Transcriptional repression is epigenetically marked by H3K9 methylation during SV40 replication
© Kallestad et al.; licensee BioMed Central Ltd. 2014
Received: 15 July 2014
Accepted: 7 October 2014
Published: 27 October 2014
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.
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.
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.
Five distinct but potentially related elements are thought to contribute to the epigenetic regulation of eukaryotic gene expression: DNA methylation, nucleosome location, histone variation, covalent histone modifications, and interactions by regulatory RNA. Of these elements, the post-translational modification of histones has been of particular interest because of the diversity of the available forms of modification and the number of target amino acids and their physical location within histones. Importantly, there is an abundance of published reports demonstrating the association between certain forms of histone modification and activation or repression of transcription in one or more well characterized biological systems.
In general, it is thought that acetylation of certain lysines in histones is associated with active biological processes like transcription while methylation of lysines or arginines may be associated with either activation or repression of gene transcription. For example, in the nucleosome core, histone H3 acetylation on lysine 9 and 14 (H3K9 and H3K14) have been shown to be associated with transcription along with methylation of lysine 4 (H3K4), while methylation of lysine 9 (H3K9) has been shown to be associated with repression[1, 2].
While the association between the methylation of H3K9 and gene repression has been well established, much less is known about the circumstances that lead to the introduction of methylated H3K9 into chromatin. What has added to this uncertainty is the complexity of methylation, including mono-, di-, and tri-methylation, and the involvement of multiple methylating enzymes.
In order to better understand the factors that contribute to epigenetic regulation, we have been investigating the role of histone modifications in the regulation of gene expression during the Simian Virus 40 (SV40) life cycle[4–9]. In a recent publication we confirmed that the mono-methylation of H3K9 in SV40 chromatin was associated with repression of transcription. Specifically, we showed that the introduction of H3K9me1 was a consequence of the repression of early transcription by the product of transcription, T-antigen, binding to a critical regulatory sequence known as Site I. Moreover, importantly, we observed that the introduction of H3K9me1 during repression required DNA replication. Repression occurring prior to the initiation of replication was not associated with the introduction of H3K9me1. Our results demonstrated that replication could serve as an epigenetic switch and that the same biological event could have different epigenetic readouts depending upon whether replication was occurring.
During the course of these studies, we investigated the introduction of all three methylated forms of H3K9 during replication. We noted that unlike the introduction of H3K9me1 which absolutely required replication, the introduction of H3K9me2 and H3K9me3 appeared to occur even when replication was substantially blocked by inhibitor. These results raised two important questions. First, was the introduction of H3K9me2 and H3K9me3 also associated with the repression of transcription in SV40 chromatin, and second, what biological factors contributed to the introduction of these epigenetic marks?
In order to address these questions in SV40 chromatin, we have investigated the effects of a general inhibition or stimulation of transcription on the introduction of H3K9 methylation in the presence or absence of active replication. Transcription was inhibited with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB), a well-characterized inhibitor of RNA Polymerase II (RNAPII) elongation, and stimulated with sodium butyrate, an inhibitor of histone deacetylase[11, 12] known to stimulate gene expression. DNA replication was inhibited with aphidicolin. We show that the introduction of H3K9me2 and H3K9me3 are the result of a general inhibition of transcription at late times in infection but not early times, and that the introduction is partially dependent upon replication but not the prior introduction of H3K9me1.
Inhibition of transcription by DRB stimulates the introduction of H3K9me2 and H3K9me3 into SV40 chromatin late in infection but not early in infection
Since we previously observed that the introduction of H3K9me2 and H3K9me3 was not directly related to repression by T-antigen binding to Site I, we hypothesized that their incorporation into SV40 chromatin might be the consequence of a more general inhibition of transcription. In order to test this hypothesis we determined the epigenetic consequences of inhibiting transcription with the RNAPII elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB). We have previously used DRB to investigate the relationship between RNA polymerase II translocation and the acetylation and deacetylation of histones during transcription. For these studies, we chose to investigate SV40 chromatin isolated at 2 hours post-infection when early transcription was occurring but prior to DNA replication, and at 48 hours post-infection when early and late transcription were occurring along with active replication.
