Counteracting FOX proteins epigenetically control the herpesvirus lytic-latent balance

Domain: neuroscience
Published: 2026-02-02
Editorial outcome: major_revision

Abstract
The lytic-latent balance is a major viral persistence strategy and obstacle to curing viral diseases, yet its mechanisms remain poorly understood. Following lytic infection in non-neuronal cells, herpes simplex virus (HSV) establishes latency specifically in neurons and is reactivated by stresses. Here we identify forkhead box (FOX) transcription factors that can strongly activate or repress replication of HSV-1 and other alphaherpesviruses, and show that neurons express activating Fox (e.g., Foxf) genes poorly but repressive Fox (Foxk) genes abundantly while non-neuronal or stressed neuronal cells exhibit higher expression of activating Fox genes. Remarkably, knockdown of Foxk1 or overexpression of activating Fox genes induces reactivation from latency in male mouse neuronal culture and in vivo. Of note, FOX proteins bind the viral genome globally and non sequence-specifically and interact with epigenetic cofactors for gene regulation. FOXF1 interacts with CBP and P300 to acetylate and open viral chromatin. FOXK1 works with SIN3A, a cofactor of histone deacetylation, and MAX to suppress HSV-1 and antagonize activating FOX proteins. Therefore, the viral lytic-latent balance is controlled by the relative abundance of counteracting host transcription factors that recruit different epigenetic regulators to the viral genome.

Introduction
Switching between productive (lytic) and latent infections is a common strategy of many viruses to persist in hosts, and a major obstacle for curing viral diseases. Herpes simplex virus 1 (HSV-1) is an ubiquitous pathogen that causes numerous human diseases. During lytic infection, after entry of the viral DNA genome into the nucleus, viral genes are expressed in the order of immediate-early (IE), early (E) and late (L) genes leading to abundant production of viral genomes and particles. In vivo, after lytic infection in non-neuronal cells of peripheral tissues, HSV establishes latent infection particularly in sensory neurons where essentially all viral genes except the locus encoding the latency-associated transcript (LAT) are silenced and production of new viral genomes and particles ceases. Sporadically, certain stresses can reactate the latent virus to resume the lytic program. HSV has evolved to strike a delicate balance between the two infection programs for optimal prevalence: while lytic infection is essential for viral propagation, toomuch of it may cause death or evoke immune responses strong enough to clear the virus; while latency facilitates immune evasion, too deep latency may preclude spread.

Because essentially all HSV-1 proteins are used for replication, the virus relies heavily on host factors for latency, among which epigenetic factors play an important role. During latency in vivo, the HSV-1 genome associates with histones with high levels of heterochromatin modifications such as H3K9me3 and H3K27me3, and inhibitors of the relevant histone demethylases can decrease reactivation. PI3K, mTOR and DNA damage response pathways, as well as the intrinsic and innate immunity mediated by PML nuclear body (PML-NB) components and type I interferons are also implicated in latency. Notably, it is becoming clearer that intrinsic features of neurons favor HSV latency. The viral activator VP16 was shown to dissociate from the viral capsid in the neuronal axons so that it cannot efficiently reach the nucleus to activate gene transcription. We showed that neuron-specific host microRNAs miR-138 and miR-9 repress the expression of multiple viral and host activators of HSV gene expression. Regarding the mechanisms of reactivation, it has been hypothesized that stress-related JNK and DLK pathways lead to an initial step of viral gene de-repression before full reactivation. The glucocorticoid receptor could activate lytic gene promoters in transfection assays, and its mutation reduced reactivation in female mice. Taken together, despite the previous work revealing upstream pathways involved, little is known about what directly acts on the viral genome to enable latency specifically in neurons and reactivation under stresses.

Our previous studies showed that FOXC1, a target of miR-138, promotes HSV-1 neuronal replication. FOXC1 belongs to the forkhead box (FOX) transcription factor family, which comprises 50 genes classified into 19 subfamilies in humans. FOX proteins share a conserved winged helix forkhead DNA binding domain (DBD). They play pivotal roles in various biological processes including development, metabolism and oncogenesis. Notably, the FOXA subfamily members are known as pioneer factors, which are transcription factors capable of accessing and then opening condensed chromatin. However, the roles of FOX proteins in viral infection remain largely unexplored. For herpesviruses, we are aware of only a few related reports, which were about regulation of human cytomegalovirus (HCMV) or Kaposi’s sarcoma-associated herpesvirus (KSHV) by FOXO or FOXK proteins. Aside from our own studies regarding FOXC1, we are unaware of any reports on roles of FOX proteins in HSV infection.

