Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection

Abstract

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.

Mapping the Location of Nucleosomes in Simian Virus 40 Chromatin Using the New England Biolabs FS Library Prep Kit

“…We have previously observed that the pattern of nucleosomes on the SV40 genome changes over the course of an infection when analyzed by MN-Seq and ChIP-Seq (2,7). In order to determine whether the pattern of nucleosome positioning when analyzed by FS-Seq also changed consistent with our previous observations, we compared results from FS-Seq to the two other techniques in SV40 chromatin isolated at 30 minutes post-infection and 48 hours postinfection.…”

“…In order to determine whether the pattern of nucleosome positioning when analyzed by FS-Seq also changed consistent with our previous observations, we compared results from FS-Seq to the two other techniques in SV40 chromatin isolated at 30 minutes post-infection and 48 hours postinfection. In our previous work with MN-Seq (7) and ChIP-Seq (2) we observed using MN-Seq that a nucleosome was present in the enhancer in chromatin from 48 hour infections which appeared to be present in much less of the chromatin in virions or at 30 minutes post infection.…”

“…Since the FS kit was likely to be inhibited by the presence of proteins in chromatin, we first determined whether the enzymes could fragment SV40 chromatin. Using real-time PCR amplification of a 300 bp SV40 fragment, we measured the relative amount of DNA in chromatin samples before digestion and after digestion at different temperatures and for different times as previously described (7). In this assay, if chromatin is digested the amount of DNA in the sample is reduced and the magnitude of the reduction is an indication of how extensively the chromatin has been fragmented.…”

“…In order to determine whether the fragmentation of SV40 chromatin found in disrupted virions by the FS kit was consistent with the generation of nucleosome fragments, we compared the sequencing results from our FS libraries to data we have previously generated using micrococcal nuclease (7) and ChIP-Seq with antibody to hyperacetylated H4 (HH4) (2) corresponding to the transcription start sites for early and late transcription respectively which appeared with all of the techniques to a greater or lesser extent. In addition to these relatively brighter bands located in the major SV40 regulatory region, there were a number of other fainter bands that appeared to coincide between FS-Seq and one or more of the other techniques.…”

“…The procedures which we used to prepare sequencing libraries by MN-Seq have been previously described in detail (6,7). For each sample of SV40, we determined the optimum extent of digestion by incubating the chromatin with diluted micrococcal nuclease in digestion buffer at 4 o for from 5 to 30 seconds.…”