Introduction of H3K9me2 and H3K9me3 following inhibition of transcription is partially dependent upon replication
Introduction of H3K9me2 and H3K9me3 following inhibition of transcription does not require the presence of H3K9me1
Introduction of H3K9me2 and H3K9me3 are associated with SV40 minichromosomes that contain RNAPII
Stimulation of transcription with sodium butyrate inhibits the incorporation of H3K9me2 and H3K9me3 into SV40 chromatin late in infection
Since the introduction of H3K9me2 and H3K9me3 appeared to be associated with repression of transcription, we then determined whether stimulation of transcription would have the opposite effect. Sodium butyrate is a well-known inhibitor of many histone deacetylases and for this reason has been used extensively to investigate the relationship between histone acetylation and various biological processes including transcription[11, 12]. We have previously shown that inhibition of histone deacetylases by sodium butyrate results in an increase in transcription of actively transcribed genes as well as an increase in histone acetylation[6, 14]. If increased transcription and/or histone acetylation prevented the introduction of H3K9me2 or H3K9me3, we would expect to see a reduction in the ratio of these two modified forms of H3 in the treated minichromosomes compared to the untreated minichromosomes.
Pools of transcribing SV40 minichromosomes are dynamic during replication
Since DRB had a major effect on transcription and the introduction of H3K9me2 and H3K9me3, we also determined whether it affected the proportion of minichromosomes carrying RNAPII and was, therefore, potentially capable of transcription. We have previously shown that with short-term treatment with DRB there was little effect on the percentage of minichromosomes carrying RNAPII. However, long-term treatment when replication was occurring could be different. In order to better understand the factors contributing to determining the pool size of minichromosomes containing RNAPII during this time frame, we also investigated the effects of aphidicolin and sodium butyrate. If the pool of minichromosomes containing RNAPII was simply related to the overall size of the pool of minichromosomes, we would expect to observe that the percentage containing RNAPII would remain constant despite the overall changes in the size of the pool following the different treatments. For this analysis, we compared the percentage of RNAPII containing minichromosomes at 24 hours post-infection when replication was beginning to 48 hours post-infection when replication was active in the presence or absence of the replication inhibitor aphidicolin, transcription inhibitor DRB, or transcription stimulator sodium butyrate.
The introduction of H3K9me2 and H3K9me3 into SV40 chromatin following repression of transcription through the inhibition of RNAPII elongation by the inhibitor DRB is consistent with previous reports showing that the presence of H3K9me2 and H3K9me3 in chromatin is associated with transcriptional repression (reviewed in[15, 16]). H3K9me3, in particular, is now considered a characteristic mark of repression by many laboratories.
H3K9me2 and H3K9me3 are typically introduced into chromatin by a member of the SET domain family of histone methyltransferases. The related proteins G9a and GLP are thought to be primarily responsible for H3K9me2 methylation in euchromatin, whereas Suv39H1 is thought to be responsible for H3K9me3 methylation in heterochromatin[17–19]. G9a/GLP also appears to be capable of introducing H3K9me3 into euchromatin. While these proteins contribute to the bulk of the H3K9me2 and H3K9me3 in a cell, there are other methyltranferases that may also play a role since deletion of these enzymes cause a significant but not complete reduction in H3K9 methylation.
There are two well-characterized mechanisms for the introduction of H3K9me2 and H3K9me3. H3K9me2 is associated with repression of nuclear hormone receptor regulated transcription (reviewed in). In this model system, the introduction of H3K9me2 is the consequence of the binding of the receptor along with the histone methyltransferase to maintain a repressive environment. Introduction of the ligand results in the selective loss of the methyltranferase and subsequent corresponding loss of H3K9me2 and activation of transcription.
The introduction of H3K9me3 into heterochromatin has also been extensively characterized (reviewed in). The introduction of H3K9me3 in heterochromatin is thought to be closely linked with replication and to occur through the targeting of the relevant methyltranferase by associated proteins including CAF1, HP1, and MBD1 in higher eukaryotes[22, 23] and Clr4 and Swi6 in fission yeast. The presence of MBD1 in higher eukaryotes links H3K9me3 to DNA methylation and suggests a possible mechanism for targeting the complex to the appropriate sites (hemi-methylated DNA) following replication.
It seems unlikely that either of these mechanisms is responsible for the introduction of H3K9me2 and H3K9me3 reported here. With respect to the introduction of H3K9me2, SV40 transcription does not appear to be regulated by a nuclear hormone type mechanism as previously described for H3K9me2. Similarly the observed increase in H3K9me2 occurred as a consequence of the inhibition of transcription by DRB not the binding of normal transcription factors. The introduction of H3K9me3 also is unlikely to occur by the same mechanism as described for heterochromatin. Notably, SV40 DNA in chromatin is not known to be methylated because of an absence of DNA methylase substrates in the DNA. Also our observed increase in H3K9me2 and H3K9me3 appeared to be only secondarily a result of DNA replication since the increase was observed on minichromosomes containing RNAPII.
