The cellular transcriptional coactivator, HCF-1, is essential for expression of the immediate early genes (IE) of the α-herpesviruses HSV and VZV11
. Both viruses utilize virion-encapsidated activators to recruit HCF-1-Set/MLL1 histone methyl-transferase (HMT) complexes9,12
to the IE promoters, resulting in histone H3-lysine 4 (H3K4) trimethylation and initiation of gene transcription12,13
. HCF-1 depletion results in increased levels of repressive histone H3-lysine 9 (H3K9) methylation, suggesting a central role for HCF-1 in modulating chromatin modifications that determine viral gene expression.
To investigate histone methylation in herpesvirus gene expression, we assessed the state of H3K4 and H3K9 methylation by chromatin immunoprecipitation (ChIP) assays using a model VZV IE promoter-reporter (). In the absence of the VZV IE activator (IE62), repressive H3K9 methylation accumulated on the promoter while in its presence, H3K9 methylation was reduced and positive H3K4 trimethylation was enhanced. This indicated that, in addition to the Set1/MLL1 H3K4 methyl-transferase, an H3K9 demethylase would also be required to modulate the levels of repressive chromatin.
LSD1 is critical for viral activator mediated transcription of VZV IE and HSV IE model promoters
Recently it has been demonstrated that the H3K9 demethylase activity of Lysine Specific Demethylase1 (LSD1) is important for nuclear hormone receptor-dependent transcription14–16
and cell fate determination17
. Therefore, we investigated the role of this enzyme in viral IE transcription. As shown in , LSD1 occupied the VZV IE promoter-reporter with HCF-1 and Set1, in the presence but not absence of the viral activator. Furthermore, depletion of LSD1 resulted in reduced induction of the reporter, demonstrating that LSD1 was important for IE62-mediated activation ().
We next asked if LSD1 recruitment was dependent upon the coactivator HCF-1. In cells depleted of HCF-1, Set1 and LSD1 occupancy was reduced and correlated with a reduction in H3K4 trimethylation and an enhancement of H3K9 methylation (8–9 fold, ). In contrast, occupancy of the activator IE62 was not affected.
The α-herpesviruses VZV and HSV-1 share similar regulatory mechanisms, including the recruitment of HCF-1 by the respective viral IE activators11
. As shown in , LSD1 depletion also reduced the viral-induced expression of an HSV IE reporter gene. Additionally, exogenous expression of wild-type LSD1 stimulated the reporter expression while a catalytic mutant had no significant impact ().
To investigate the role of these factors during viral infection, HCF-1 depleted cells were infected with VZV. In non-depleted cells (), promoter occupancy by Set1, MLL1, and LSD1 were substantial with a high level of H3K4 trimethylation and near background level of H3K9 methylation. In contrast, promoter occupancy by Set1, MLL, and LSD1 were decreased in HCF-1 depleted cells with a correlating decrease in H3K4 trimethylation and increase in repressive H3K9 methylation (). The requirement for LSD1 was addressed by infection of an inducible LSD1-RNAi cell line (). Depletion of 60% of the cellular LSD1 reduced the levels of the IE protein by 66% and mRNA by 78%; indicating that LSD1 was critical for IE gene expression during viral infection.
An HCF-1/LSD1 complex is essential for α-herpesvirus IE gene transcription
In an analogous manner, HCF-1 and LSD1 occupied the HSV-1 IE promoter during infection with the correlating high level of H3K4 and low level of H3K9 methylation (). Furthermore, HCF-1 depletion resulted in a concomitant decrease in the recruitment of LSD1 (). Importantly, depletion of LSD1 resulted in reduced viral IE proteins and mRNAs () and the levels of IE gene expression were recovered by exogenous expression of wild-type LSD1 but not an LSD1 catalytic mutant or a mutant lacking the amine oxidase domain (). Strikingly, depletion of LSD1- resulted in accumulation of nucleosomes bearing repressive H3K9 methylation on the viral IE promoter (). Together, the results demonstrate that the HCF-1-dependent recruitment of LSD1 plays an important role in the initiation of both VZV and HSV-1 infection, likely via modulation of the levels of repressive H3K9 chromatin marks at the IE promoters.