In this work, we identified multiple FOX proteins that can strongly activate or repress neuronal replication of HSV-1 and other alpha-herpesviruses. These proteins are expressed in ways that correlate with the viral phenotypes of latency or lytic replication, and perturbation of their expression profoundly impacts the lytic-latent balance in neuronal culture and in vivo. Mechanistically, the FOX proteins widely associate with viral DNA and cooperate with epigenetic regulators to modulate viral chromatin. These results lead to a new model about how the viral lytic-latent balance is controlled.

Results
Identification of FOX proteins that can strongly activate or repress HSV-1 neuronal replication
To identify FOX family proteins that can regulate HSV-1 infection, we first selected one member from each subfamily and transfected their human genes before infecting mouse neuroblastoma Neuro-2a cells (Fig. 1a, Supplementary Fig. 1a). If a Fox gene from a subfamily had an effect, other members of the subfamily were also tested. In case the viral E3 ubiquitin ligase ICP0 interfered with these functions, both wild-type (WT) strain KOS and its ICP0-null mutant (7134 virus) were used. Proteins with significant impacts on both viruses at a low multiplicity of infection (MOI) were selected. Thus, FOXA1, A2, A3, C1, C2, E1, F1 and F2 that could substantially increase viral yields are designated activating FOX proteins, and FOXK1 and K2 with opposite effects are designated repressive FOX proteins (Fig. 1a). The effects were generally greater on ICP0-null than WT HSV-1 suggesting that ICP0 indeed interfered with FOX protein effects. To test whether this could be due to degradation of FOX proteins induced by ICP0, we examined FOXA1, C1, F1 and K1 expression in human embryonic kidney 293T cells infected with either WT or ICP0-null virus. Expression of these FOX proteins was similar following infection by the two viruses, with the possible exception of less FOXK1 expression in WT infected cells (Supplementary Fig. 1b), implying that instead of targeting FOX proteins for degradation, ICP0 might interfere with downstream steps regulated by the FOX proteins. To avoid such confounding interference, some mechanistic studies described below used ICP0-null virus.

For representative activating FOXF1 and repressive FOXK1, their impacts were confirmed by growth curves (Fig. 1b) and by using Neuro-2a cells stably transduced with these genes for doxycycline (DOX)-inducible expression (Fig. 1c, Supplementary Fig. 1a). Even at a high MOI, FOXK1 could also suppress both viruses, although FOXF1 could stimulate ICP0-null but not WT HSV-1 (Fig. 1d). Mouse FOXF1 and FOXK1 had the same effects as their human counterparts (Supplementary Fig. 1a, c), providing support for using mice or mouse cells to study the human proteins. The natural sites of HSV-1 infection and latency are human sensory neurons. Therefore, we employed human induced pluripotent stem cells (iPSCs)-derived sensory neurons (iNeurons), and found that lentivirus transduced FOXA1, C1, E1 and F1 all substantially increased WT HSV-1 yields while FOXK1 decreased them (Fig. 1e). However, in 293T cells, transfected FOXF1 had no effect on either virus although FOXK1 decreased yields of both viruses (Fig. 1f). In human foreskin fibroblast (HFF) cells, transduced FOXF1 increased yields of ICP0-null but not WT virus while FOXK1 had no significant effect on either virus (Fig. 1g). Therefore, the effects of FOX proteins are cell-type specific but conserved in human and mouse neuronal cells.

