SP1 binding in SV40 chromatin in vitro and in vivo was characterized in order to better understand its role during the initiation of early transcription. We observed that chromatin from disrupted virions, but not minichromosomes, was efficiently bound by HIS-tagged SP1 in vitro, while the opposite was true for the presence of endogenous SP1 introduced in vivo. Using ChIP-Seq to compare the location of SP1 to nucleosomes carrying modified histones, we found that SP1 could occupy its whole binding site in virion chromatin but only the early side of its binding site in most of the minichromosomes carrying modified histones due to the presence of overlapping nucleosomes. The results suggest that during the initiation of an SV40 infection, SP1 binds to an open region in SV40 virion chromatin but quickly triggers chromatin reorganization and its own removal by a hit and run mechanism.

No abstract

We have recently shown that at late times in an SV40 infection a nucleosome containing modified histones is positioned over the enhancer region consistent with repression of early transcription and activation of late transcription. However, this nucleosome appears to slide toward the late region during virion formation consistent with a role in the activation of early transcription and repression of late transcription in a subsequent infection. Because the transcription factor SP1 plays a major role in regulating SV40 early and late transcription and its binding should be sensitive to nucleosome positioning, we measured the ability of HIS‐tagged SP1 to bind to various forms of SV40 chromatin in vitro and compared this to the presence of SP1 in SV40 chromatin derived in vivo. We found that HIS‐tagged SP1 bound well to chromatin from virions and very poorly to other forms of SV40 chromatin in vitro, while cellular SP1 was associated with minichromosomes isolated from infected cells and was not found in chromatin from virions. In order to determine whether the location of nucleosomes and associated histone modifications plays a role in the activation and continuation of early transcription during the initiation of an infection, we have mapped the location of RNAPII and nucleosomes containing a number of histone modifications in chromatin from virions and minichromosomes isolated 30 minutes and 2 hours post‐infection using ChIP‐Seq. During the first two hours of infection we found that RNAPII first associates with the miRNA site in the SV40 genome (at 30 minutes post‐infection) and then shifts to the early regulatory region around the enhancer (2 hours post‐infection). We next compared the location of RNAPII at the two time points to the location of nucleosomes carrying one of nine different specific histone modifications in the chromatin from virions, and minichromosomes isolated at these times. Chromatin from virions was used because it is the substrate for the activation of transcription, while chromatin from the minichromosomes would indicate any changes occurring as a consequence of transcriptional activation or extension. Based upon this rationale, we identified acetylated H3 as the most likely histone modification in SV40 predicting subsequent binding by RNAPII. We also identified acetylated H4 and the shifting of nucleosomes away from the RNAPII binding site as changes which result from activation of transcription. Together these results indicate that positioned nucleosomes carrying certain histone modifications may control accessibility of transcription factors and also serve as marks for either the binding of RNAPII during initiation of transcription or subsequent re‐initiation during active transcription. Support or Funding Information NIH 1R03AI127969‐01NIH 1R03AI142011‐01

No abstract

SP1 binding in SV40 chromatin in vitro and in vivo was characterized in order to better understand its role during the initiation of early transcription. We observed that chromatin from disrupted virions, but not minichromosomes, was efficiently bound by HIS-tagged SP1 in vitro, while the opposite was true for the presence of endogenous SP1 introduced in vivo. Using ChIP-Seq to compare the location of SP1 to nucleosomes carrying modified histones, we found that SP1 could occupy its whole binding site in virion chromatin but only the early side of its binding site in most of the minichromosomes carrying modified histones due to the presence of overlapping nucleosomes. The results suggest that during the initiation of an SV40 infection, SP1 binds to an open region in SV40 virion chromatin but quickly triggers chromatin reorganization and its own removal by a hit and run mechanism.