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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 introduction of H3K9me2 and H3K9me3 is inhibited by sodium butyrate stimulation of transcription during active replication. Wild-type SV40 minichromosomes were isolated at 12 hr post-infection with or without treatment from the initiation of infection with sodium butyrate and at 48 hr post-infection with or without treatment with sodium butyrate from 24 to 48 hr post-infection. Intact minichromosomes isolated at 12 hr post-infection were subjected to ChIP analyses with antibodies to methylated H3K9. Intact minichromosomes isolated at 48 h post-infection were subjected to ChIP analyses with antibodies to methylated H3K4 and methylated H3K9. The percentage of treated and untreated minichromosomes containing each form of methylated H3K4 or H3K9 was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold change resulting from treatment was then calculated from the percentages.
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Fig5: The introduction of H3K9me2 and H3K9me3 is inhibited by sodium butyrate stimulation of transcription during active replication. Wild-type SV40 minichromosomes were isolated at 12 hr post-infection with or without treatment from the initiation of infection with sodium butyrate and at 48 hr post-infection with or without treatment with sodium butyrate from 24 to 48 hr post-infection. Intact minichromosomes isolated at 12 hr post-infection were subjected to ChIP analyses with antibodies to methylated H3K9. Intact minichromosomes isolated at 48 h post-infection were subjected to ChIP analyses with antibodies to methylated H3K4 and methylated H3K9. The percentage of treated and untreated minichromosomes containing each form of methylated H3K4 or H3K9 was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold change resulting from treatment was then calculated from the percentages.

Mentions: SV40 wild-type infected cells were harvested at 24 hours post-infection, 48 hours post-infection, or 48 hours post-infection following treatment with 50 μM sodium butyrate from 24 to 48 hours post-infection to determine the effects of sodium butyrate during the period of active replication. In a parallel analysis, infected cells were harvested at 12 hours post-infection and 12 hours following a 12-hour treatment with sodium butyrate to determine the effects of sodium butyrate in the absence of replication. The minichromosomes were purified and subjected to ChIP analyses with antibodies to methylated H3K4 and H3K9 with minichromosomes isolated late in infection but only methylated H3K9 when isolated at very early times. The reason for this was our previous observation that relatively little of the SV40 chromatin contained methylated H3K4 at the very early times[9] and because there was no change in the levels of methylated H3K4 following DRB treatment. As shown in Figure 5, we observed ratios close to 1 for H3K9me1 (0.9 ± 0.1) and H3K9me3 (1.5 ± 0.8) in minichromosomes isolated at 12 hours post-infection. We saw very low levels of H3K9me2 in both treated and untreated minichromosomes, which were too variable to quantitate at this time. In contrast, for minichromosomes isolated at 48 hours post-infection, we observed significant inhibition of H3K9 methylation. The ratios were 0.46 ± 0.13 for H3K9me1, 0.08 ± 0.04 for H3K9me2, and 0.39 ± 0.2 for H3K9me3. While H3K4me2 (0.75 ± 0.13) and H3K4me3 (1.26 ± 0.38) did not seem to be affected by sodium butyrate treatment at late times, H3K4me1 (0.47 ± 0.27) appeared to be moderately inhibited (data not show). The decrease in the levels of H3K9me2 and H3K9me3 when transcription was stimulated were consistent with the idea that these modifications were being introduced as a consequence of a general repression of transcription at late times.Figure 5


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

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

The introduction of H3K9me2 and H3K9me3 is inhibited by sodium butyrate stimulation of transcription during active replication. Wild-type SV40 minichromosomes were isolated at 12 hr post-infection with or without treatment from the initiation of infection with sodium butyrate and at 48 hr post-infection with or without treatment with sodium butyrate from 24 to 48 hr post-infection. Intact minichromosomes isolated at 12 hr post-infection were subjected to ChIP analyses with antibodies to methylated H3K9. Intact minichromosomes isolated at 48 h post-infection were subjected to ChIP analyses with antibodies to methylated H3K4 and methylated H3K9. The percentage of treated and untreated minichromosomes containing each form of methylated H3K4 or H3K9 was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold change resulting from 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

Fig5: The introduction of H3K9me2 and H3K9me3 is inhibited by sodium butyrate stimulation of transcription during active replication. Wild-type SV40 minichromosomes were isolated at 12 hr post-infection with or without treatment from the initiation of infection with sodium butyrate and at 48 hr post-infection with or without treatment with sodium butyrate from 24 to 48 hr post-infection. Intact minichromosomes isolated at 12 hr post-infection were subjected to ChIP analyses with antibodies to methylated H3K9. Intact minichromosomes isolated at 48 h post-infection were subjected to ChIP analyses with antibodies to methylated H3K4 and methylated H3K9. The percentage of treated and untreated minichromosomes containing each form of methylated H3K4 or H3K9 was determined by real-time PCR amplification of the intact SV40 genomic DNA with primers recognizing the promoter region. The fold change resulting from treatment was then calculated from the percentages.
Mentions: SV40 wild-type infected cells were harvested at 24 hours post-infection, 48 hours post-infection, or 48 hours post-infection following treatment with 50 μM sodium butyrate from 24 to 48 hours post-infection to determine the effects of sodium butyrate during the period of active replication. In a parallel analysis, infected cells were harvested at 12 hours post-infection and 12 hours following a 12-hour treatment with sodium butyrate to determine the effects of sodium butyrate in the absence of replication. The minichromosomes were purified and subjected to ChIP analyses with antibodies to methylated H3K4 and H3K9 with minichromosomes isolated late in infection but only methylated H3K9 when isolated at very early times. The reason for this was our previous observation that relatively little of the SV40 chromatin contained methylated H3K4 at the very early times[9] and because there was no change in the levels of methylated H3K4 following DRB treatment. As shown in Figure 5, we observed ratios close to 1 for H3K9me1 (0.9 ± 0.1) and H3K9me3 (1.5 ± 0.8) in minichromosomes isolated at 12 hours post-infection. We saw very low levels of H3K9me2 in both treated and untreated minichromosomes, which were too variable to quantitate at this time. In contrast, for minichromosomes isolated at 48 hours post-infection, we observed significant inhibition of H3K9 methylation. The ratios were 0.46 ± 0.13 for H3K9me1, 0.08 ± 0.04 for H3K9me2, and 0.39 ± 0.2 for H3K9me3. While H3K4me2 (0.75 ± 0.13) and H3K4me3 (1.26 ± 0.38) did not seem to be affected by sodium butyrate treatment at late times, H3K4me1 (0.47 ± 0.27) appeared to be moderately inhibited (data not show). The decrease in the levels of H3K9me2 and H3K9me3 when transcription was stimulated were consistent with the idea that these modifications were being introduced as a consequence of a general repression of transcription at late times.Figure 5

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