SV40 Minichromosomes Research Articles

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. Acetyl-H3 acted like epigenetic memory by being present at sites subsequently bound by RNAPII, while H3K9me1 and H3K27me3 were reorganized to the late side of the SV40 regulatory region apparently to repress late transcription. The organization of acetyl-H3 and H3K9me1 but not H3K27me3 required active transcription.

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. In virions from the mutant cs1085, which does not repress early transcription, we found that these two nucleosomes were significantly reduced compared to wild-type virions confirming a repressive role for them. In chromatin from cells infected for only 30min with wild-type virus, we observed a significant reduction in the nucleosomes at nt 5223 and nt 363 indicating that the potential repression by these nucleosomes appeared to be relieved very early in infection.

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

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)

Understanding How Proteins Shape DNA Using Energy Minimization

Several proteins are known to produce significant changes in DNA shape by locally deforming the double helix or by mediating the formation a loop. For example, the binding of the bacterial proteins HU or Hbb produces large bends in DNA and the tetrameric Lac repressor protein induces the formation of a loop between two distant DNA operators. In order to understand how such proteins sculpt DNA, we have developed a novel optimization method for DNA at the base-pair level. Our method accounts for the sequence-dependent elasticity of DNA and can be applied to a DNA fragment in which the first and last base pairs are spatially constrained. Moreover, our approach makes it possible to constrain intervening parts of the DNA in order to model the presence of bound proteins. We can therefore compute the energy landscape for a wide variety of protein-DNA systems. For example, we studied the effect of the presence of multiple HU or Hbb proteins on DNA minicircles and identified the most likely configurations for given DNA chain lengths and numbers of bound proteins. We also investigated how the presence of HU can enhance or diminish the apparent flexibility of DNA. In addition, we performed a detailed analysis of the energy landscape for loops induced by the Lac repressor. We focused on the binding of the Lac repressor protein on DNA minicircles and studied the competition between large and small loops on supercoiled molecules. We also found that changes in the repressor structure can lead to different loop topologies. Our method makes it possible to conduct thorough analyses of the geometry and mechanics of protein-DNA systems by computing the associated energy landscape and paves the way for the study of other biologically relevant system such as the SV40 minichromosome.

Replication and transcription of SV40 direct distinct epigenetic signaling

We have characterized how transcriptional regulatory sequences direct histone modifications during replication and transcription of Simian Virus 40. The role of DNA replication in directing histone modification was determined by analyzing the introduction of histone modifications (H3K4me1,me2,me3 and H3K9me1,me2,me3) from 24 hours to 48 hours post‐infection and following aphidicolin inhibition of DNA replication. Notably, only the introduction of H3K9me1 and H3K4me1 appeared to be totally dependent upon DNA replication. Since the repressive regulatory sequence, Site I, results in the introduction of H3K9me1 during replication, we determined its effect on transcriptional repression early in infection. Consistent with repression, we observed a reduction in SV40 minichromosomes containing RNA polymerase II from 2 hours to 8 hours post‐infection. Unexpectedly, we also observed a reduction in the presence of H3K9me1, H3K9me2, and H3K9me3 indicating that these modifications were not being introduced along with repression. We also analyzed SV40 recombinants containing either one or two copies of Site I and confirmed that the introduction of H3K9me1 was only associated with Site I during replication. Taken together these results indicate that transcriptional regulatory sequences can alter the epigenetic profile of a gene depending upon whether they function during replication or transcription.

Effect of magnesium ions and temperature on the sequence-dependent curvature of DNArestriction fragments

Transient electric birefringence has been used to quantify the curvature of two DNArestriction fragments, a 199-base-pair fragment taken from the origin of replication of theM13 bacteriophage and a 207-base-pair fragment taken from the VP1 gene inthe SV40 minichromosome. Stable curvature in the SV40 and M13 restrictionfragments is due to a series of closely spaced A tracts, runs of 4–6 contiguousadenine residues located within 40 or 60 base pair ‘curvature modules’ near thecenter of each fragment. The M13 and SV40 restriction fragments exhibit bends of ∼ 45° in solutions containing monovalent cations and ∼ 60° in solutionscontaining Mg2 + ions. The curvature is not localized at a single site but is distributed over the various Atracts in the curvature modules. Thermal denaturation studies indicate that thecurvature in the M13 and SV40 restriction fragments remains constant up to30 °C in solutions containing monovalent cations, and up to40 °C in solutionscontaining Mg2 + ions, before beginning to decrease slowly with increasing temperature. Hence, stable curvaturein these DNA restriction fragments exists at the biologically important temperature of37 °C.

