In order to test whether the repression of early transcription which occurs prior to replication was also associated with the same forms of histone methylation observed when replication was occurring, we used two distinct strategies. First, we determined whether there were changes in histone methylation during the first 8 h post-infection in a wild-type infection consistent with what we previously reported for repression of early gene expression late in infection during DNA replication (Milavetz et al., 2012
). We hypothesized that if transcriptional repression occurring at early times was associated with mono-methylation (me1) of H3K9 as observed during DNA replication, we would observe an increase in H3K9me1 over the first hours of an infection perhaps approaching the 20% value seen at late times when transcriptional repression was occurring. In contrast if early transcriptional repression was not associated with mono-methylation of H3K9 we would expect no effect on the levels of H3K9me1. Since we previously reported that the fraction of SV40 minichromosomes containing RNAPII decreased during the first hours of infection consistent with the repression of early transcription (Balakrishnan and Milavetz, 2006
), we first confirmed that this was the case. SV40 wild-type minichromosomes were isolated 2, 4, 6, and 8 h post-infection and analyzed by ChIP for the presence of RNAPII. As shown in Figure
, we observed a slow and continual decrease in the percentage of RNAPII bound to SV40 minichromosomes between 2 and 8 h post-infection. We next determined the percentage of minichromosomes isolated at 30 min, 2, 4, and 8 h which contained H3K9me1. We did not analyze for the presence of methylated H3K4 at these times because we have previously shown that minichromosomes contain very low levels of methylated H3K4 (Milavetz et al., 2012
). As shown in Figure
, we did not observe an increase in the level of H3K9me1 as expected if it was associated with transcriptional repression. H3K9me1 remained present in approximately 1% or less of the minichromosomes at this time which was similar to the level that we previously reported present in the SV40 virus particles, 2.9 ± 1% (Milavetz et al., 2012
), which was used for the infection.
FIGURE 2 Repression of active early transcription does not result in an increase in H3K9me1. SV40 wild-type minichromosomes were isolated between 30 min and 8 h post-infection, and subsequently subjected to ChIP analyses with antibodies to either RNA polymerase (more ...)
Secondly, we determined whether infection by the mutant cs1085 which lacks Site I and fails to repress early transcription resulted in a changed pattern of histone methylation compared to wild-type virus during the same time. Again, we focused only on the methylated forms of H3K9 at this time because we have previously shown that there is very little if any methylated H3K4 at the very early times in question (Milavetz et al., 2012
). SV40 minichromosomes were prepared at the indicated times, subjected to ChIP analyses and the percentage of minichromosomes containing each methylated form of H3K9 determined by real-time PCR. The data is represented as the percentage of minichromosomes containing the modification present at 8 h of infection divided by the percentage present at 30 min of infection. A ratio less than 1 indicates that the percentage of minichromosomes carrying a particular methylated H3 is reduced over this period. As shown in Figure
, we observed that for both the wild-type and cs1085 mutant we observed a reduction in the relative amount of H3K9me1 and H3K9 tri-methylation (H3K9me3) present in minichromosomes between 30 min and 8 h post-infection. However, while the amount of H3K9 di-methylation (H3K9me2) was reduced during this period in the wild-type virus, the amount was significantly increased in the cs1085 mutant. These results suggest that repression of early gene expression during active transcription occurs by a process in which the levels of H3K9me2 are kept low.
FIGURE 3 H3K9me2 is significantly increased during active early transcription in the site I deletion mutant cs1085. Wild-type and cs1085 SV40 minichromosomes were isolated from appropriately infected cells at 30 min and 8 h post-infection. Isolated minichromosomes (more ...)
In order to independently confirm that Site I was responsible for the introduction of H3K9me1 at late times but not early times, we compared the level of H3K9me1 in an SV40 recombinant containing two copies of Site I (pBM131-1) to a parental recombinant containing only a single copy of Site I (pBM129-1). We hypothesized that if Site I was responsible for the introduction of H3K9me1 in a replication dependent manner, we would observe an increase in the percentage of H3K9me1 in the recombinant compared to the parental virus at late times but not at early times when replication was not occurring. For these studies we used recombinant viruses originally prepared to study the ability of SV40 regulatory sequences to phase nucleosomes and generate nucleosome free regions in SV40 chromatin. The parental recombinant and its construction as well as the recombinant containing two copies of Site I have been previously described (Hermansen et al., 1996
). The structures of both of these constructs are shown in Figure
. The parental construct pBM129-1 has a single copy of Site I in the regulatory region as in the wild-type virus (Figure
). pBM131-1 has two copies of Site I, one located as in pBM129-1 and a second copy present in the reporter region as shown in Figure
. The results of this analysis are graphically represented in Figure
. As shown at 8 h post-infection when Site I should be active down-regulating early transcription we observed a ratio of 0.50 ± 0.35 indicating that there was less methylation of H3K9me1 at this time in the recombinant carrying two copies of Site I than in the parental recombinant with only one copy. In contrast at 48 h post-infection when replication is occurring we observed a ratio of 1.66 ± 0.37 confirming that Site I is capable of directing the introduction of H3K9me1 when SV40 is replicated. Interestingly it is also apparent that the second copy of Site I can function during replication outside of its normal location within the virus genome.