FOX proteins can also regulate other alphaherpesviruses
To test whether similar neurotropic viruses are regulated by FOX proteins, transfection-infection experiments were conducted for some other alphaherpesviruses, including HSV-2, pseudorabies virus (PRV), and bovine herpesvirus 1 (BoHV-1) (Supplementary Fig. 2a–c). HSV-2 and PRV, but not BoHV-1, efficiently infected Neuro-2a cells. TCID50 assays of BoHV-1 stocks in Neuro-2a cells showed slow development of cytopathic effects but TCID50 titers similar to PFU titers in bovine kidney MDBK cells indicating efficient entry but inefficient replication. Regardless, sufficient BoHV-1 yields were obtained in Neuro-2a cells by using a high MOI (Supplementary Fig. 2c). For all three viruses, FOXF1 or K1 significantly increased or decreased viral yields, respectively (Supplementary Fig. 2a–c). We found that varicella-zoster virus (VZV) cannot infect Neuro-2a cells, but its gene expression could be detected after the genome was transfected. Co-transfecting FOXF1 with the VZV genome significantly increased IE61 (homolog of HSV ICP0) and IE62 (homolog of HSV ICP4) mRNA levels, while FOXK1 decreased IE61 but not IE62 mRNA levels (Supplementary Fig. 2d). Thus, the role of FOX proteins in regulating neuronal viral replication maybe conserved in at least some alphaherpesviruses.

Fox gene expression profiles in neurons favor suppression of HSV-1
To learn about the expression patterns of Fox genes, we turned to RNA-seq resources including reported data for Neuro-2a, 293T and Vero cells (Gene Expression Omnibus GSE182742), and the database for some other cell lines (https://www.proteinatlas.org/), as well as RNA-seq data that we generated for mock-infected human iNeurons (Fig. 2a). Normalized transcript levels showed that Foxk1 and k2 are expressed abundantly in all cell lines examined whereas the activating Fox genes display differences between neuronal and non-neuronal cells. All the non-neuronal cells expressed multiple activating Fox genes abundantly with particular high expression of one or more of them. However, the neurons and neuronal cell lines all showed almost undetectable Foxe or Foxf mRNAs, and low to medium levels of certain Foxa or Foxc mRNAs. In particular, all the activating Fox genes were weakly expressed in iNeurons. Therefore, neuronal cells generally express activating Fox genes poorly but repressive Fox genes abundantly, which, based on our results above, should favor suppression of HSV-1.

Acute infection and stresses upregulate certain activating Fox genes
Next, we examined whether Fox gene expression is altered during infection or other stresses. In our RNA-seq data from HSV-1 infected iNeurons, although each experiment lacked replicates, two experiments, one with infection at MOI 1 and one at MOI 10, both hinted at upregulation of Foxc1 (Supplementary Fig. 3a). We then infected mouse trigeminal ganglia (TG) in vivo following corneal inoculation with WT HSV-1. Relative to mock-infection, acute infection upregulated Foxa3, c1 and f1, and possibly Foxa1 (P = 0.056) at 3 days post-infection (dpi) without affecting repressive Fox genes (Fig. 2b, Supplementary Fig. 3b). Latent infection upregulated both activating (Foxc2, f1 and f2) and repressive (Foxk1 and k2) Fox genes at 30 dpi (Fig. 2c, Supplementary Fig. 3c). Explant of latently infected TG, which mimics neural damage that causes HSV-1 recurrences clinically, can trigger reactivation. Relative to TG without explant, explant for only 6 h caused upregulation of Foxa2, Foxa3 and Foxf1 by ~2-, 100- and 6.6-fold, respectively, although these changes were independent of infection since mock-infected TG were affected similarly by explant (Fig. 2c, Supplementary Fig. 3c). Hyperthermia can trigger reactivation too at least in mice. In Neuro-2a cells treated with hyperthermia (43 °C for 1 h), Foxa3, c2 and f1 were upregulated by 12-, 6- and 3-fold, respectively (Fig. 2d). Neither explant nor hyperthermia affected Foxk1 or k2 mRNA levels. Thus, acute infection and reactivation-related stresses upregulate certain activating Fox genes without affecting repressive Fox genes.