Histone H4 lysine 20 mono- and tri-methylation define distinct biologically 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. In contrast, H4K20me2 did not appear to be present and H4K20me3 appeared to be present only in minichromosomes obtained 30 minutes post-infection. The presence of H4K20me1 late in infection in replicating minichromosomes and its relative enrichment in virions suggested that it played a role in the encapsidation process. In contrast, the presence of H4K20me3 at the earliest stages of the infection and its subsequent relatively rapid loss along with SV40 chromatin suggested that it was functioning during the uncoating process.

Temporal Control of Histone H4 methylation in SV40 minichromosomes during lytic infection

Acetylation and methylation of histones are known to play critical roles in many biological processes. We have previously shown that in the coding region of a transcribing SV40 minichromosome, histone hyperacetylation is a dynamic process regulated by the interplay of p300, a histone acetyltransferase and a histone deacetylase, co migrating along with the transcription complex. Our current studies are focused on histone H4 methylation in SV40 minichromosomes. Histone H4 is known to undergo mono, di, or tri methylation of lysine 20 (H4K20me1, H4K20me2, and H4K20me3 respectively) with differing biological effects. Using various chromatin immunoprecipitation procedures we have determined that minichromosomes containing H4K20me1 were present throughout an SV40 infection, and minichromosomes containing H4K20me3 were present only during the first hours of infection. Minichromosomes containing H4K20me2 were not present in infected cells. Combining ChIP analysis with tritiated thymidine pulse radiolabeling of chromatin we observed direct evidence for conservation of histone methylation in daughter minichromosomes following replication. In contrast to our results with histone hyperacetylation in SV40 minichromosomes, methylation of H4K20 appears to define a small conserved stable subclass of minichromosomes with the potential to participate in various biological functions.

Dual agarose magnetic (DAM) ChIP

BackgroundChromatin 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.FindingsProtein A agarose and protein G magnetic beads bound with different IgGs have been combined in a single Chromatin Immunoprecipitation (ChIP) assay to analyze for the presence of two epitopes on the same chromatin at the same time. This procedure has been used with non-immune rabbit IgG bound to either the agarose or beads in order to include an internal negative control for non-specific binding of chromatin. The procedure has also been used with various antibodies including those targeting RNA Polymerase II and replication protein A 70 to determine whether specific forms of modified histones are present in either transcribing or replicating forms of SV40 minichromosomes respectively.ConclusionsThe DAM ChIP procedure is a rapid, simple, and sensitive technique to characterize two epitopes located in the same chromatin. It should be particularly useful for the rapid screening of epigenetic modifications present in biologically active chromatin.

DEK Proto-Oncogene Expression Interferes with the Normal Epithelial Differentiation Program

Overexpression of the DEK gene is associated with multiple human cancers, but its specific roles as a putative oncogene are not well defined. DEK transcription was previously shown to be induced by the high-risk human papillomavirus (HPV) E7 oncogene via E2F and Rb pathways. Transient DEK overexpression was able to inhibit both senescence and apoptosis in cultured cells. In at least the latter case, this mechanism involved the destabilization of p53 and the decreased expression of p53 target genes. We show here that DEK overexpression disrupts the normal differentiation program in a manner that is independent of either p53 or cell death. DEK expression was distinctly repressed upon the differentiation of cultured primary human keratinocytes, and stable DEK overexpression caused epidermal thickening in an organotypic raft model system. The observed hyperplasia involved a delay in keratinocyte differentiation toward a more undifferentiated state, and expansion of the basal cell compartment was due to increased proliferation, but not apoptosis. These phenotypes were accompanied by elevated p63 expression in the absence of p53 destabilization. In further support of bona fide oncogenic DEK activities, we report here up-regulated DEK protein levels in both human papilloma virus-positive hyperplastic murine skin and a subset of human squamous cell carcinomas. We suggest that DEK up-regulation may contribute to carcinoma development at least in part through increased proliferation and retardation of differentiation.

