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

Kallestad L, Christensen K, Woods E, Milavetz B - Clin Epigenetics (2014)

Bottom Line: The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.Our data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication.The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a 'proof reading' mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, School of Medicine and Health Sciences, University of North Dakota, 501 N Columbia Road, Grand Forks, ND 58203 USA.

ABSTRACT

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. The role of DNA replication was determined by concomitant inhibition of replication with aphidicolin.

Results: We observed that H3K9me2/me3 was specifically introduced when transcription was inhibited during active replication. The introduction of H3K9me2/me3 that occurred when transcription was inhibited was partially blocked when replication was also inhibited. The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.

Conclusions: Our data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication. The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a 'proof reading' mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.

No MeSH data available.


Related in: MedlinePlus

Inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) stimulates the incorporation of H3K9me2 and H3K9me3 into SV40 chromatin late in infection. Wild-type SV40 minichromosomes were isolated at 2 hr post-infection with or without incubation with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from minus 2 hr until isolation and at 48 hr post-infection with or without incubation with DRB from 24 to 48 hr post-infection. The percentages of intact minichromosomes containing methylated H3K4 and H3K9 were determined by ChIP analyses followed by purification of the intact genomic DNA and PCR amplification with primers recognizing the promoter region. For each form of histone modification, the ratio of the percentage present in the treated minichromosomes compared to the untreated minichromosomes was calculated. The effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from minus 2 hr to isolation at 2 hr post-infection is shown in (A). The corresponding effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from 24 to 28 hr post-infection are shown in (B).
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Fig1: Inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) stimulates the incorporation of H3K9me2 and H3K9me3 into SV40 chromatin late in infection. Wild-type SV40 minichromosomes were isolated at 2 hr post-infection with or without incubation with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from minus 2 hr until isolation and at 48 hr post-infection with or without incubation with DRB from 24 to 48 hr post-infection. The percentages of intact minichromosomes containing methylated H3K4 and H3K9 were determined by ChIP analyses followed by purification of the intact genomic DNA and PCR amplification with primers recognizing the promoter region. For each form of histone modification, the ratio of the percentage present in the treated minichromosomes compared to the untreated minichromosomes was calculated. The effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from minus 2 hr to isolation at 2 hr post-infection is shown in (A). The corresponding effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from 24 to 28 hr post-infection are shown in (B).

Mentions: SV40 minichromosomes were isolated and purified from cells infected with wild-type 776 virus following treatment with DRB or no treatment. When minichromosomes were isolated at 2 hours, cells were pretreated with DRB for 2 hours prior to infection, while for minichromosomes isolated at 48 hours post-infection infected cells were treated from 24 to 48 hours. In these experiments, we observed a 112 ± 35 fold increase in the size of the pool of SV40 chromosomes present in glycerol gradient fractions following purification from cells infected for 48 hours compared to 24 hours post-infection. While the increase was somewhat variable we always observed at least a tenfold increase. This increase was reduced 10 ± 6 fold following treatment of the SV40 infected cells with DRB from 24 to 48 hours post-infection. The latter analysis was determined by comparing the increase in the presence and absence of inhibitor and indicates that on average there was about a tenfold reduction in the pool of SV40 minichromosomes following inhibition. As expected, treatment with DRB substantially inhibited the generation of mRNA (data not shown).The effect of DRB on the introduction of methylated H3K4 and H3K9 was determined by subjecting treated and untreated samples of intact SV40 minichromosomes obtained at 2 hours and 48 hours post-infection to ChIP analyses with antibodies that recognize mono-, di-, or tri-methylated H3K4 or H3K9. Intact SV40 minichromosomes were used because they are easily obtained in relatively large amounts and yield the maximum PCR signal compared to fragmented chromatin. Because the SV40 genome was intact, this analysis only yielded information relative to changes in the numbers of minichromosomes carrying a particular modification following treatment. No information was obtained concerning the location of any specific histone modifications. The results of these analyses are graphically shown in Figure 1. The data is displayed as the ratio of the percentage of minichromosomes that contain a modification following treatment divided by the percentage of untreated minichromosomes containing the same modification. If treatment had no effect on the introduction of a particular modification the ratio will be one. Ratios greater than one indicate that treatment results in an increase in the minichromosomes containing the modification while ratios less than one indicate that treatment resulted in a decrease in the presence of a modification.As shown in Figure 1A and B, DRB treatment had no significant effect on the methylation of H3K4 at either 2 hours or 48 hours post-infection. For each form of methylation, the ratio of treated sample to untreated sample was approximately one. In contrast, DRB treatment had a significant effect on the presence of H3K9me2 and H3K9me3 at 48 hours post-infection and little effect at 2 hours post-infection (Figure 1A and B). We observed a 6 ± 4 fold increase for H3K9me2 with a range from 3 to 12 for four independent samples and a 6 ± 2 fold increase for H3K9me3 with a range of 3 to 8 for four independent samples. As expected from our previous published work, DRB treatment had no significant effect on the presence of H3K9me1 at either time point.Figure 1


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

Kallestad L, Christensen K, Woods E, Milavetz B - Clin Epigenetics (2014)

Inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) stimulates the incorporation of H3K9me2 and H3K9me3 into SV40 chromatin late in infection. Wild-type SV40 minichromosomes were isolated at 2 hr post-infection with or without incubation with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from minus 2 hr until isolation and at 48 hr post-infection with or without incubation with DRB from 24 to 48 hr post-infection. The percentages of intact minichromosomes containing methylated H3K4 and H3K9 were determined by ChIP analyses followed by purification of the intact genomic DNA and PCR amplification with primers recognizing the promoter region. For each form of histone modification, the ratio of the percentage present in the treated minichromosomes compared to the untreated minichromosomes was calculated. The effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from minus 2 hr to isolation at 2 hr post-infection is shown in (A). The corresponding effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from 24 to 28 hr post-infection are shown in (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: Inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) stimulates the incorporation of H3K9me2 and H3K9me3 into SV40 chromatin late in infection. Wild-type SV40 minichromosomes were isolated at 2 hr post-infection with or without incubation with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from minus 2 hr until isolation and at 48 hr post-infection with or without incubation with DRB from 24 to 48 hr post-infection. The percentages of intact minichromosomes containing methylated H3K4 and H3K9 were determined by ChIP analyses followed by purification of the intact genomic DNA and PCR amplification with primers recognizing the promoter region. For each form of histone modification, the ratio of the percentage present in the treated minichromosomes compared to the untreated minichromosomes was calculated. The effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from minus 2 hr to isolation at 2 hr post-infection is shown in (A). The corresponding effects of DRB treatment on the introduction of methylated H3K4 and H3K9 from 24 to 28 hr post-infection are shown in (B).
Mentions: SV40 minichromosomes were isolated and purified from cells infected with wild-type 776 virus following treatment with DRB or no treatment. When minichromosomes were isolated at 2 hours, cells were pretreated with DRB for 2 hours prior to infection, while for minichromosomes isolated at 48 hours post-infection infected cells were treated from 24 to 48 hours. In these experiments, we observed a 112 ± 35 fold increase in the size of the pool of SV40 chromosomes present in glycerol gradient fractions following purification from cells infected for 48 hours compared to 24 hours post-infection. While the increase was somewhat variable we always observed at least a tenfold increase. This increase was reduced 10 ± 6 fold following treatment of the SV40 infected cells with DRB from 24 to 48 hours post-infection. The latter analysis was determined by comparing the increase in the presence and absence of inhibitor and indicates that on average there was about a tenfold reduction in the pool of SV40 minichromosomes following inhibition. As expected, treatment with DRB substantially inhibited the generation of mRNA (data not shown).The effect of DRB on the introduction of methylated H3K4 and H3K9 was determined by subjecting treated and untreated samples of intact SV40 minichromosomes obtained at 2 hours and 48 hours post-infection to ChIP analyses with antibodies that recognize mono-, di-, or tri-methylated H3K4 or H3K9. Intact SV40 minichromosomes were used because they are easily obtained in relatively large amounts and yield the maximum PCR signal compared to fragmented chromatin. Because the SV40 genome was intact, this analysis only yielded information relative to changes in the numbers of minichromosomes carrying a particular modification following treatment. No information was obtained concerning the location of any specific histone modifications. The results of these analyses are graphically shown in Figure 1. The data is displayed as the ratio of the percentage of minichromosomes that contain a modification following treatment divided by the percentage of untreated minichromosomes containing the same modification. If treatment had no effect on the introduction of a particular modification the ratio will be one. Ratios greater than one indicate that treatment results in an increase in the minichromosomes containing the modification while ratios less than one indicate that treatment resulted in a decrease in the presence of a modification.As shown in Figure 1A and B, DRB treatment had no significant effect on the methylation of H3K4 at either 2 hours or 48 hours post-infection. For each form of methylation, the ratio of treated sample to untreated sample was approximately one. In contrast, DRB treatment had a significant effect on the presence of H3K9me2 and H3K9me3 at 48 hours post-infection and little effect at 2 hours post-infection (Figure 1A and B). We observed a 6 ± 4 fold increase for H3K9me2 with a range from 3 to 12 for four independent samples and a 6 ± 2 fold increase for H3K9me3 with a range of 3 to 8 for four independent samples. As expected from our previous published work, DRB treatment had no significant effect on the presence of H3K9me1 at either time point.Figure 1

Bottom Line: The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.Our data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication.The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a 'proof reading' mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, School of Medicine and Health Sciences, University of North Dakota, 501 N Columbia Road, Grand Forks, ND 58203 USA.

ABSTRACT

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. The role of DNA replication was determined by concomitant inhibition of replication with aphidicolin.

Results: We observed that H3K9me2/me3 was specifically introduced when transcription was inhibited during active replication. The introduction of H3K9me2/me3 that occurred when transcription was inhibited was partially blocked when replication was also inhibited. The introduction of H3K9me2/me3 did not require the presence of H3K9me1 since similar results were obtained with the mutant cs1085 whose chromatin contains very little H3K9me1.

Conclusions: Our data suggest that methylation of H3K9 can occur either as a consequence of a specific repressive event such as T-antigen binding to Site I or as a result of a general repression of transcription in the presence of active replication. The results suggest that the nonproductive generation of transcription complexes as occurs following DRB treatment may be recognized by a 'proof reading' mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.

No MeSH data available.


Related in: MedlinePlus