FOXK1 suppresses lytic replication and is important for HSV-1 latency
Given their abundance, we examined the role of endogenous FOXK proteins. Knockout (KO) of FOXK1 in Neuro-2a cells increased ICP0-null virus yields, which were reduced again by transfected FOXK1 (Fig. 3a). In the FOXK1 KO cells, siRNAs that effectively knocked down FOXK2 (Supplementary Table 1) increased viral yields further (Fig. 3b). We then employed a mouse primary TG neuron model of latency and reactivation, which usually relies on chemical triggers to induce reactivation. Considering possible functional redundancy between FOXK1 and K2, we attempted dual knockdown. We selected one shRNA for FOXK1 and one for FOXK2 that effectively knocked down the respective proteins (Supplementary Fig. 1a). Remarkably, even without chemical triggers, dual knockdown of FOXK1 and K2 caused reactivation from latency in 50% of the wells, significantly higher than the 14% of control wells reactivated (Fig. 3c).

To examine the role of FOXK1 in vivo, we generated FOXK1f/f mice with LoxP sites flanking the Foxk1 gene and crossed them with Advillin-Cre (AvilCre+/−) mice which express Cre specifically in sensory neurons. The resulting FOXK1-conditional knockout (cKO) (AvilCre+/−Foxk1f/f) mice expressed less FOXK1 in TG than the control AvilCre−/−Foxk1f/f mice (Fig. 3d). After corneal inoculation, viral titers in tear films from FOXK1-cKO mice were not different from those in control mice at 1 and 3 dpi (Fig. 3e) but were significantly higher in TG at 3 dpi (Fig. 3f) consistent with FOXK1 specifically repressing HSV-1 replication in sensory neurons. Accordingly, viral genome levels in FOXK1-cKO mice were also higher at 3 and 5 dpi (Fig. 3g). However, latent viral genome levels at 30 dpi were essentially identical between FOXK1-cKO and control mice (Fig. 3h). Nevertheless, following explant, the virus reactivated from latently infected TG of FOXK1-cKO mice faster than those of control mice (Fig. 3i). Therefore, FOXK1 not only suppresses acute viral replication to expedite latency establishment, but is also important for latency maintenance.

Activating Fox proteins are sufficient to stimulate reactivation from latency
To investigate the effects of activating Fox proteins on latency, we first utilized previously constructed a recombinant HSV-1 expressing FOXC1 (HSV1FOXC1) and, as controls, two viruses, each of which expresses an inactive FOXC1 mutant with both N-terminal and C-terminal activating domains of FOXC1 deleted (HSV1FOXC1delAD and HSV1FOXC1delADb). In our previous study, HSV1FOXC1 exhibited increased acute titers and similar latent viral loads in mouse TG relative to the control viruses, but reactivation was not tested. Here we found that after corneal inoculation and latent infection of mice in vivo, HSV1FOXC1 reactivated significantly faster than both control viruses during explant of the latently infected TG (Fig. 3j). We then employed the mouse neuronal model of HSV-1 latency mentioned above, and found that even without chemical inducers, transduced Foxa1, c1, e1 and f1 could all lead to 40–60% of wells reactivated, significantly higher than the <10% of control wells reactivated (Fig. 3k). Therefore, increased expression of activating Fox genes is sufficient to trigger reactivation from latency.

Widespread occupancy of FOXF1 on the HSV-1 genome
To understand how FOX proteins regulate HSV-1 infection, we first asked whether these proteins associate with viral DNA. The following studies focused mainly on FOXF1 and FOXK1 as examples of an activator and repressor, respectively, of viral replication and reactivation. We first performed chromatin immunoprecipitation (ChIP)-qPCR using an anti-FOXF1 antibody to analyze DNA association in HSV-1-infected 293T cells, where endogenous FOXF1 is abundant (Fig. 4a). As positive controls, regions in host Gpr20 and Kit gene enhancers, which are known to associate with FOXF1, were significantly enriched in DNA precipitated by the FOXF1 antibody relative to the negative control IgG. For both WT and ICP0-null viruses, FOXF1 associated strongly not only with regions within viral UL22 and UL50 genes that contain the DNA motif for FOXF1 association, but also with ICP8 and ICP27 promoters that contain no such sequence. An antibody against FOXP1, which does not regulate HSV-1 (Fig. 1a), also precipitated DNA enriched with these regions simply implying that DNA association is insufficient to confer gene regulation. Additionally, we observed that some FOXC1 co-localized with viral replication compartments (Supplementary Fig. 4a), also consistent with association of FOX proteins with viral DNA.

