Histone Hyperacetylation during SV40 Transcription Is Regulated by p300 and RNA Polymerase II Translocation

Abstract

The effects of the RNA polymerase II (RNAPII) translocation inhibitors alpha amanitin and 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) and an siRNA targeting p300 on the presence of RNAPII, p300, hyperacetylated H4 and H3 and unmodified H4 and H3 in transcribing simian virus 40 (SV40) minichromosomes were determined. Following treatment with alpha amanitin we observed a profound reduction in the occupancy of the promoter by RNAPII, the loss of p300 from chromatin fragments containing RNAPII, and an increase in the amount of unmodified H4 and H3 associated with the RNAPII. Treatment with DRB had little effect on the presence of RNAPII or p300 but also resulted in a significant increase in the amount of unmodified H4 and H3 present in chromatin fragments associated with RNAPII. Following treatment with a p300 small interfering RNA (siRNA), we observed a significant decrease in late transcription and a corresponding reduction in the amounts of p300 and hyperacetylated histones associated with the transcribing SV40 minichromosomes. We conclude that in transcribing SV40 minichromosomes histone hyperacetylation and deacetylation is dependent upon the presence of p300 and an as yet unknown histone deacetylase associated with the RNAPII complex that occurs coordinately as the RNAPII complex moves through a nucleosome.

Virion-mediated transfer of SV40 epigenetic information

“…Following standard PCR amplifications, amplification products were separated on 2.4% submerged agarose gels (Sigma) by electrophoresis. [12][13][14] Amplification products were visualized by staining with ethidium bromide and digitally recorded using a UVP GDS8000 gel documentation system (Ultra Violet Products).…”

“…Simian virus 40 (SV40) is a DNA tumor virus that is organized into chromatin in the intracellular minichromosome and virion, and undergoes hyperacetylation of H3 and H4 during transcription [12][13][14] and methylation of H4K20 during an infection. 15 Because of its similarity to cellular chromatin, we hypothesized that SV40 should be able to transfer epigenetic information recapitulating the conditions of an infection to a subsequent infection through the infecting virions.…”

Viral Epigenetics

“…Correspondingly repression also results in a significant reduction in the presence of hyperacetylated H3 and H4 located in the early region. Not surprisingly this reduction is reversed in the mutant confirming the direct relationship between the presence of RNAPII and hyperacetylated histones [30]. …”

“…Like early transcription, late transcription is also closely linked to histone hyperacetylation with the hyperacetylated histones preferentially associated with regions of chromatin which contain RNAPII [11, 30]. Also like early transcription, late transcription is dynamic with p300 bound to RNAPII and responsible for histone hyperacetylation [30]. P300 was shown to be required to direct histone hyperacetylation for active late transcription to occur using siRNA knockdown of p300 [30].…”

“…Also like early transcription, late transcription is dynamic with p300 bound to RNAPII and responsible for histone hyperacetylation [30]. P300 was shown to be required to direct histone hyperacetylation for active late transcription to occur using siRNA knockdown of p300 [30]. …”