Differential regulation by SV40 t-antigen binding at site I defines two distinct classes of nucleosome-free promoter

et al. 2018

Preprint

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40Simian Virus 40 (SV40) exists as chromatin throughout its life cycle, and 41 undergoes typical epigenetic regulation mediated by changes in nucleosome 42 location and associated histone modifications. In order to investigate the role of 43 epigenetic regulation during the encapsidation of late stage minichromosomes 44 into virions, we have mapped the location of nucleosomes containing acetylated 45 or methylated lysines in the histone tails of H3 and H4 present in the chromatin 46 from 48-hour post-infection minichromosomes and disrupted virions. In 47 minichromosomes obtained late in infection, nucleosomes were found carrying 48 various histone modifications primarily in the regulatory region with a major 49 nucleosome located within the enhancer and other nucleosomes at the early and 50 late transcriptional start sites. The nucleosome found in the enhancer would be 51 expected to repress early transcription by blocking access to part of the SP1 52 binding sites and the left side of the enhancer in late stage minichromosomes 53 while also allowing late transcription. In chromatin from virions, the principal 54 nucleosome located in the enhancer was shifted ~ 70 bases in the late direction 55 from what was found in minichromosomes and the level of modified histones was 56 increased throughout the genome. The shifting of the enhancer-associated 57 nucleosome to the late side would effectively serve as a switch to relieve the 58 repression of early transcription found in late minichromosomes while likely also 59 repressing late transcription by blocking access to necessary regulatory 60 3 sequences. This epigenetic switch appeared to occur during the final stage of 61 virion formation. 62 63 Introduction 64 Simian Virus 40 (SV40), a member of the Polyomavirus family of DNA 65 viruses, consists of a 5243 base-pair (bp) double-strand closed-circular genome 66(1). Like many double-strand DNA viruses, SV40 exists as chromatin with the 67 usual assortment of histones and most of the epigenetic marks typical of 68 eukaryotic chromatin when found within an infected cell (2, 3). However, unlike 69 most DNA viruses, SV40 also exists with a typical chromatin structure when 70 found within the virion (3). The organization of SV40 DNA into chromatin 71 throughout its life cycle raises the interesting possibility that epigenetics plays a 72 critical role in the regulation of the various stages of the SV40 life cycle. In 73 support of this hypothesis, we have previously shown that cellular treatments that 74 affect the level of certain histone modifications in the SV40 virus produced during 75 the course of an infection can result in changes in the infectivity of the virus (4). 76Moreover, we have also shown that the organizations of nucleosomes in SV40 77 minichromosomes at 30 minutes and 48 hours post-infection differs significantly 78 from the organization of nucleosomes in the chromatin from SV40 virions (5). 79Since SV40 virion chromatin differs in organization from late stage 80 minichromosomes and also carries informati...

Section: Resultsmentioning

confidence: 99%

Directed Nucleosome Sliding in SV40 Minichromosomes During the Formation of the Virus Particle Exposes DNA Sequences Required for Early Transcription

Kumar

Kasti

et al. 2018

Preprint

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“…In order to confirm that the regulatory nucleosomes containing H3K9me1 were involved in the repression of early transcription, we compared the organization of H3K9me1 in wild-type virion chromatin, where repression has occurred, to the organization of H3K9me1 in the SV40 mutant cs1085, which lacks T-antigen binding site I and as a consequence does not undergo repression of early transcription (15,16). We have used this mutant extensively to study the epigenetic regulation of SV40 molecular biology (4,5,8,9,14,17,18) and have shown that the organization of total nucleosomes changes in the regulatory region of the SV40 mutant cs1085, along with a reduction in the proportion of SV40 chromatin containing H3K9me1 (4,5,8,14). It should be noted that in our prior studies (4), although there was a significant reduction in the percentage of SV40 chromatin containing H3K9me1, the percentage never actually reached zero, indicating that some nucleosomes containing H3K9me1 remained in the SV40 chromatin.…”

Section: Organization Of Histone Modifications In Nucleosomes Presentmentioning

confidence: 99%

Directed Nucleosome Sliding during the Formation of the Simian Virus 40 Particle Exposes DNA Sequences Required for Early Transcription

Kumar

Kasti

et al. 2019

J Virol

Self Cite

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 mapped the locations of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-hpostinfection 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 ϳ70 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. IMPORTANCE For a virus to complete infection, it must produce a new virus particle in which the genome is able to support a new infection. This is particularly important for viruses like simian virus 40 (SV40), which exist as chromatin throughout their life cycles, since chromatin structure plays a major role in the regulation of the life cycle. In order to determine the role of SV40 chromatin structure late in infection, we mapped the locations of nucleosomes and their histone tail modifications in SV40 minichromosomes and in the SV40 chromatin found in virions using chromatin immunoprecipitation-DNA sequencing (ChIP-Seq). We have identified a novel viral transcriptional control mechanism in which a nucleosome found in the regulatory region of the SV40 minichromosome is directed to slide during the formation of the virus particle, exposing transcription factor binding sites required for early transcription that were previously blocked by the presence of the nucleosome.

“…Simian virus 40 (SV40), a member of the polyomavirus family of small DNA tumor viruses, has a closed‐circular double‐stranded DNA genome that is 5243 bp in length in the wild‐type laboratory strain (Tooze & Acheson, ). Like cellular DNA, SV40 DNA is organized with cellular proteins into typical chromatin structure and is subject to typical epigenetic regulation (Cereghini & Yaniv, ; Coca‐Prados & Hsu, ; Kube & Milavetz, ; Milavetz et al., ). Unlike most other viral DNAs (such as adenovirus and herpesvirus DNAs), SV40 DNA is found as chromatin in both the virus particle and intracellular minichromosomes, and as a consequence undergoes epigenetic regulation during an infection that can be transmitted to a subsequent infection (Milavetz et al., ).…”

Section: Introductionmentioning

confidence: 99%

Epigenetic Analysis of SV40 Minichromosomes

Milavetz

2017

CP Microbiology

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Simian Virus 40 (SV40) is one of the best characterized members of the polyomavirus family of small DNA tumor viruses. It has a small genome of 5243 base pairs and utilizes cellular proteins for its molecular biology with the exception of the T-antigen protein, which is coded by the virus and is involved in regulating transcription and directing replication. Importantly, SV40 exists as chromatin in both the virus particle and intracellular minichromosome. These facts combined with high yields of virus and minichromosomes following infection, and ease of manipulation, have made SV40 an extremely useful model to study all aspects of eukaryotic molecular biology. This protocol consists of two parts. The first part describes the procedures for working with SV40 and preparing SV40 chromatin from infected cells and virus particles. The second part describes how the SV40 chromatin can be used to study epigenetic regulation.