HDAC inhibitors stimulate viral transcription by multiple mechanisms

BackgroundThe effects of histone deacetylase inhibitor (HDACi) treatment on SV40 transcription and replication were determined by monitoring the levels of early and late expression, the extent of replication, and the percentage of SV40 minichromosomes capable of transcription and replication following treatment with sodium butyrate (NaBu) and trichostatin A (TSA).ResultsThe HDACi treatment was found to maximally stimulate early transcription at early times and late transcription at late times through increased numbers of minichromosomes which carry RNA polymerase II (RNAPII) transcription complexes and increased occupancy of the transcribing minichromosomes by RNAPII. HDACi treatment also partially relieved the normal down-regulation of early transcription by T-antigen seen later in infection. The increased recruitment of transcribing minichromosomes at late times was correlated to a corresponding reduction in SV40 replication and the percentage of minichromosomes capable of replication.ConclusionThese results suggest that histone deacetylation plays a critical role in the regulation of many aspects of an SV40 lytic infection.

Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication

Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase α-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein–protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end.

Kinetic behavior of the reaction between hydroxyl radical and the SV40 minichromosome

Aqueous solutions containing the minichromosomal form of the virus SV40 and the radical scavenger DMSO were subjected to γ-irradiation, and the resulting formation of single-strand breaks (SSB) was quantified. Under the irradiation conditions, most SSBs were produced as a consequence of hydroxyl radical ( OH) reactions. By controlling the competition between DMSO and the viral DNA substrate for OH, we are able to estimate the rate coefficient for the reaction of OH with the SV40 minichromosome. The results cannot be described adequately by homogeneous competition kinetics, but it is possible to describe the rate coefficient for the reaction as a function of the scavenging capacity of the solution. The experimentally determined rate coefficient lies in the range 1×10 9–2×10 9 L mol −1 s −1 at 10 7 s −1, and increases with increasing scavenging capacity.

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. The absence of unacetylated H4 and H3 at sites containing RNAPII was correlated with the specific association of the histone acetyl transferase p300 with the RNAPII. In contrast, the presence of unacetylated H4 and H3 at sites lacking RNAPII was shown to result from the action of a histone deacetylase based upon the effects of the inhibitor sodium butyrate. These results suggest that the extent of hyperacetylation of H4 and H3 during transcription alternates between hyperacetylation directed by an RNAPII associated histone acetyl transferase and deacetylation directed by a histone deacetylase at other sites.

Ataxia-telangiectasia-mutated (ATM) Is a T-antigen Kinase That Controls SV40 Viral Replication in Vivo

The structurally related ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases fulfill overlapping yet non-redundant functions as key regulators of cellular DNA damage responses. We recently showed that ATM phosphorylates the cyclic AMP response element-binding protein, CREB, following exposure to ionizing radiation (IR) and other DNA-damaging stimuli. Here, we show that a phospho-specific antibody recognizing the major ATM phosphorylation site in CREB cross-reacts with SV40 large tumor antigen (LTag), a multifunctional oncoprotein required for replication of the SV40 minichromosome. The relevant IR-induced phosphorylation site in LTag recognized by phospho-CREB antibody was mapped to Ser-120. IR strongly induced the phosphorylation of Ser-120 in an ATM-dependent manner in mouse embryo fibroblasts. Infection of African green monkey CV1 cells with SV40 resulted in the activation of ATM and phosphorylation of LTag and endogenous ATM substrates. Infection-induced LTag phosphorylation correlated with the onset of DNA replication, was ATM-dependent, and peaked when viral DNA levels reached their maximum. SV40 replication in CV1 cells required an intact LTag Ser-120 phosphorylation site and was inhibited following transfection with ATM small interfering RNA suggesting that ATM is required for optimal SV40 replication in primate cells. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.

