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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

The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 is partially dependent upon ongoing DNA Replication. Wild-type SV40 minichromosomes were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) and aphidicolin from 24 to 48 hr post-infection. The treated and untreated intact minichromosomes were subjected to ChIP analyses with antibodies to H3K9me2 and H3K9me3 and the percentages of the minichromosomes containing the modified H3K9s determined by real-time PCR amplification of the bound intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase in the percentages of minichromosomes containing H3K9me2 and H3K9me3 was then calculated.
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Fig2: The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 is partially dependent upon ongoing DNA Replication. Wild-type SV40 minichromosomes were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) and aphidicolin from 24 to 48 hr post-infection. The treated and untreated intact minichromosomes were subjected to ChIP analyses with antibodies to H3K9me2 and H3K9me3 and the percentages of the minichromosomes containing the modified H3K9s determined by real-time PCR amplification of the bound intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase in the percentages of minichromosomes containing H3K9me2 and H3K9me3 was then calculated.

Mentions: Since we have previously shown that the introduction of H3K9me2 and H3K9me3 into SV40 chromatin late in infection does not require DNA replication[8], we next tested whether replication played a role in the enhanced introduction of these modifications following DRB inhibition of transcription. SV40 minichromosomes were obtained from infected cells that were untreated or treated from 24 to 48 hours post-infection with a combination of DRB and aphidicolin to inhibit both transcription and replication. The minichromosomes were then subjected to ChIP analyses with antibody to H3K9me2 and H3K9me3 with the results shown in Figure 2. The data is again shown as the ratio between the percentages of input minichromosomes containing the modification of interest in the treated sample compared to the untreated sample. Treatment with aphidicolin and DRB resulted in approximately a 99% reduction in the amount of SV40 minichromsomes obtained at 48 hours post-infection compared to the amount obtained from untreated cells. Following inhibition we observed no increases in the ratio for H3K9me2 (1 ± 0.5) and a small increase in the ratio for H3K9me3 (2 ± 1). These increases were significantly lower than the values obtained when transcription was inhibited while DNA replication was occurring as shown in Figure 1. These results suggest that ongoing replication plays at least a small role in the introduction of H3K9me2 and H3K9me3 at late times in infection.Figure 2


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 is partially dependent upon ongoing DNA Replication. Wild-type SV40 minichromosomes were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) and aphidicolin from 24 to 48 hr post-infection. The treated and untreated intact minichromosomes were subjected to ChIP analyses with antibodies to H3K9me2 and H3K9me3 and the percentages of the minichromosomes containing the modified H3K9s determined by real-time PCR amplification of the bound intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase in the percentages of minichromosomes containing H3K9me2 and H3K9me3 was then calculated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 is partially dependent upon ongoing DNA Replication. Wild-type SV40 minichromosomes were isolated at 48 hr post-infection with or without treatment with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB) and aphidicolin from 24 to 48 hr post-infection. The treated and untreated intact minichromosomes were subjected to ChIP analyses with antibodies to H3K9me2 and H3K9me3 and the percentages of the minichromosomes containing the modified H3K9s determined by real-time PCR amplification of the bound intact SV40 genomic DNA with primers recognizing the promoter region. The fold increase in the percentages of minichromosomes containing H3K9me2 and H3K9me3 was then calculated.
Mentions: Since we have previously shown that the introduction of H3K9me2 and H3K9me3 into SV40 chromatin late in infection does not require DNA replication[8], we next tested whether replication played a role in the enhanced introduction of these modifications following DRB inhibition of transcription. SV40 minichromosomes were obtained from infected cells that were untreated or treated from 24 to 48 hours post-infection with a combination of DRB and aphidicolin to inhibit both transcription and replication. The minichromosomes were then subjected to ChIP analyses with antibody to H3K9me2 and H3K9me3 with the results shown in Figure 2. The data is again shown as the ratio between the percentages of input minichromosomes containing the modification of interest in the treated sample compared to the untreated sample. Treatment with aphidicolin and DRB resulted in approximately a 99% reduction in the amount of SV40 minichromsomes obtained at 48 hours post-infection compared to the amount obtained from untreated cells. Following inhibition we observed no increases in the ratio for H3K9me2 (1 ± 0.5) and a small increase in the ratio for H3K9me3 (2 ± 1). These increases were significantly lower than the values obtained when transcription was inhibited while DNA replication was occurring as shown in Figure 1. These results suggest that ongoing replication plays at least a small role in the introduction of H3K9me2 and H3K9me3 at late times in infection.Figure 2

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