DNA was prepared. In initial experiments, immunoprecipitation using antimethyl-lysine histone H3 antibody was performed before the crosslinks were reversed. However, the IPs were not required for ORF enrichment.