Analysis of nuclease hypersensitive‐equivalent sites in SV40 by Next Generation Sequencing

Abstract

Chromatin fragments obtained by sonication or Micrococcal nuclease digestion of SV40 minichromosomes or disrupted virions were analyzed by Next Generation Sequencing (NGS) to determine the location of regions of the SV40 genome differentially sensitive to either treatment. Both treatments yielded samples which upon analysis allowed us to identify regions of hypersensitivity and moderate sensitivity. With sonication all of the samples contained regions of hypersensitivity centered on nucleotides 141 and 221 and regions of moderate sensitivity surrounding these sites which included the 21 base pair repeat regions and enhancers. Following Micrococcal nuclease treatment Hypersensitive sites were found in minichromosomes centered on nucleotides 30 and 92 and in chromatin from disrupted virions centered at nucleotide 106. Micrococcal nuclease also generated a pattern of peaks and valleys based upon the number of reads at each nucleotide consistent with the location of nucleosomes. While the pattern was very similar throughout most of the SV40 genome in minichromosomes and chromatin from disrupted virions, the pattern differed significantly in the regulatory region. In minichromosomes we observed a nucleosome‐free region extending from approximately nucleotide 5000 to nucleotide 250 encompassing most of the early and late transcription regulatory sequences and the origin of replication. In chromatin from disrupted virions we observed a very small open region which at most included only the enhancers.Support or Funding InformationThis work was supported by grants from NIH AI094441 (to BM) and NIH GM098328 (to LB)