Mapping Nucleosome Location Using FS-Seq.

The organization of nucleosomes in eukaryotic chromatin is thought to play a critical role in the regulation of the biological function of the chromatin. Because of this potential role in regulation, a number of techniques have been developed, which combine chromatin fragmentation around nucleosomes with next-generation sequencing to map the location of nucleosomes in chromatin. In this section, a procedure using a kit from New England Biolabs (NEB NEXT Ultra II FS DNA library prep Kit) to fragment chromatin in preparation for next-generation sequencing is described and compared to other available procedures for mapping nucleosome location.

Early in an SV40 infection, histone modifications correlate with the presence or absence of RNAPII and direction of transcription.

Since epigenetic regulation seemed likely to be involved in SV40 early transcription following infection, we have analyzed the organization of nucleosomes carrying histone modifications (acetyl-H3, acetyl-H4, H3K9me1, H3K9me3, H3K4me1, H3K4me3, H3K27me3, H4K20me1) at 30 min and 2 h post infection in SV40 minichromosomes prepared in the absence or presence of the transcription inhibitor dichloro-1-beta-d-ribofuranosyl benzimidazole. The former condition was used to determine how SV40 chromatin structure changed during early transcription, and the latter was used to determine the role of active transcription. The location of RNAPII was used as a marker to identify where histone modifications were most likely to be involved in regulation.

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.

Comparing a new method for mapping nucleosomes in simian virus 40 chromatin to standard procedures.

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 (Assay for Transposase-Accessible Chromatin 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.

Directed Nucleosome Sliding during the Formation of the Simian Virus 40 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 mapped the locations of nucleosomes containing acetylated or methylated lysines in the histone tails of H3 and H4 present in the chromatin from 48-h-postinfection 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.

Epigenetic Regulation of Viral Biological Processes.

It is increasingly clear that DNA viruses exploit cellular epigenetic processes to control their life cycles during infection. This review will address epigenetic regulation in members of the polyomaviruses, adenoviruses, human papillomaviruses, hepatitis B, and herpes viruses. For each type of virus, what is known about the roles of DNA methylation, histone modifications, nucleosome positioning, and regulatory RNA in epigenetic regulation of the virus infection will be discussed.

Epigenetic Analysis of SV40 Minichromosomes.

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 bp 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 minichromosomes.

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

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.

Viral epigenetics.

DNA tumor viruses including members of the polyomavirus, adenovirus, papillomavirus, and herpes virus families are presently the subject of intense interest with respect to the role that epigenetics plays in control of the virus life cycle and the transformation of a normal cell to a cancer cell. To date, these studies have primarily focused on the role of histone modification, nucleosome location, and DNA methylation in regulating the biological consequences of infection. Using a wide variety of strategies and techniques ranging from simple ChIP to ChIP-chip and ChIP-seq to identify histone modifications, nuclease digestion to genome wide next generation sequencing to identify nucleosome location, and bisulfite treatment to MeDIP to identify DNA methylation sites, the epigenetic regulation of these viruses is slowly becoming better understood.

Transcriptional repression is epigenetically marked by H3K9 methylation during SV40 replication.

Background: We have recently shown that T-antigen binding to Site I results in the replication-dependent introduction of H3K9me1 into SV40 chromatin late in infection. Since H3K9me2 and H3K9me3 are also present late in infection, we determined whether their presence was also related to the status of ongoing transcription and replication. Transcription was either inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) or stimulated with sodium butyrate and the effects on histone modifications early and late in infection determined.

Transcription and replication result in distinct epigenetic marks following repression of early gene expression.

Simian virus 40 (SV40) early transcription is repressed when the product of early transcription, T-antigen, binds to its cognate regulatory sequence, Site I, in the promoter of the SV40 minichromosome. Because SV40 minichromosomes undergo replication and transcription potentially repression could occur during active transcription or during DNA replication. Since repression is frequently epigenetically marked by the introduction of specific forms of methylated histone H3, we characterized the methylation of H3 tails during transcription and replication in wild-type SV40 minichromosomes and mutant minichromosomes which did not repress T-antigen expression.

Virion-mediated transfer of SV40 epigenetic information.

In eukaryotes, epigenetic information can be encoded in parental cells through modification of histones and subsequently passed on to daughter cells in a process known as transgenerational epigenetic regulation. Simian Virus 40 (SV40) is a well-characterized virus whose small circular DNA genome is organized into chromatin and, as a consequence, undergoes many of the same biological processes observed in cellular chromatin. In order to determine whether SV40 is capable of transgenerational epigenetic regulation, we have analyzed SV40 chromatin from minichromosomes and virions for the presence of modified histones using various ChIP techniques and correlated these modifications with specific biological effects on the SV40 life cycle.

An improved 1-D gel electrophoresis image analysis system.

Images obtained through the gel electrophoresis technique contain important genetic information. However, due to degradations and abnormalities from which these images suffer, extracting this information can be a tedious task and may lead to reproducibility issues. Image processing techniques that are commonly used to analyze gel electrophoresis images require three main steps: band detection, band matching, and quantification.

Decoding the histone H4 lysine 20 methylation mark.

The molecular biology of histone H4 lysine 20 (H4K20) methylation, like many other post-translational modifications of histones, has been the subject of intensive interest in recent years. While there is an emerging consensus linking H4K20me1, H4K20me2, and H4K20me3 to transcription, repair, and constitutive heterochromatin, respectively, the specific details of these associations and the biological mechanisms by which the methylated histones are introduced and function are now the subject of active investigation. Although a large number of methylases capable of methylating H4K20 have been identified and characterized; there is no known demethylase of H4K20, though the search is ongoing.