To investigate this in neuronal cells, where FOXF1 is poorly expressed, we conducted ChIP-seq by pulling down transfected FOXF1 in Neuro-2a cells infected with ICP0-null virus. The positions of viral and host peaks and their corresponding genes are listed in Supplementary Data 1. Validating the results, many host peaks are positioned on FOXF1 target sites reported previously in a database (Datasource: Cistrome Data Browser, GSM2062274) and a published report, including those within Etv1, Gpr20, Fos, Sfi1 and Bcl10 genes. Regarding the viral genome, the data reveal widespread occupancies of FOXF1 on numerous sites that were highly consistent between two biological replicates (Fig. 4b). The fraction of viral reads in total reads was 16-fold higher in the immunoprecipitated samples than input samples further indicating high enrichment of viral DNA associated with FOXF1 (Fig. 4c). The viral peaks showed little or no enrichment around transcription start sites (TSS), contrasting with clear enrichment of host peaks at TSS (Supplementary Fig. 4b) indicating differential occupancy patterns between viral and host DNA. Motif search identified the known DNA motif for FOXF1 as well as some de novo motifs that all conform to a RTAAACA sequence (R = purine) (Fig. 4c). Indeed, all RTAAACA sequences in the viral genome have peaks associated with them. However, many other viral sequences also show peaks despite lacking this consensus sequence, and ChIP-qPCR results showed that besides associating with the sites containing RTAAACA (motif+ sites) in the UL2, UL22 and UL55 genes, FOXF1 also associated with the ICP27 and ICP8 promoters lacking RTAAACA (motif− sites) (Fig. 4d). Thus, FOXF1 extensively associates with the HSV-1 genome in both sequence-specific and non sequence-specific (non-specific) manners.

Non-specific DNA interactions are crucial for HSV-1 regulation by FOX proteins
We also performed ChIP-qPCR for repressive FOXK1 and found robust association with the same set of viral sites as for FOXF1 above (Fig. 4e). FOX proteins comprise well-defined DBDs as well as other domains that are well-defined for FOXA and C proteins but less so for FOXE, F or K proteins (Supplementary Fig. 5a, b). By using plasmids expressing truncated proteins (Supplementary Fig. 5c, d), we found that HSV-1 stimulation by FOXF1 required its DBD and the region between aa200–300 (Supplementary Fig. 5e) while efficient suppression by FOXK1 required its DBD, the C-terminus and possibly N-terminus (Supplementary Fig. 5f) suggesting that the DBDs were necessary but insufficient for virus regulation. We further constructed plasmids expressing proteins with the DBD of FOXC1 replaced with that of FOXA1, E1, F1 or K1 (Supplementary Fig. 5c, d). Remarkably, the chimeric proteins, even including the one with the DBD of repressive FOXK1, could all strongly stimulate viral replication (Fig. 4f) indicating that DBDs of different FOX proteins function similarly but are insufficient to determine activation or repression. Based on a crystal structure, the FOXC2 DBD contacts DNA mainly through an asparagine-histidine (NH) site that interacts with DNA bases, and arginine-arginine (RR) and serine-tryptophan (SW) sites that interact with DNA backbones (Fig. 4g). Surprisingly, mutating the corresponding NH site in FOXC1 had no effect on virus stimulation, mutating the RR site had partial effects, while mutating both residues or just the tryptophan of the SW site eliminated stimulation (Supplementary Fig. 5g). Similar mutagenesis studies of FOXF1 showed that among the three sites, only the corresponding tryptophan in the HW site (the serine in the FOXC SW site becomes histidine in FOXF1) is essential for stimulation (Fig. 4h). Moreover, substitution of this tryptophan eliminated virus stimulation by FOXA2 and E1 and attenuated suppression by FOXK1 (Fig. 4i, j, Supplementary Fig. 5d). The substitution also greatly reduced the association of FOXF1 with the HSV-1 promoters (Fig. 4k). Therefore, non-specific DNA backbone interactions, mediated by the conserved tryptophan, are critical for HSV-1 regulation by FOX proteins.

