A Genomics Analysis of RNA polymerase II modification and chromatin architecture related to 3' end RNA polyadenylation

Abstract

Genomic analyses have been applied extensively to analyze the process of transcription initiation in mammalian cells, but much less to the events associated with transcript 3'end formation and transcription termination. We have used a novel approach to prepare 3' end fragment libraries from polyadenylated RNA of several cell types, and globally mapped the position of the poly(A) addition site using oligonucleotide arrays tiling one percent of the human genome. This approach revealed more 3' ends than had been previously annotated. The distribution of these ends relative to DNA sites bound by RNA polymerase II and the distributions with those for di- and trimethylated lysine 4 and lysine 36 of histone 3, was compared by ChIP-chip analysis. We found that a substantial fraction of unannotated 3' ends of RNA are intronic and reside antisense to the embedding gene. Poly(A) ends of annotated messages lie at a variable distance averaging approximately two kb upstream of the end of RNA polymerase binding (termination). Near the sites of RNA polymerase termination, as well as in some internal sites, there is an accumulation of both unphosphorylated and carboxy-terminal domain (CTD) serine 2 phosphorylated large subunit of polymerase II, suggesting pausing of the polymerase and perhaps dephosphorylation prior to release. Lysine 36 trimethylation occurs across the body of many transcribed genes, sometimes alternating with stretches of DNA in which lysine36 dimethylation is relatively more prominent. Lysine 36 methylation often decreases beginning at or near the site of polyadenylation, sometimes disappearing before disappearance of phosphorylated RNA polymerase II and release of RNA polymerase from DNA. Our results suggest that transcription termination may involve the separable events of loss of histone3 lysine 36 methylation and later release of RNA polymerase. The latter is often associated with polymerase pausing before release. Thus, overall our study reveals extensive sites of poly(A) addition across the human genome and provides insights into the events that may occur during 3' end formation.

• chromatin
• transcription termination