Reorganization of RNA polymerase II on the SV40 genome occurs coordinately with the early to late transcriptional switch

Abstract

The pattern of organization of RNA polymerase II (RNAPII) in wild-type and mutant cs1085 SV40 chromosomes isolated between 30 min and 48 h post-infection was determined using a combination of chromatin immunoprecipitation (ChIP) techniques. During the course of a wild-type infection, we observed a slow but significant decline in the relative occupancy of RNAPII at the early region and a corresponding increase in occupation in the late region. In the promoter, occupancy began high, decreased to a minimum at 8 h post-infection, and then increased to a high level by 48 h post-infection. In the mutant cs1085, which does not down-regulate early transcription, we observed high occupancy of the early region and the promoter throughout the infection. The changing organization of RNAPII on the wild-type SV40 but not the mutant cs1085 genome appears to be a result of the switch from early to late transcription.

Histone Hyperacetylation in the Coding Region of Chromatin Undergoing Transcription in SV40 Minichromosomes Is a Dynamic Process Regulated Directly by the Presence of RNA Polymerase II

“…We expected that the immune selected SV40 chromatin would consist of chromosomes with RNAPII located in the promoter which were undergoing initiation, chromosomes with RNAPII located in coding regions which were undergoing transcriptional elongation, and chromosomes with RNAPII located in either the promoter or coding regions in which the RNAPII was paused. We confirmed that antibody to RNAPII could be used to immune select transcribing SV40 chromosomes using a modified ChIP procedure that we refer to as immune selection and fragmentation (ISF) (6,7) . In the ISF procedure the immune selected transcribing SV40 chromosomes were fragmented by sonication into bound chromatin fragments that contained RNAPII and soluble chromatin fragments that lacked RNAPII.…”

“…At 8 hours post-infection in SV40 chromosomes from wild-type virus, we observed that RNAPII occupancy of the promoter and early region was relatively low consistent with down-regulation, while in a mutant of SV40 (cs1085) in which the T-antigen binding site was lacking and down-regulation could not occur the occupancy of the promoter and early region was very high. Since the occupancy of the SV40 genome during the course of infection correlated directly with the known pattern of transcription, we concluded that we were immune selecting transcribing SV40 chromosomes using antibody to RNAPII (7) .…”

“…In this publication (7) we also demonstrated the feasibility of directly analyzing chromatin undergoing transcription for the presence of hyperacetylated histones. Because the immune selected chromatin undergoing transcription was fragmented into chromatin fragments that either contained RNAPII or lacked RNAPII, we were able to determine whether a particular hyperacetylated histone was present along with the RNAPII or independent of the RNAPII at any given site in the genome.…”

“…We have been using the simian virus 40 (SV40) chromosome as a eukaryotic model to investigate two aspects of chromatin structure, nucleosome phasing and histone hyperacetylation (6)(7)(8)(9) . The SV40 chromosome is particularly well suited for this function since it utilizes host cell proteins for its transcription and replication and has been extensively studied as a model for both eukaryotic processes (10)(11)(12)(13) .…”

“…In the ISF procedure the immune selected transcribing SV40 chromosomes were fragmented by sonication into bound chromatin fragments that contained RNAPII and soluble chromatin fragments that lacked RNAPII. Using this strategy we measured the relative occupancy of RNAPII in various regions of the SV40 genome in chromosomes undergoing transcription during the course of an infection and correlated these results to the well characterized early to late shift in SV40 transcription (7) . Specifically, we observed that at early times RNAPII was preferentially associated with the promoter and early region of the genome while at late times RNAPII was preferentially associated with the promoter and late region of the genome as expected.…”