FIGURE 4 Two copies of Site I directs the incorporation of more H3K9me1 compared to one copy of Site I in SV40 minichromosomes isolated at 48 h post-infection but not at 8 h post-infection. SV40 minichromosomes were prepared from cells infected with pBM129-1 (one (more ...)
Since the effect of repression on H3K9me1 was only seen at late times in infection, it seemed likely that it was either directly or indirectly related to the replication of SV40 DNA which was occurring at this time. In order to test his hypothesis we determined the effect of the inhibition of replication on the introduction of methylated H3K4 and H3K9. SV40 minichromosomes were prepared at 24 h post-infection when replication was beginning and at 48 h post-infection in the presence or absence of aphidicolin, a specific inhibitor of eukaryotic DNA replication (Ohashi et al., 1978
). SV40 minichromosomes were then subjected to ChIP analysis with antibodies to methylated H3K4 and H3K9. We first investigated the introduction of methylated H3 during the increase in SV40 chromatin resulting from replication between 24 and 48 h post-infection. Since we generally observe a 50- to 200-fold increase in the pool size of SV40 minichromosomes between 24 and 48 h post-infection, we compared the increase in a particular form of modification to the increase in the amount of SV40 minichromosomes. We expected that this ratio would be 1 if both the SV40 minichromosomes and form of modification were increasing at the same rate, greater than 1 if the newly replicated minichromosomes were more likely to contain the form of modification, or less than 1 if the minichromosomes were increasing faster than the introduction of the modified histone H3. The results of this analysis are graphically represented in Figure
. Based upon the observed ratios, all methylated forms of H3K4 and H3K9 were being introduced into the newly replicated minichromosomes at a rate faster than the increase in SV40 chromatin. However, H3K4me2 and H3K9me3 appeared to be introduced at rates close to the rate of increase of chromatin (1.74 and 1.23, respectively), while the other methylated forms of H3 were introduced at rates much greater than 1.
FIGURE 5 H3K4me1 and H3K9me1 are introduced into wild-type SV40 minichromosomes primarily during active replication. Wild-type SV40 minichromosomes were isolated at 24, 48, and 48 h post-infection following treatment with the DNA replication inhibitor aphidicolin (more ...)
Next, we determined whether the introduction of a particular form of methylated H3 was actually dependent upon ongoing DNA replication. If ongoing DNA replication was necessary for the introduction of a particular methylated form of H3, inhibition of replication with aphidicolin should also block the introduction of the methylated form of H3. In contrast if the introduction of a methylated form of H3 was due to some other biological process, one would expect little if any effect on the introduction of the methylated form of H3 following inhibition of replication. SV40 minichromosomes were isolated from cells treated with aphidicolin from 24 to 48 h post-infection or from untreated cells at 48 h post-infection and subjected to ChIP analysis and real-time PCR. For each methylated form of H3, we then calculated the ratio of the decrease in methylated H3 to the decrease in the amount of SV40 minichromosomes following inhibition of replication. A ratio of 1 or greater would indicate that the introduction of methylated H3 was equal to or even greater than the reduction in the amount of SV40 chromatin, while a ratio near 0 would indicate that the introduction of methylated H3 was independent of DNA replication. The results of this analysis are graphically represented in Figure . As shown in the figure the ratios for H3K9me1 (1.75) and H3K4me1 (0.92) were similar to or greater than 1 indicating that the introduction of these two methylated forms of H3 into SV40 chromatin were directly dependent upon DNA replication. The ratios for three of the methylated forms of H3 were very low including H3K4me2 (0.15), H3K4me3 (0.10), and H3K9me3 (0.17) indicating that these methylated forms of H3 were being introduced in the absence of direct DNA replication. The ratio for H3K9me2 (0.47) was intermediate between the other forms of methylated H3 suggesting that it was at least in part dependent upon replication. While we believe that the changes observed following aphidicolin treatment are primarily a result of the extensive inhibition of replication, we cannot exclude the possibility that indirect effects on transcription or induction of the DNA damage response following aphidicolin might also be contributing to changes in histone modifications.