The location of nucleosomes in SV40 virions and minichromosomes isolated during infection were determined by next generation sequencing (NGS). The patterns of reads within the regulatory region of chromatin from wild-type virions indicated that micrococcal nuclease-resistant nucleosomes were specifically positioned at nt 5223 and nt 363, while in minichromosomes isolated 48 h post-infection we observed nuclease-resistant nucleosomes at nt 5119 and nt 212. The nucleosomes at nt 5223 and nt 363 in virion chromatin would be expected to repress early and late transcription, respectively. In virions from the mutant cs1085, which does not repress early transcription, we found that these two nucleosomes were significantly reduced compared to wild-type virions confirming a repressive role for them. In chromatin from cells infected for only 30 min with wild-type virus, we observed a significant reduction in the nucleosomes at nt 5223 and nt 363 indicating that the potential repression by these nucleosomes appeared to be relieved very early in infection.

Simian virus 40 (SV40) early transcription is repressed when the product of early transcription, T-antigen, binds to its cognate regulatory sequence, Site I, in the promoter of the SV40 minichromosome. Because SV40 minichromosomes undergo replication and transcription potentially repression could occur during active transcription or during DNA replication. Since repression is frequently epigenetically marked by the introduction of specific forms of methylated histone H3, we characterized the methylation of H3 tails during transcription and replication in wild-type SV40 minichromosomes and mutant minichromosomes which did not repress T-antigen expression. While repressed minichromosomes following replication were clearly marked with H3K9me1 and H3K4me1, minichromosomes repressed during early transcription were not similarly marked. Instead repression of early transcription was marked by a significant reduction in the level of H3K9me2. The replication dependent introduction of H3K9me1 and H3K4me1 into wild-type SV40 minichromosomes was also observed when replication was inhibited with aphidicolin. The results indicate that the histone modifications associated with repression can differ significantly depending upon whether the chromatin being repressed is undergoing transcription or replication.

Background: We 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.

We have recently shown that the repression of early transcription during a Simian Virus 40 (SV40) infection results in different histone modifications depending upon the replicative status of the chromatin (Kallestad, et al., 2013, Frontiers in Genetics). In order to test whether replication affected other aspects of transcriptional regulation, we have analyzed SV40 chromosomes for the presence of the methylated forms of H3K4 and H3K9 following dysregulation of transcription by DRB which inhibits RNA Polymerase II elongation, and sodium butyrate which increases transcription from genes committed to transcription. SV40 chromosomes from treated and control infections were isolated between 2 and 12 hours post‐infection when only transcription was occurring or 48 hours post‐infection when transcription and replication were both occurring, purified, and subjected to ChIP analyses with antibodies to mono‐, di‐, and tri‐methylated H3K4 and H3K9 followed by real‐time PCR quantitation. Consistent with our previous published results we observed that dysregulation of transcription yielded different epigenetic readouts depending upon whether replication was occurring. Treatment with DRB resulted in a large increase in the levels of H3K9me2 and me3 in the presence of replication but had little effect on the levels of these modfications in the absence of replication. Treatment with sodium butyrate had the opposite effects. There was a significant reduction in H3K9me3 during replication and transcription but not when only transcription occurred. Taken together these results indicate that replication acts as a switch to epigenetically define the chromatin structure of newly replicated chromatin which will become new virus particles. Grant Funding Source: Supported by National Institutes of Health grant AI094441 to B.M.

We have previously shown that during the Simian Virus 40 (SV40) life‐cycle, DNA replication serves as an epigenetic switch by introducing H3K9me1 into newly replicated SV40 minichromosomes in order to repress early transcription in these newly‐replicated minichromosomes. While these studies demonstrated that epigenetics played a critical role in regulation of SV40 transcription, they did not directly address the exact mechanism of how the introduction of H3K9me1 repressed early transcription. We have now analyzed the location of nucleosomes in SV40 wild‐type 776 and the repression‐defective mutant cs1085 using a ChIP‐Seq like approach and confirmed that there is a significant increase in minichromosomes lacking a nucleosome over the early regulatory region in the cs1085 repression‐defective mutant minchromosomes compared to the wild‐type minichromosomes. In addition, we have shown that nucleosomes containing H3K9me1 are preferentially found within the early regulatory region in the wild‐type minichromosomes. These results suggest replication‐dependent repression of early transcription occurs by a mechanism in which nucleosomes specifically containing H3K9me1 are targeted to locations within the early regulatory region including the binding site of TBP and RNA Polymerase II. These specifically located nucleosomes would serve as a physical barrier to prevent the binding of the factors necessary for initiation of early transcription.