To explain our results we propose the following model. As DNA replication takes place, some of the newly replicated minichromosomes are committed to transcription through the binding of specific and general transcription factors. Concurrently, for reasons as yet unknown, some of the preexisting transcribing minichromosomes stop transcription. These nonfunctioning minichromosomes are recognized as being repressed, and H3K9me2 and H3K9me3 are introduced into their chromatin as a mark of repression. We propose that following replication and the activation for new transcription there is a ‘proofreading mechanism,’ which ensures that the newly initiated minichromosomes are capable of productive transcription. In the event that this is not the case, the minichromosomes are again recognized as repressed, as with the nonfunctioning minichromosomes, and marked by H3K9me2 and H3K9me3. Since treatment with the inhibitor DRB results in a large increase in the number of minichromosomes in which transcription is stopped, presumably at initiation, there is a corresponding increase in the incorporation of H3K9me2 and H3K9me3.
This proofreading mechanism could be specific to SV40 and other similar viruses in which case it could function to ensure that the size of the pool of transcribing minichromosomes is tied to the level of replication and encapsidation. Conversely, this mechanism could be true for higher eukaryotes in general and serve as a way to ensure that when genes are activated for transcription, only those genes correctly activated are allowed to persist in the activated state.
Assuming that the introduction of H3K9me2 and H3K9me3 following the inhibition of elongation by RNAPII reflects a normal biological process for SV40, we believe it is likely to play a role in two aspects of normal SV40 molecular biology. First, the introduction of H3K9me2 and H3K9me3 could play a critical role in regulating the pool size of transcriptionally competent minichromosomes during an infection. We have presented evidence that the pool size is dynamic with new transcribing minichromosomes entering the pool as a result of replication and old minichromosomes leaving the pool. We believe that old minichromosomes leaving the pool of transcribing minichromosomes would be labeled with H3K9me2 and H3K9me3 in order to prevent their reactivation.
Second, H3K9me2 and H3K9me3 could also play a critical role during the initiation of a subsequent infection. We have shown that minichromosomes in virions contain significant amounts of H3K9me2/3. If the minichromosomes, which contain these modifications, result primarily from the minichromosomes that originally transcribed the late genes following replication, it seems desirable that they be silenced during a new infection when only the transcription of the early genes is required. Allowing transcription of the late genes during the establishment of an infection would seem to be very undesirable for the virus.
Because DRB treatment blocks RNAPII elongation, we believe that it is a way to model the effects of repression of transcription in order to characterize the subsequent changes in epigenetic modifications. However, we recognize that the DRB treatment, as well as the treatment with the other inhibitors themselves, may be responsible for the observed effects on the levels of H3K9me2 and H3K9me3 and may not accurately reflect the epigenetic changes occurring in vivo.
Methylation of H3K9 can occur during active expression 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. Moreover, these results suggest that the nonproductive transcription complexes which are generated by DRB treatment may be recognized by a ‘proof reading’ mechanism, which leads to the specific introduction of H3K9me2 and H3K9me3.
Cells and viruses
Wild-type and mutant SV40 minichromosomes were prepared in the monkey kidney BSC-1 cell line (ATCC) using either wild-type 776 virus (from Dr. Daniel Nathans) or cs1085 virus (from Dr. Daniel Nathans).
Cell culture and infections
BSC-1 cells were maintained and infected as previously described[5–7]. An RNA polymerase II inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidizole (DRB), was used at a concentration of 200 μM. For 2-hour infections, DRB was added two hours prior to infection and re-applied at 30 minutes post-infection when the media was replaced by fresh media without virus. For 48-hour infections, DRB was added at 24 hours post-infection. Sodium butyrate was used at a concentration of 250 μM. Sodium butyrate was added at infection and along with fresh media following removal of unbound virus for 12-hour infections and at 24 hours post-infection for 48-hour infections. Replication was inhibited with aphidicolin added at 24 hours post-infection at a final concentration of 6 μM as previously described.
Preparation of SV40 minichromosomes
SV40 minichromosomes were isolated at the indicated times from 2 hours to 48 hours post-infection as described for each of the analyses using our standard purification protocols[5–7] with one minor modification. After transferring the lysed cells to a 15-ml centrifuge tube, an additional one ml of nuclei preparation buffer was used to rinse the flask and was subsequently added to the centrifuge tube in order to maximize the yield of minichromosomes from each infection.
