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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 associated with minichromosomes containing RNAPII. Wild-type SV40 minichromosomes were isolated from cells 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 an ISFIP ChIP analysis in which minichromosomes were first immune selected with antibody to RNAPII. The minichromosomes bound by antibody to RNAPII were sonicated and the soluble chromatin fraction subjected to a second ChIP with antibody to either H3K9me2 or H3K9me3. The percentage of the treated and untreated chromatin containing H3K9me2 and H3K9me3 was determined by real-time PCR using primers that recognize the early region of the genome and the fold increase resulting from treatment calculated.
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Fig4: The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 is partially associated with minichromosomes containing RNAPII. Wild-type SV40 minichromosomes were isolated from cells 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 an ISFIP ChIP analysis in which minichromosomes were first immune selected with antibody to RNAPII. The minichromosomes bound by antibody to RNAPII were sonicated and the soluble chromatin fraction subjected to a second ChIP with antibody to either H3K9me2 or H3K9me3. The percentage of the treated and untreated chromatin containing H3K9me2 and H3K9me3 was determined by real-time PCR using primers that recognize the early region of the genome and the fold increase resulting from treatment calculated.

Mentions: In order to determine whether H3K9me2 and H3K9me3 were being added to minichromosomes that contain RNAPII following treatment with DRB, we analyzed SV40 minichromosomes that contain RNAPII for the presence of the two methylated forms of H3K9 following treatment with DRB using our ISFIP procedure[4, 5]. In this procedure, SV40 minichromosomes containing RNAPII were immune-selected with antibody to RNAPII bound to protein A agarose in a standard ChIP assay. Following purification of the bound chromatin and prior to elution, the minichromosomes bound to agarose were sonicated to fragment the chromatin into nucleosome-sized pieces and the bound fragments separated from the released fragments. The released fragments were then subjected to a second ChIP with antibodies to H3K9me2 and H3K9me3 to determine whether H3K9me2 and H3K9me3 were present in the minichromosomes containing RNAPII and if so whether the amount changed upon treatment with DRB.As shown in Figure 4 we observed increases in the percentage of the RNAPII containing minichromosomes which also contained H3K9me (2 ± 1 fold) and H3K9me3 (3 ± 2 fold) although not as significant as those observed in Figure 1. These results suggest that minichromosomes containing RNAPII may serve as the substrate for H3K9 methylation.Figure 4


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 associated with minichromosomes containing RNAPII. Wild-type SV40 minichromosomes were isolated from cells 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 an ISFIP ChIP analysis in which minichromosomes were first immune selected with antibody to RNAPII. The minichromosomes bound by antibody to RNAPII were sonicated and the soluble chromatin fraction subjected to a second ChIP with antibody to either H3K9me2 or H3K9me3. The percentage of the treated and untreated chromatin containing H3K9me2 and H3K9me3 was determined by real-time PCR using primers that recognize the early region of the genome and the fold increase resulting from treatment calculated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB)-stimulated introduction of H3K9me2 and H3K9me3 is partially associated with minichromosomes containing RNAPII. Wild-type SV40 minichromosomes were isolated from cells 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 an ISFIP ChIP analysis in which minichromosomes were first immune selected with antibody to RNAPII. The minichromosomes bound by antibody to RNAPII were sonicated and the soluble chromatin fraction subjected to a second ChIP with antibody to either H3K9me2 or H3K9me3. The percentage of the treated and untreated chromatin containing H3K9me2 and H3K9me3 was determined by real-time PCR using primers that recognize the early region of the genome and the fold increase resulting from treatment calculated.
Mentions: In order to determine whether H3K9me2 and H3K9me3 were being added to minichromosomes that contain RNAPII following treatment with DRB, we analyzed SV40 minichromosomes that contain RNAPII for the presence of the two methylated forms of H3K9 following treatment with DRB using our ISFIP procedure[4, 5]. In this procedure, SV40 minichromosomes containing RNAPII were immune-selected with antibody to RNAPII bound to protein A agarose in a standard ChIP assay. Following purification of the bound chromatin and prior to elution, the minichromosomes bound to agarose were sonicated to fragment the chromatin into nucleosome-sized pieces and the bound fragments separated from the released fragments. The released fragments were then subjected to a second ChIP with antibodies to H3K9me2 and H3K9me3 to determine whether H3K9me2 and H3K9me3 were present in the minichromosomes containing RNAPII and if so whether the amount changed upon treatment with DRB.As shown in Figure 4 we observed increases in the percentage of the RNAPII containing minichromosomes which also contained H3K9me (2 ± 1 fold) and H3K9me3 (3 ± 2 fold) although not as significant as those observed in Figure 1. These results suggest that minichromosomes containing RNAPII may serve as the substrate for H3K9 methylation.Figure 4

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