Date of Award
Doctor of Philosophy (PhD)
Simian Virus 40 (SV40) is a well-characterized virus whose small circular DNA genome is organized into chromatin and, as a consequence, undergoes many of the same biological processes observed in cellular chromatin. 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. We have subsequently 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.
In order to determine the capacity of SV40 epigenetic regulation, we have analyzed SV40 chromatin from minichromosomes and virions for the presence of modified histones using various ChIP techniques and correlated these modifications with specific biological effects on the SV40 life cycle. 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.
We 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.
Our results demonstrate that, like its cellular counterpart, SV40 chromatin is capable of passing biologically relevant transgenerational epigenetic information between infections. Our 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.
Kallestad, Les, "Using Simian Virus 40 As A Model To Determine The Effects Of Replication And Transcription On Histone Methylation" (2016). Theses and Dissertations. 2034.