ChIP kits were obtained from Millipore (Temicula, California, USA) and the protocol was followed as previously described. The antibodies used included: H3K4me1 (07 = 436, Millipore (Temicula, California, USA), H3K4me2 (39141, Active Motif Carlsbad, California, USA), H3K4me3 (04 = 745, Millipore Temicula, California, USA), H3K9me1 (ab9045, Abcam, Cambridge Massachusetts USA), H3K9me2 (ab1220, Abcam, Cambridge, Massachusetts, USA), H3K9me3 (ab8898, Abcam, Cambridge, Massachusetts, USA), and RNA PII 905 = 623, Millipore,Temicula, California, USA). All antibodies were ChIP validated by the respective vendors. A total of 100 μl of Protein A agarose, 800 μl chip dilution buffer, and 7.5 μl of each antibody were combined in a protein low-bind tube. The mixture was rotated for 5 hours at 4°C on an end to end rotator in a refrigerator to bind the antibody to Protein A agarose. Following binding of the antibody, the Protein A agarose was centrifuged at 2,000 rpms for 2 minutes and the supernatant discarded. Next, 800 μl of fresh ChIP dilution buffer was added, and 100 μl of the SV40 chromatin to be analyzed was added. The samples containing antibody bound to Protein A agarose and chromatin were incubated with end to end rotation for a further 7 hours at 4°C. The chromatin bound to Protein A agarose was washed according to the manufacturer’s protocol and eluted as previously described.
Immune selection fragmentation immunoprecipitation
ChIPs were performed as previously described[5–7] with the following changes. The amount of input chromatin added was increased to 200 μl and the amount of Protein A agarose was doubled to 200 μl. Following the final wash of the protein A agarose containing antibody and bound chromatin, the agarose was resuspended in 200 μl of buffer and transferred to a clean low-bind Eppendorf centrifuge tube. The suspension was sonicated using a Branson Digital Sonifier for 6 minutes with the amplitude set at 50%. The sonicated samples were centrifuged at 2,000 rpms for 2 minutes to separate the chromatin that remained bound to the agarose from the fragmented chromatin in the supernatant and the supernatant chromatin saved. The chromatin that remained bound to agarose following sonication was removed with lysis buffer according to the ChIP kit’s instructions. The supernatant was then used as the starting material for a subsequent ChIP.
Preparation of DNA: Samples were prepared for PCR using an MP Bioscience (Solon, Ohio, USA) Geneclean Spin Kit (#111101-200) with the following modifications. The glassmilk reagent (100 μl) was mixed with 50 μl of sample in a 1.5-ml centrifuge tube. The tube was mixed by repeated inversion at 2 minutes and again at 4 minutes of incubation. Following 5 minutes of room temperature incubation, the samples were centrifuged at 6,000 rpm for 30 seconds in a Micro One (Tomy) to pellet the glass. The supernatant was discarded and 200 μl of the wash buffer was added to the tube. As the wash buffer was being added, the pipette tip was used to break up the pellet by physically rubbing the pellet and vigorously pipetting up and down. The samples were inverted twice and centrifuged at 6,000 rpm for 30 seconds to again pellet the glass. The supernatant was discarded and the pellets were dried in a vacuum for 5 minutes. The glass pellet with bound DNA was resuspended in 25 μl Tris EDTA (TE) buffer.
For most of the amplifications, DNA was amplified from the promoter region of the SV40 genome using the primers 5′ TTG CAA AAG CCT CCA AA 3′ and 5′ TGA CCT ACG AAC CTT AAC CGA GGG 3′ in a CFX Connect Real Time System thermal cycler (Bio-Rad, Ipswich, Massachusetts, USA) using ‘SSO Advanced DNA polymerase (Bio-Rad, Ipswich, Massachusetts, USA). In the immune selection fragmentation experiment, DNA was amplified from the early region using the primers 5′TGCTCCCATTCATCAGTTCC3′ and 5′CTGACTTTGGAGGCTTCTGG3′ because the promoter region is extremely sensitive to sonication. Immediately before use, the primers and DNAse free water were added, and 28 μl of the mix was used per sample. 2 μl of the resuspended glass milk in TE buffer was added per sample. Samples were amplified by PCR in triplicate with a melt curve applied afterwards to ensure specific amplification. All sample preparation for PCR was done in either a Nuaire biological safety cabinet Model NU_425-400 or an AirClean 600 PCR Workstation (ISC BioExpress).
Statement on the use of human subjects
This research did not involve the use of any human subjects, materials, or data.
This work was supported by grants from the National Institutes of Health GM074811 and AI070193 to BM
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