Biology subjects

Milavetz, B.

Publications and source records attributed to Milavetz, B..

3 recordsLinked to original sources

bioRxiv2020

Differential SP1 Interactions in SV40 Chromatin from Virions and Minichromosomes

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.

molecular biology↗

bioRxiv2020

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

The location of nucleosomes in chromatin significantly impacts many biological processes including DNA replication, repair and gene expression. A number of techniques have been developed for mapping nucleosome locations in chromatin including MN-Seq (micrococcal nuclease digestion followed by next generation sequencing), ATAC-Seq (Tagamet chromatin fragmentation followed by next generation sequencing), and ChIP-Seq (chromatin immunoprecipitation and fragmentation followed by next generation sequencing). All of these techniques have been successfully used, but each with its own limitations. Recently, New England Biolabs has marketed a new kit, the NEBNext UltraII FS Library Prep kit, for preparing libraries for next generation sequencing from purified genomic DNA. This kit is based on a novel proprietary DNA fragmentation procedure which appears to cleave DNA that is not bound by proteins. Because DNA is fragmented directly in the FS kit, we tested whether the kit might also be useful for mapping the location of nucleosomes in chromatin. Using Simian Virus 40 (SV40) chromatin isolated at different times in an infection, we have compared nucleosome mapping using the NEB FS kit (FS-Seq) to MN-Seq, ATAC-Seq, and ChIP-Seq. Mapping nucleosomes using FS-Seq generated nucleosome profiles similar to those generated by ATAC-Seq and ChIP-Seq in regulatory regions of the SV40 genome. We conclude that FS-Seq is a simple, robust, cost-effective procedure for mapping nucleosomes in SV40 chromatin that should be useful for other forms of chromatin as well. We also present evidence that the FS kit may be useful for mapping the location of transcription factors in chromatin when sequencing reads between 75 and 99 base pairs in size are analyzed.

molecular biology↗

bioRxiv2018

Directed nucleosome sliding during the formation of the SV40 virus particle exposes DNA sequences required for early transcription

Simian Virus 40 (SV40) exists as chromatin throughout its life cycle, and undergoes typical epigenetic regulation mediated by changes in nucleosome location and associated histone modifications. In order to investigate the role of epigenetic regulation during the encapsidation of late stage minichromosomes into virions, we have mapped the location of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-hour post-infection minichromosomes and disrupted virions. In minichromosomes obtained late in infection, nucleosomes were found carrying various histone modifications primarily in the regulatory region with a major nucleosome located within the enhancer and other nucleosomes at the early and late transcriptional start sites. The nucleosome found in the enhancer would be expected to repress early transcription by blocking access to part of the SP1 binding sites and the left side of the enhancer in late stage minichromosomes while also allowing late transcription. In chromatin from virions, the principal nucleosome located in the enhancer was shifted ~ 70 bases in the late direction from what was found in minichromosomes and the level of modified histones was increased throughout the genome. The shifting of the enhancer-associated nucleosome to the late side would effectively serve as a switch to relieve the repression of early transcription found in late minichromosomes while likely also repressing late transcription by blocking access to necessary regulatory sequences. This epigenetic switch appeared to occur during the final stage of virion formation.

molecular biology↗