Our present study has focused on the functional identification of host factors that maintain retroviral silencing and further testing of the hypothesis that silencing is a component of an antiviral response. We reasoned that siRNA-mediated knockdown of individual host proteins that maintain retroviral silencing would result in reactivation and that this approach could be used to identify specific silencing factors. siRNA-based methods are quite powerful but are prone to a variety of artifacts, including off-target effects (4
). Furthermore, siRNA transfection or target knockdown could produce secondary effects, such as a cellular stress response. As retroviruses and transposable elements can be reactivated by various forms of cellular stress, it seemed possible that “off-target” or nonspecific responses to siRNA transfection might produce a high background in the assay. As such, numerous controls were used to unequivocally demonstrate specificity. Our initial attempt to apply and validate the use of the siRNA methodology began with the prediction that the knockdown of one or more HDACs would phenocopy the effects of HDIs. We show that challenge of a GFP-silent cell population (or clones derived thereof) with siRNAs targeted to HDAC1 resulted in significant and specific reactivation of the GFP retroviral reporter gene. We describe rigorous control experiments, including the use of an siRNA-resistant HDAC1 mutant. As the HDAC1 siRNA and HDIs produce the same phenotype, we conclude that HDAC1 is the relevant target for HDIs in this system. Remarkably, there seems to be no redundancy in HDAC-mediated silencing in this system. This finding indicates that the silencing of viral vectors could be reversed by highly specific siRNA reagents.
We previously identified a role for the transcriptional repressor Daxx in the initiation of retroviral silencing and hypothesized that this protein participated in an antiviral response (20
). We also found that the association of HDACs with retroviral DNA was dependent on Daxx, consistent with an overall model whereby the repressive activities of Daxx are mediated by HDAC binding partners (25
). Subsequent studies by others have shown that Daxx also represses hCMV early gene expression (7
). An antiviral role for Daxx has now been supported by the finding that the hCMV pp71 protein acts as a viral countermeasure that targets Daxx for proteasomal degradation (49
). The avian adenovirus Gam1 and hCMV IE1/IE2 proteins inhibit HDACs and may also act as viral countermeasures to overcome HDAC-mediated repression (9
). Here, we have shown that expression of pp71, Gam1, or IE2 results in GFP reactivation, consistent with their proposed roles as Daxx and HDAC inhibitors. We note that Daxx siRNA or Daxx inhibitor pp71 expression resulted in the reactivation of silent GFP in long-term-passage cells. This finding revealed a role for Daxx in the maintenance of silencing, in addition to the earlier identified role in the initiation of silencing. However, in several cell clones, the knockdown of Daxx did not result in detectable reactivation (Fig. ). It is possible that the relevant HDAC1 activity in these clones is associated with a different binding partner or complex.
Based on our previous studies (20
) and those of others, it seems likely that the roles of HDAC1 and Daxx in retroviral silencing are direct, i.e., that these factors interact with integrated viral chromatin. Here, we provide evidence that Daxx is physically associated with the viral locus during the long-term maintenance of silencing (Fig. ). With respect to HDAC1, we detected rapid histone H4 hyperacetylation and the concomitant induction of GFP mRNA within several hours after TSA treatment of TI-C cells (data not shown). Therefore, the effects of TSA appear to be direct, and the relevant target of inhibition by TSA is likely HDAC1 resident at the silent viral locus. There is additional evidence to support a direct role for HDAC activity in silencing maintenance. It is now clear that HDACs play a role in the repression of a variety of retroviruses (10
), large DNA viruses (9
), and viral transgenes (8
), as measured by reversal of silencing by HDI treatment, and such treatment can produce a transition from a hypo- to a hyperacetylated histone state in the vicinity of the viral promoter (44
). Although the simplest model is that HDACs maintain silencing at the viral loci via deacetylation of histone tails, the acetylation-deacetylation cycle is a common regulatory mechanism of cellular processes, and HDACs have broad substrate activity on nonhistone substrates, including transcription factors (19
). However, the fact that HDAC1 is required to maintain the silencing of two different promoters (hCMV and the ASV LTR) suggests that the critical substrate(s) is a general factor (e.g., histones) rather than a specific factor (e.g., transcription factors). Irrespective of the precise mechanism of action, we have shown that siRNAs provide a highly specific means of reversing epigenetic silencing.
