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 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 does not require the presence of H3K9me1. SV40 minichromosomes from the mutant virus cs1085 were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from 24 to 48 hr post-infection. Intact minichromosomes were subjected to ChIP analyses with antibody recognizing H3K9me2 and H3K9me3 and the intact SV40 genomic DNA present in the bound fraction quantitated by real-time PCR with primers recognizing the promoter region. The percentages of minichromosomes in the treated and untreated samples were determined and the fold increase in the percentage in the treated samples compared to the untreated calculated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 does not require the presence of H3K9me1. SV40 minichromosomes from the mutant virus cs1085 were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from 24 to 48 hr post-infection. Intact minichromosomes were subjected to ChIP analyses with antibody recognizing H3K9me2 and H3K9me3 and the intact SV40 genomic DNA present in the bound fraction quantitated by real-time PCR with primers recognizing the promoter region. The percentages of minichromosomes in the treated and untreated samples were determined and the fold increase in the percentage in the treated samples compared to the untreated calculated.

Mentions: Since H3K9me1 can be present in as much as 22% of the SV40 minichromosomes present at late times[9], it seemed reasonable that the H3K9me1 containing minichromosomes might serve as substrates for the introduction of H3K9me2 and H3K9me3. To test this possibility we characterized the effect of DRB treatment on the mutant cs1085 SV40 virus. This mutant lacks T-antigen binding Site I and as a consequence fails to down-regulate early transcription. We have previously shown that minichromosomes from this mutant contain very low levels of H3K9me1in contrast to parental wild-type viral chromatin[9]. If the minichromosomes containing H3K9me1 served as a substrate for the introduction of H3K9me2 and H3K9me3, we would expect that inhibition of transcription of cs1085 with DRB would not result in a large increase in the amounts of H3K9me2 and H3K9me3 in the treated minichromosomes compared to the untreated minichromosomes.Cells infected with SV40 cs1085 virus were treated with DRB from 24 to 48 hours post-infection or left untreated and the minichromosomes present in the cells isolated and purified. The minichromosomes were subjected to ChIP analyses with antibodies to H3K9me2 and H3K9me3 with the results shown in Figure 3. Treatment with DRB resulted in significant increases in the amounts of H3K9me2 (3 ± 1 fold) and H3K9me3 (5 ± 3 fold) present in the cs1085 minichromosomes indicating that the presence of H3K9me1 in the minichromosomes was not necessary for the introduction of the higher levels of methylated H3K9.Figure 3


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

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

The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 does not require the presence of H3K9me1. SV40 minichromosomes from the mutant virus cs1085 were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from 24 to 48 hr post-infection. Intact minichromosomes were subjected to ChIP analyses with antibody recognizing H3K9me2 and H3K9me3 and the intact SV40 genomic DNA present in the bound fraction quantitated by real-time PCR with primers recognizing the promoter region. The percentages of minichromosomes in the treated and untreated samples were determined and the fold increase in the percentage in the treated samples compared to the untreated calculated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 does not require the presence of H3K9me1. SV40 minichromosomes from the mutant virus cs1085 were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) from 24 to 48 hr post-infection. Intact minichromosomes were subjected to ChIP analyses with antibody recognizing H3K9me2 and H3K9me3 and the intact SV40 genomic DNA present in the bound fraction quantitated by real-time PCR with primers recognizing the promoter region. The percentages of minichromosomes in the treated and untreated samples were determined and the fold increase in the percentage in the treated samples compared to the untreated calculated.
Mentions: Since H3K9me1 can be present in as much as 22% of the SV40 minichromosomes present at late times[9], it seemed reasonable that the H3K9me1 containing minichromosomes might serve as substrates for the introduction of H3K9me2 and H3K9me3. To test this possibility we characterized the effect of DRB treatment on the mutant cs1085 SV40 virus. This mutant lacks T-antigen binding Site I and as a consequence fails to down-regulate early transcription. We have previously shown that minichromosomes from this mutant contain very low levels of H3K9me1in contrast to parental wild-type viral chromatin[9]. If the minichromosomes containing H3K9me1 served as a substrate for the introduction of H3K9me2 and H3K9me3, we would expect that inhibition of transcription of cs1085 with DRB would not result in a large increase in the amounts of H3K9me2 and H3K9me3 in the treated minichromosomes compared to the untreated minichromosomes.Cells infected with SV40 cs1085 virus were treated with DRB from 24 to 48 hours post-infection or left untreated and the minichromosomes present in the cells isolated and purified. The minichromosomes were subjected to ChIP analyses with antibodies to H3K9me2 and H3K9me3 with the results shown in Figure 3. Treatment with DRB resulted in significant increases in the amounts of H3K9me2 (3 ± 1 fold) and H3K9me3 (5 ± 3 fold) present in the cs1085 minichromosomes indicating that the presence of H3K9me1 in the minichromosomes was not necessary for the introduction of the higher levels of methylated H3K9.Figure 3

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