Transcriptional repression is epigenetically marked by H3K9 methylation during SV40 replication.

Les Kallestad,Kendra Christensen,Barry Milavetz,Emily Woods

doi:10.1186/1868-7083-6-21

Abstract

BackgroundWe have recently shown that T-antigen binding to Site I results in the replication-dependent introduction of H3K9me1 into SV40 chromatin late in infection. Since H3K9me2 and H3K9me3 are also present late in infection, we determined whether their presence was also related to the status of ongoing transcription and replication. Transcription was either inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) or stimulated with sodium butyrate and the effects on histone modifications early and late in infection determined. The role of DNA replication was determined by concomitant inhibition of replication with aphidicolin.ResultsWe observed that H3K9me2/me3 was specifically introduced when transcription was inhibited during active replication. The introduction of H3K9me2/me3 that occurred when transcription was inhibited was partially blocked when replication was also inhibited. The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.ConclusionsOur data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication. The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a ‘proof reading’ mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.

Highlights

IntroductionIntroduction ofH3K9me and H3K9me following inhibition of transcription is partially dependent upon replication Since we have previously shown that the introduction of H3K9me and H3K9me into Simian Virus 40 (SV40) chromatin late in infection does not require DNA replication [8], we tested whether replication played a role in the enhanced introduction of these modifications following DRB inhibition of transcription
In order to address these questions in Simian Virus 40 (SV40) chromatin, we have investigated the effects of a general inhibition or stimulation of transcription on the introduction of H3K9 methylation in the presence or absence of active replication
We chose to investigate SV40 chromatin isolated at 2 hours post-infection when early transcription was occurring but prior to DNA replication, and at 48 hours post-infection when early and late transcription were occurring along with active replication

Summary

Introduction

Introduction ofH3K9me and H3K9me following inhibition of transcription is partially dependent upon replication Since we have previously shown that the introduction of H3K9me and H3K9me into SV40 chromatin late in infection does not require DNA replication [8], we tested whether replication played a role in the enhanced introduction of these modifications following DRB inhibition of transcription. These results suggest that ongoing replication plays at least a small role in the introduction of H3K9me and H3K9me at late times in infection. H3K9me and H3K9me following inhibition of transcription does not require the presence of H3K9me Since H3K9me can be present in as much as 22% of the SV40 minichromosomes present at late times [9], it seemed reasonable that the H3K9me containing minichromosomes might serve as substrates for the introduction of H3K9me and H3K9me3 To test this possibility we characterized the effect of DRB treatment on the mutant cs1085 SV40 virus. What has added to this uncertainty is the complexity of methylation, including mono-, di-, and tri-methylation, and the involvement of multiple methylating enzymes [3]

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