Our results provide some insight into general models for the initiation and maintenance of retroviral silencing. Examination of cell clones harboring silent retroviruses revealed that, in each case, silencing was controlled by HDAC1, indicating that this factor can mediate silencing at numerous integration sites (Fig. ). Our previous studies showed that ca. 40% of ASV integration events occur in protein-encoding genes (43
), yet HDAC-mediated silencing and repression occurred at high frequency (28
). It is unlikely, therefore, that the HDAC-mediated effects are limited to rare integration events into preexisting hypoacetylated heterochromatin. Relevant studies using HDIs have been interpreted to mean that HATs and HDACs are unable to access heterochromatin in the absence of DNA replication (reviewed in reference 53
). In independent studies, we have observed that TSA-mediated reactivation of silent retroviral reporter genes is DNA replication independent (28
; also data not shown). This behavior therefore provides further support for the idea that the location of these silent proviruses is not restricted to preexisting heterochromatin sites. Lastly, we do not believe that levels of repressive factors (e.g., HDAC1) in individual cells determine the frequency of silencing in our system, as a matched HIV-based GFP vector was uniformly resistant to silencing (28
). Taken together, our results support a model whereby repressive factors are able to nucleate on viral chromatin independently of the integration site (Fig. ) (28
), although their repressive activities are not fully penetrant and may be modulated by the integration site (28
The studies described here focus on, but are not limited to, retroviral constructs in which the silent GFP gene is under the control of the strong hCMV IE promoter. This promoter is capable of driving high-intensity GFP expression and thus provides an excellent on-off dynamic range (Fig. ). These characteristics initially led us to identify cells harboring silent viral DNA (28
). We also reasoned that repression of this strong promoter likely signified roles for potent silencing factors. After identifying these factors (HDAC1, etc.) by use of the hCMV IE promoter system, we went on to ask if they were promoter specific by testing the silent GFP genes under the control of the native viral LTR (Fig. ) and the EF1-α cellular promoter (28
; also data not shown). We tentatively conclude that the promoters are not a major determinant in shaping the constellation of repressive factors that we have identified. It is possible that viral sequences, including the native retroviral LTR, which is retained in all of viral constructs, provide a common determinant.
The HP1 family members typically concentrate in silent pericentric heterochromatin via binding to a specific histone modification associated with repressive chromatin, H3K9 methylation. This modification is written and erased by activities of histone methyltransferases and demethylases, respectively (50
). The recognition of H3K9 methylation by HP1 is mediated by the HP1 chromodomain, while the multimerization of HP1, which is believed to drive chromatin compaction, is mediated by the HP1 chromoshadow domain (Fig. ). These properties of HP1 provide a strong paradigm for how histone code modifications may drive heterochromatin formation and silencing. HP1α and HP1β localize to heterochromatin, while HP1γ is found in both heterochromatin and euchromatin (39
). We found that knockdown of the HP1γ but not the HP1α or HP1β isoform was sufficient for reactivation. Assuming that reactivation is the direct result of HP1γ depletion, our results suggest that HP1γ contributes to retroviral gene silencing at numerous loci, possibly within euchromatin. Consistent with this interpretation, HP1 proteins are known to mediate the silencing of specific euchromatic genes as part of sequence-specific repressor complexes (45
In addition to roles for HP1 and H3K9 methylation in heterochromatin formation/maintenance, several studies have identified an association of heterochromatic markers with a subset of transcribed genes. These findings indicate that heterochromatin can also serve as a platform for various activities, including transcription (21
). More-recent studies have indicated that HP1γ itself is regulated by a modification subcode; specifically, phosphorylation of HP1γ Ser83 is associated with actively transcribed genes (34
). Our findings are more consistent with the classical view that HP1γ participates in gene silencing. The fact that the HP1γ isoform mediates silencing in our system is also consistent with our current hypothesis, namely, that the chromosomal location of silent or partially repressed retroviruses is not limited to constitutive heterochromatin (28
). More-definitive studies, including chromosomal mapping of integration sites and identification of the forms of HP1 that are physically associated with these integrated viral genomes, are required to test this model. We also considered that our cell-sorting strategy to isolate GFP-silent cells may have introduced a bias in terms of the constellation of factors that maintain silencing (i.e., HDAC1, HP1γ). Although this bias cannot be ruled out, our previous study (28
) indicated that HDAC-mediated repression and silencing occurred at a very high frequency in this system. Thus, the selected cells used in this study likely do not represent a rare subset. Furthermore, our findings are consistent with those in other systems that indicate roles for HDACs (10
), as well as HP1γ (15
), in retroviral silencing. Therefore, we suggest that the function identified for HP1γ in HIV type 1 DNA latency (15
) may signify a more generic role.
In summary, we describe compelling evidence for roles for specific host factors in retroviral reporter gene silencing. Furthermore, the factors we have identified are not uniquely dedicated to the silencing of retroviruses and may even represent a more general system to silence foreign DNA. This work has also established that siRNAs provide a powerful tool to dissect the function of host proteins in this process. Reversal of epigenetic silencing by HDIs and other compounds may be a useful component of both cancer (6
) and HIV (31
) therapies. It will be important to develop more-specific therapeutics to inhibit individual HDACs and other mediators of silencing to avoid broad effects on the epigenetic state of treated cells. Our findings indicate that such approaches may be possible.