FOXF1 can activate HSV-1 genes and modulate HSV-1 chromatin globally at early times post-infection
We next investigated which viral genes were regulated by FOX proteins and at what stage. At both 3 and 6 hpi with ICP0-null HSV-1, transfected FOXF1 and FOXK1 did not affect viral DNA levels (Fig. 5a), but significantly increased and decreased, respectively, ICP27 (IE gene), TK (E gene) and gC (L gene) mRNA levels regardless of the presence of ACV (Fig. 5b) demonstrating that FOX proteins regulate viral gene transcription at an early stage independent of DNA synthesis.

Regulation of gene transcription is often related to chromatin remodeling. Therefore, we analyzed the association of viral DNA with histone H3 and its modifications by ChIP-qPCR. The occupancies of repressive H3K9me3 and activating H3K18ac and H3K27ac were normalized to those of H3 to assess the fractions of H3 that were modified. Both motif− and motif+ sites were examined. At 2 hpi in N2AFOXF1 cells, DOX-induced FOXF1 expression already significantly decreased total H3 on most sites (Supplementary Fig. 6a, left panel) and decreased H3K9me3/H3 on the ICP8 site while increasing H3K18ac/H3 and H3K27ac/H3 on the ICP27 and UL22 sites (Supplementary Fig. 6a, other panels). At 5 hpi, FOXF1 decreased total H3 on all sites (Fig. 5c, left panel) and H3K9me3/H3 on most sites but increased H3K18ac/H3 and H3K27ac/H3 on all sites (Fig. 5c, other panels). We also analyzed host FOXF1 target sites in Fos and Etv1 genes identified in our ChIP-seq data. Although FOXF1 did not reduce H3 or H3K9me3/H3 on these sites, it did increase H3K18ac/H3 and H3K27ac/H3 on the Etv1 site, and possibly on the Fos site (a trend without significance) (Supplementary Fig. 6b) suggesting that inducing histone acetylation maybe a FOXF1 function applicable to both viral and host genes. We then tested the effects on the viral chromatin further by transfection in Neuro-2a cells and observed decreases in total H3 but increases in H3K27ac/H3 on all the viral sites tested induced by transfected FOXF1 relative to both the empty vector and an irrelevant FOX protein, FOXP1 (Fig. 5d).

The above results strongly indicate that, starting very early during infection, FOXF1 could induce the removal of H3 and H3K9me3 from HSV-1 chromatin while promoting acetylation of the remaining H3. Since all these effects are consistent with chromatin opening, we then performed assays for transposase-accessible chromatin with sequencing (ATAC-seq). At 3 h after ICP0-null virus infection of Neuro-2a cells, transfected FOXF1 increased viral reads relative to total reads associated with accessible chromatin (Fig. 5e), and increased accessibility in all viral promoter regions (Fig. 5f). However, signals were increased globally (Supplementary Fig. 6c) along the viral genome without specific peaks like those in the ChIP-seq data suggesting that even though DNA association is partially sequence-specific, the effects on accessibility are non-specific and global.

Such effects made us wonder whether PML-NBs that colocalize with the HSV-1 genome might be affected. Indeed, while basically all control cells showed clear ATRX puncta representing PML-NBs, overexpressed FOXF1 but not FOXK1 caused almost total dispersal of the puncta in ICP0-null virus infected Neuro-2a cells (Fig. 5g). Although these results may be an artifact of FOXF1 overexpression, we note that because PML-NBs contain multiple repressive chromatin remodelers, such as ATRX and DAXX, dispersal of PML-NBs is also in line with activation of viral chromatin.

Activating FOX proteins generally associate with CBP/P300
To understand how FOX proteins modulate viral chromatin, we first considered the observation that FOXA1 binds core histones. Therefore, we pulled down transfected FOXA1, C1, E1, F1 and K1 after Benzonase treatment to digest DNA (Supplementary Fig. 7a) and observed co-immunoprecipitation of histone H3 with all of them (Supplementary Fig. 7b). An H3 antibody also immunoprecipitated transfected FOXF1 (Supplementary Fig. 7c). The association with FOXF1 depended on aa200–380 but not the DBD (Supplementary Fig. 7d) consistent with the observation that FOXA1 uses a non-DBD domain to interact with H3. However, since both activating and repressive FOX proteins associate with H3, this association cannot explain the activating or repressive activities.