Curved DNA molecules migrate anomalously slowly in free solution

The electrophoretic mobility of a curved DNA restriction fragment taken from the VP1 gene in the SV40 minichromosome has been measured in polyacrylamide gels and free solution, using capillary electrophoresis. The 199 bp restriction fragment has an apparent bend angle of 46 ± 2° located at SV40 sequence position 1922 ± 2 bp [Lu Y.J., Weers B.D. and Stellwagen N. C. (2005) Biophys. J., 88, 1191–1206]. The ‘curvature module’ surrounding the apparent bend center contains five unevenly spaced A- and T-tracts, which are responsible for the observed curvature. The parent 199 bp fragment and sequence mutants containing at least one A-tract in the curvature module migrate anomalously slowly in free solution, as well as in polyacrylamide gels. Hence, the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due in part to their anomalously slow mobilities in free solution. Analysis of the gel and free solution mobility decrements indicates that each A- or T-tract contributes independently, but not equally, to the curvature of the 199 bp fragment and its A-tract mutants. The relative contribution of each A- or T-tract to the observed curvature depends on its spacing with respect to the first A-tract in the curvature module.

Role of DNA organisation and environmental scavenging capacity in the evolution of radiobiological damage: models and simulations

Theoretical models and Monte Carlo simulations were developed, aimed to investigate the role played by the organisation of interphase DNA and the environmental scavenging capacity conditions in the induction of radiobiological damage. The induction of single- and double-strand breaks by gamma rays impinging on different DNA structures (e.g. linear DNA, SV40 minichromosome and cellular DNA) was simulated as a function of the environment scavenging capacity. Furthermore, yields of chromosome aberrations (CA) induced by gamma rays and light ions were simulated with a purposely developed MC code that explicitly takes into account the DNA higher-order organisation as chromosome territories. Simulations performed with the PARTRAC code allowed quantification of the dependence of dsb and ssb both on the target structure, and on the scavenging capacity. The results relative to CA showed the importance of DNA damage complexity (nanometre scale) and interphase chromosome domains (micrometre scale) in the process of aberration formation. Very good agreement was found between the model predictions on ssb, dsb and CA and available experimental data.

D(GA x TC)(n) microsatellite DNA sequences enhance homologous DNA recombination in SV40 minichromosomes.

The genomic distribution of the abundant eukaryotic d(GA x TC)(n) DNA microsatellite suggests that it could contribute to DNA recombination. Here, it is shown that this type of microsatellite DNA sequence enhances DNA recombination in SV40 minichromosomes, the rate of homologous DNA recombination increasing by as much as two orders of magnitude in the presence of a d(GA x TC)(22) sequence. This effect depends on the region of the SV40 genome at which the d(GA x TC)(22) sequence is cloned. It is high when the sequence is located proximal to the SV40 control region but no effect is observed when located 3.5 kb away from the SV40 ori. These results indicate that the recombination potential of d(GA x TC)(n) sequences is likely linked to DNA replication and/or transcription. The potential contribution of the structural properties of d(GA x TC)(n) sequences to this effect is discussed.

Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins

We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.

Transcriptional and structural analyses of isolated SV40 chromatin.

AbstractAn ideal biochemical source of a defined chromatin template assembled in vivo is the SV40 minichromosomes (see Note 1). At all stages in the viral lytic cycle, SV40 DNA is complexed with cellular histone and nonhistone proteins to form the episomal chromatin structure called a minichromosome (MC). MCs are therefore the viral template for both the host replication and host transcription machinery. The chromatin properties of MCs reflect transcriptionally competent host chromatin in every respect that has been examined. They contain more highly acetylated histones, high-mobility group proteins, and DNase I hypersensitive sites (1–4). For these reasons, SV40 MCs have long been used as a model system for transcriptionally active chromatin. Because of the transcriptionally competent nature of these templates, they provide chromatin with characteristics distinct from, but complementary to, most in vitro reconstituted chromatin. SV40 MCs are especially useful in approaching questions regarding stages in transcriptional activation from a potentially competent to a fully active state. Furthermore, because of the utility of SV40 as a viral vector for exogenous promoters (5,6), fully functional cellular promoters in a native chromatin context can also be isolated and studied in this manner.KeywordsViral StockMicrococcal NucleaseBasal Transcription FactorEthanol BathSV40 MinichromosomesThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.