Recruitment of LSD1 is dependent on HCF-1 (, ). Therefore, we determined if this reflected an interaction by coimmunoprecipitation assays. Both Set1- and LSD1-specific antibodies efficiently coimmunoprecipitated HCF-1 (, top). In addition, immunoprecipitation of either HCF-1 or LSD1 resulted in coimmunoprecipitation of RbBP5 (, bottom), a common core subunit of the Set/MLL HMTs18
; suggesting that LSD1 was associated with the HCF-1/HMT complex.
LSD1 has both repressive (H3K4 demethylation) and activating (H3K9 demethylation) activities. Demethylation of H3K4 is mediated by the CoREST/LSD1 complex and targeting or specificity is determined by the associated components19,20
. As shown in Supplementary Fig. 1
, CoREST factors were present in the LSD1 immunoprecipitate but absent from the HCF-1/LSD1 complex. Based on these data, we propose that HCF-1 couples the demethylase LSD1 with the Set1/MLL1 HMT in a novel complex, providing both specificities to promote IE gene transcription ().
Mechanistically, LSD is unique relative to other identified demethylases, including the members of the Jmjd family, as it demethylates lysine residues via a flavin-adenine-dinucleotide-dependent reaction21,22
. This reaction is inhibited by monoamine oxidase inhibitors (MAOIs)15,23–25
; pharmaceuticals used in the treatment of psychiatric disorders (clinical depression, anxiety), Parkinson’s, and migraines. Furthermore, select MAOIs inhibit LSD1 at levels comparable to inhibition of the clinical mitochondrial MAO targets23,24
. Therefore, we investigated the impact of the MAOIs Pargyline and Tranylcypromine (TCP) on the initiation of VZV and HSV infection. For both viruses, treatment with either MAOI resulted in a dose dependent decrease in viral IE mRNA and proteins (). The levels of cellular protein controls were unaffected and no significant cellular toxicity was seen (Supplementary Fig. 2
). TCP also reduced viral yields from HSV-infected cells nearly 1000-fold (). Strikingly, analogous to LSD1 RNAi-mediated depletions (), nucleosomes bearing repressive marks accumulated on the HSV-1 IE promoter in the presence of either MAOI (). The results support the model whereby LSD1 prevents accumulation of H3K9 methylation, thereby allowing productive infection by both α-herpesviruses.
Inhibition of LSD1 with MAOIs blocks α-herpesviral lytic gene expression
In addition to mono- and di-methyl H3K9, inhibition of LSD1 resulted in increased H3K9-trimethylation and occupancy by the heterochromatin protein 1 (Supplementary Fig. 3a
). As LSD1 only removes mono- and di-methyl modifications, the increased H3K9 trimethylation in the absence of LSD1 may reflect (i) increased levels of dimethyl substrates that accumulate during chromatin assembly or (ii) the requirement for an additional H3K9 demethylase(s) of the Jmjd family, in conjunction with LSD1, to provide the specificity required to remove tri-methylation26,27
. The latter is supported by the observation that even in the presence of LSD1, H3K9 trimethylation was detected on the IE promoters during initial stages of HSV-1 infection (Supplementary Fig. 3b
). Irrespective, the requirement for LSD1 to promote viral IE expression identifies it as an essential control component and a target for inhibition of α-herpesvirus infection.
In addition to lytic replication, α-Herpesviruses establish latent infections and cycles of reactivation in sensory neurons. In HSV-1, latency and reactivation correlate with alterations in chromatin modifications on the viral IE promoters5,28–31
. Significantly, HCF-1 is (i) sequestered in the cytoplasm of unstimulated sensory neurons; (ii) rapidly transported to the nucleus upon reactivation stimuli32,33
; and (iii) recruited to the viral IE promoters at the outset of reactivation34
. Therefore, given the impact of LSD1 on viral IE gene expression and its association with HCF-1, we investigated the potential role of LSD1 in viral reactivation by tissue explant of HSV latently infected trigeminal ganglia (TGs) in the presence and absence of TCP (). Strikingly, TCP significantly reduced the reactivation of HSV-1 (P
= 0.0043 and 0.0011 for days 2 and 4, respectively). Due to variance in viral loads of individual animals, these results were confirmed by studies in which each half of a TG was explanted in the presence and absence of TCP (P
= 0.0002; ). Moreover, reduced levels of the inhibitor also effectively blocked viral reactivation (). Finally, as shown in , potential TCP toxicity was not responsible for the suppression of viral reactivation as high viral titers were recovered following drug reversal.