Histone H4 lysine 20 mono- and tri-methylation define distinct biological processes in SV40 minichromosomes.

The methylation profile of histone h4 on lysine 20 in sV40 chromatin during an infection was investigated using ChIp analyses with antibodies to monomethyl (h4K20me1), dimethyl (h4K20me2), and trimethyl (h4K20me3) histone h4. h4K20me1 was found in late-transcribing, uncoating, encapsidating and replicating minichromosomes as well as in the sV40 chromatin present in virions. Its prevalence was greatest in virions and least in minichromosomes present between 4 and 24 hours post-infection.

Dual agarose magnetic (DAM) ChIP.

Background: Chromatin immunoprecipitation (ChIP) has become a very popular technique to study epigenetic regulation because it can be used to identify proteins and protein modifications present at specific locations in chromatin. While techniques have been developed to investigate epigenetic modifications present in chromatin during a specific biological function such as transcription, they depend upon the ability of the ChIP to analyze two epitopes on the same chromatin and are generally time consuming, difficult to perform, and not very sensitive. The Dual Agarose Magnetic (DAM) ChIP procedure described here is designed to address these shortcomings.

Histone hyperacetylation during SV40 transcription is regulated by p300 and RNA polymerase II translocation.

The effects of the RNA polymerase II (RNAPII) translocation inhibitors alpha amanitin and 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) and an siRNA targeting p300 on the presence of RNAPII, p300, hyperacetylated H4 and H3 and unmodified H4 and H3 in transcribing simian virus 40 (SV40) minichromosomes were determined. Following treatment with alpha amanitin we observed a profound reduction in the occupancy of the promoter by RNAPII, the loss of p300 from chromatin fragments containing RNAPII, and an increase in the amount of unmodified H4 and H3 associated with the RNAPII. Treatment with DRB had little effect on the presence of RNAPII or p300 but also resulted in a significant increase in the amount of unmodified H4 and H3 present in chromatin fragments associated with RNAPII.

Histone hyperacetylation in the coding region of chromatin undergoing transcription in SV40 minichromosomes is a dynamic process regulated directly by the presence of RNA polymerase II.

SV40 chromosomes undergoing transcription operationally defined by the presence of RNA polymerase II (RNAPII) were immune-selected with antibody to RNAPII and subjected to secondary chromatin immunoprecipitation with antibodies to hyperacetylated or unacetylated H4 or H3. Immune selection fragmentation and immunoprecipitation was used to determine the hyperacetylation status of histones independent of the location of the RNAPII and Re chromatin immunoprecipitation was used to determine their hyperacetylation status when associated with RNAPII. While hyperacetylated H4 and H3 were found in the coding regions regardless of the location of RNAPII, unacetylated H4 and H3 were found only at sites lacking RNAPII.

Sexually transmitted Human Papillomavirus type variations resulting in high grade cervical dysplasia in North-East North Dakota and North-West Minnesota.

Background: A review of Pap smear diagnoses from a reference laboratory in Grand Forks, North Dakota over a 3-year period (07/00 to 10/03) revealed a two-fold higher rate of high grade squamous intraepithelial lesion in a community in northwest Minnesota (Roseau, 0.486%) than in northeast North Dakota (Grand Forks, 0.249%), in spite of both having similar rates of low-grade squamous intraepithelial lesion (1.

Reorganization of RNA polymerase II on the SV40 genome occurs coordinately with the early to late transcriptional switch.

The pattern of organization of RNA polymerase II (RNAPII) in wild-type and mutant cs1085 SV40 chromosomes isolated between 30 min and 48 h post-infection was determined using a combination of chromatin immunoprecipitation (ChIP) techniques. During the course of a wild-type infection, we observed a slow but significant decline in the relative occupancy of RNAPII at the early region and a corresponding increase in occupation in the late region. In the promoter, occupancy began high, decreased to a minimum at 8 h post-infection, and then increased to a high level by 48 h post-infection.

Programmed remodeling of hyperacetylated histone H4 and H3 organization on the SV40 genome during lytic infection.

The presence of nucleosomes containing hyperacetylated histone H4 and H3 on the early, late, and promoter regions of the SV40 genome in chromosomes isolated 30 min, 8 h, and 48 h post-infection was determined by chromatin immunoprecipitation (ChIP) analysis with PCR amplification of fragmented SV40 chromatin using two complementary strategies. In chromosomes isolated at 30 min post-infection hyperacetylated H4 was found intermittently in all the three regions with no preference for one region over the other. In contrast, hyperacetylated H3 was organized primarily within the promoter region and occasionally elsewhere.

Expression of two succinyl-CoA synthetases with different nucleotide specificities in mammalian tissues.

For nearly 50 years, succinyl-CoA synthetase in animals was thought to be specific for guanine nucleotides. Recently, we purified and characterized both an ADP-forming succinyl-CoA synthetase from pigeon breast muscle and the GDP-forming enzyme from liver (Johnson, J. D.

Hyperacetylation and differential deacetylation of histones H4 and H3 define two distinct classes of acetylated SV40 chromosomes early in infection.

SV40 chromosomes undergoing encapsidation late in infection and SV40 chromatin in virions are hyperacetylated on histones H4 and H3. However, the fate of the SV40 chromosomes containing hyperacetylated histones in a subsequent round of infection has not been determined. In order to determine if SV40 chromosomes undergo changes in the extent of histone acetylation during early infection, we have analyzed SV40 chromosomes isolated 30 min and 3 h postinfection by quantitative ChIP assays, depletion ChIP assays, competitive ChIP assays, and ChIP assays combined with restriction endonuclease sensitivity using antibodies to hyperacetylated histones H4 and H3.