Limits...
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

The pool size of SV40 minichromosomes containing RNAPII is dynamic and dependent upon replication and rate of transcription. SV40 minichromosomes were isolated from cells infected with wild-type virus at 24 and 48 hr post-infection. Infected cells were either untreated or treated from 24 to 48 hr post-infection with DRB, sodium butyrate (NaBt), or aphidicolin. Intact minichromosomes were subjected to ChIP analyses with antibody to RNAPII and the percentage of untreated and treated minichromosomes containing RNAPII was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase or decrease from 24 to 48 hr post-infection with or without treatment was then calculated from the percentages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4230732&req=5

Fig6: The pool size of SV40 minichromosomes containing RNAPII is dynamic and dependent upon replication and rate of transcription. SV40 minichromosomes were isolated from cells infected with wild-type virus at 24 and 48 hr post-infection. Infected cells were either untreated or treated from 24 to 48 hr post-infection with DRB, sodium butyrate (NaBt), or aphidicolin. Intact minichromosomes were subjected to ChIP analyses with antibody to RNAPII and the percentage of untreated and treated minichromosomes containing RNAPII was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase or decrease from 24 to 48 hr post-infection with or without treatment was then calculated from the percentages.

Mentions: As indicated above, between 24 and 48 hours post-infection we observed a 112 ± 35 fold increase in the size of the minichromosome pool. Following treatment with aphidicolin the pool size at 48 hours post-infection was essentially the same as at 24 hours post-infection indicating substantial inhibition by the drug. Following treatment with DRB we again observed a substantial increase in the pool of minichromosomes although the increase was 10 ± 6 fold less than observed in the absence of the inhibitor. Treatment with sodium butyrate was seen to increase the size of the pool of minichromosomes by 10 ± 4 fold above the level of increase without the added sodium butyrate. The latter two results indicate that the inhibitors had a modest effect on pool sizes when replication was occurring.SV40 minichromosomes from treated or untreated infections were subjected to ChIP analysis with antibody to RNAPII with the results shown in Figure 6. We observed that the ratio of the percentage of minichromosomes containing RNAPII was only 0.14 ± 0.04 comparing minichromosomes isolated at 48 hours post-infection to 24 hours post-infection indicating that only a small fraction of the newly replicated minichromosomes became associated with RNAPII. Following treatment with aphidicolin to block replication the ratio was reduced even further. Compared to untreated controls at 48 hours the ratio was 0.64 ± 0.16. Since there was no increase in the overall pool size this indicated that the actual amount of SV40 chromatin containing RNAPII following inhibition of replication was lower in the treated samples than was present at 24 hours post-infection. Following treatment with DRB from 24 to 48 hours post-infection the ratio of minichromosomes containing RNAPII increased to 1.2 ± 0.5, indicating that the pool size of transcribing minichromosomes was increasing along with replication in the presence of DRB. Finally, we observed that treatment with sodium butyrate during replication also had a profound effect. The ratio of RNAPII containing minichromosomes was 0.39 ± 0.20 following treatment with sodium butyrate from 24 to 48 hours post-infection. This indicated that compared to untreated controls a larger fraction of minichromosomes were capable of transcription.Figure 6


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

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

The pool size of SV40 minichromosomes containing RNAPII is dynamic and dependent upon replication and rate of transcription. SV40 minichromosomes were isolated from cells infected with wild-type virus at 24 and 48 hr post-infection. Infected cells were either untreated or treated from 24 to 48 hr post-infection with DRB, sodium butyrate (NaBt), or aphidicolin. Intact minichromosomes were subjected to ChIP analyses with antibody to RNAPII and the percentage of untreated and treated minichromosomes containing RNAPII was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase or decrease from 24 to 48 hr post-infection with or without treatment was then calculated from the percentages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230732&req=5

Fig6: The pool size of SV40 minichromosomes containing RNAPII is dynamic and dependent upon replication and rate of transcription. SV40 minichromosomes were isolated from cells infected with wild-type virus at 24 and 48 hr post-infection. Infected cells were either untreated or treated from 24 to 48 hr post-infection with DRB, sodium butyrate (NaBt), or aphidicolin. Intact minichromosomes were subjected to ChIP analyses with antibody to RNAPII and the percentage of untreated and treated minichromosomes containing RNAPII was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase or decrease from 24 to 48 hr post-infection with or without treatment was then calculated from the percentages.
Mentions: As indicated above, between 24 and 48 hours post-infection we observed a 112 ± 35 fold increase in the size of the minichromosome pool. Following treatment with aphidicolin the pool size at 48 hours post-infection was essentially the same as at 24 hours post-infection indicating substantial inhibition by the drug. Following treatment with DRB we again observed a substantial increase in the pool of minichromosomes although the increase was 10 ± 6 fold less than observed in the absence of the inhibitor. Treatment with sodium butyrate was seen to increase the size of the pool of minichromosomes by 10 ± 4 fold above the level of increase without the added sodium butyrate. The latter two results indicate that the inhibitors had a modest effect on pool sizes when replication was occurring.SV40 minichromosomes from treated or untreated infections were subjected to ChIP analysis with antibody to RNAPII with the results shown in Figure 6. We observed that the ratio of the percentage of minichromosomes containing RNAPII was only 0.14 ± 0.04 comparing minichromosomes isolated at 48 hours post-infection to 24 hours post-infection indicating that only a small fraction of the newly replicated minichromosomes became associated with RNAPII. Following treatment with aphidicolin to block replication the ratio was reduced even further. Compared to untreated controls at 48 hours the ratio was 0.64 ± 0.16. Since there was no increase in the overall pool size this indicated that the actual amount of SV40 chromatin containing RNAPII following inhibition of replication was lower in the treated samples than was present at 24 hours post-infection. Following treatment with DRB from 24 to 48 hours post-infection the ratio of minichromosomes containing RNAPII increased to 1.2 ± 0.5, indicating that the pool size of transcribing minichromosomes was increasing along with replication in the presence of DRB. Finally, we observed that treatment with sodium butyrate during replication also had a profound effect. The ratio of RNAPII containing minichromosomes was 0.39 ± 0.20 following treatment with sodium butyrate from 24 to 48 hours post-infection. This indicated that compared to untreated controls a larger fraction of minichromosomes were capable of transcription.Figure 6

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