Inhibition of LSD1 with MAOIs blocks HSV-1 reactivation from latency
These studies suggested that MAOI inhibition of LSD1 prevented viral reactivation. However, it remained possible that TCP inhibited lytic spread of the virus but not the initiating reactivation events. Therefore, ganglia were explanted in the presence of DMSO (control), Acyclovir (to prevent viral DNA replication/spread35,36
), or TCP. Sections were probed with antibodies to an HSV lytic antigen (ICP8) ( and Supplementary Fig. 4a
). In control treated ganglia, clusters of ICP8+
neurons were detected in multiple sections of 13 of 16 ganglia, representing initiating neurons as well as infected neurons and support cells from lytic spread. In the presence of Acyclovir (ACV), distinct ICP8+
neurons were detected in sections of 9 of 12 ganglia, representing primary neurons undergoing viral reactivation. Strikingly, in the presence of TCP, only a single ICP8+
neuron was detected in 1 of 15 ganglia, clearly demonstrating that TCP inhibited the initiation of reactivation rather than inhibiting lytic spread (P
= .00002). As additional evidence that TCP inhibits IE gene expression and consequently, reactivation of HSV from latency, viral IE mRNAs could be readily detected by nested RT-PCR from ganglia explanted in the presence of DMSO or ACV but were not detected in the presence of TCP ( and Supplementary Fig. 4c
Together the data support the model () whereby the genomes of infecting α-herpesviruses are subject to cell-directed accumulation of repressive chromatin. For productive infection, α-herpesviruses recruit HCF-1-dependent modification complexes containing LSD1 and the H3K4 HMTs Set1/MLL1 to prevent the accumulation of repressive chromatin marks and install positive marks. It should be noted that the encapsidated HSV-1 genome is devoid of nucleosomes which are deposited during the initial stage of infection37,38
. As positive chromatin marks are installed and viral gene transcription is activated, the levels of associated nucleosomes decrease; likely due to targeted chromatin remodeling. In contrast, inhibition of the HCF-1 complex components results in accumulation of nucleosomes bearing H3K9 methylation and repression of viral gene expression.
As LSD1 can demethylate both histone H3K4 and H3K9, the coupling of this protein in the HCF-1 Set/MLL methyltransferase complex may enhance H3K9 demethylation or preferentially target it to this substrate; although additional histone modifications and modification activities may also contribute to the H3K4 or H3K9 recognition and specificity. Moreover, the presence of the Set1/MLL1 H3K4 methyltransferase components in the complex could function to maintain the levels of H3K4 methylation, even in the presence of LSD1 H3K4 demethylase activity.
The recruitment of HCF-1 complex(es) during the initiation of infection emphasizes the mechanism by which these viruses escape the host cell-directed assembly of repressive chromatin. Interestingly, LSD1 has also been recently shown to demethylate non-histone proteins39,40
, raising the possibility that components involved in the viral IE gene transcription machinery may also be modulated by LSD1-dependent demethylation.
With respect to the cycle of latency and reactivation established by these viruses, signals that lead to viral reactivation result in rapid nuclear transport32,33
and occupancy of viral IE promoters by HCF-134
. Coupled with the data presented here that inhibition of the HCF-1 associated demethylase LSD1 blocks viral reactivation, the observations strongly support the model that HCF-1 modification complexes play a critical role in determining the latency-reactivation state of these viruses.
The dependence of viral pathogens on host cell chromatin machinery highlights a potential for therapeutic intervention. As LSD1 is a well defined target of MAOIs and these pharmaceuticals are widely used therapeutically, these observations identify a novel therapeutic target for herpesvirus infections and enhances the importance of ongoing efforts to develop additional LSD1 inhibitors.