To explore binding partners involved in the regulatory activities, we conducted proximity-labeling using FOX proteins fused to TurboID to label proximal proteins by biotin (Fig. 6a, Supplementary Fig. 8a), which were pulled down using streptavidin beads and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). FOXF1-TurboID were compared with three negative controls, FOXK1-TurboID, FOXF1Δ200–380-TurboID, and unfused TurboID. Immunofluorescence assays demonstrated nuclear localization of all TurboID-FOX fusion proteins (Supplementary Fig. 8b). After transfection and infection of Neuro-2a cells, comparisons with the three controls commonly identified 53 potential interactors (Fig. 6b, Supplementary Fig. 8c). Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis shows that these proteins are predominantly related to ATP-dependent chromatin remodeling (Supplementary Fig. 8d). Application of a more stringent threshold resulted in 11 proteins (Fig. 6b), which form an interaction network according to the STRING database (Supplementary Fig. 8e). Interestingly, homologous histone acetyltransferases CBP and P300, known to stimulate gene transcription by histone acetylation, were both among these 11 proteins. Therefore, we tested their association with FOX proteins further. After Benzonase treatment, co-transfected FOXF1-HA and CBP-Flag were both co-immunoprecipitated by HA and Flag antibodies (Fig. 6c). Immunoprecipitation of transfected FOXF1 pulled down endogenous CBP, too (Fig. 6d). Transfected CBP also co-immunoprecipitated with transfected FOXA1, C1 and E1, but not with FOXK1 or FOXF1Δ200–300 (Fig. 6e, Supplementary Fig. 9). Therefore, activating but not repressive FOX proteins generally associate with CBP/P300.

FOXF1 recruits CBP/P300 to acetylate histones in HSV-1 chromatin and stimulate viral gene transcription
Next, we examined the effects of these FOXF1 proximal proteins. We knocked down each of these except TTN, which is not implicated in gene regulation (NCBI database), and found that siRNAs that effectively knocked down BCL9L, CREBBP (CBP), EP300 (P300) or ARID1A (Supplementary Table 1) decreased viral yields in FOXF1 overexpressing cells, and a second set of siRNAs confirmed the effects of CBP, P300 and ARID1A but not BCL9L (Fig. 6f). We then focused on CBP/P300. Surprisingly, in the absence of FOXF1, both dual knockdown of CBP and P300 and a specific CBP/P300 inhibitor (A-485) substantially increased yields of both ICP0-null and WT HSV-1 at 48 hpi indicating that CBP/P300 normally suppresses HSV-1 replication perhaps indirectly (Fig. 6g, h, Supplementary Fig. 10a, b), such that the differences between viral yields in the presence and absence of FOXF1 were eliminated or greatly reduced. This suggests that FOXF1 requires CBP/P300 to efficiently activate HSV-1 replication. Even at 3 hpi, the dual knockdown greatly attenuated the stimulation of viral gene expression by FOXF1 (Supplementary Fig. 10c) suggesting that CBP/P300 acts at the early stage of transcription.

To test whether CBP/P300 mediates stimulation of viral chromatin acetylation by FOXF1 (Fig. 5c), we used A-485 in ChIP-qPCR assays. At 5 hpi, in the absence of FOXF1, A-485 had little effect on the baseline H3K27ac/H3 levels on the viral sites (Fig. 6i) and even increased H3K18ac/H3 on some sites (Supplementary Fig. 10d) suggesting that CBP/P300 does not acetylate HSV-1 chromatin without FOXF1. Remarkably, in the presence of FOXF1, A-485 significantly reduced H3K27ac/H3 on all viral sites tested, with stronger impacts on motif+ than motif− sites. A-485 also decreased H3K18ac/H3 on the motif+ but not motif− sites. Regardless, the increases in histone acetylation on viral sites caused by FOXF1 were largely eliminated by A-485 for both modifications, which strongly suggests that FOXF1 recruits CBP/P300 to acetylate histones associated with the HSV-1 genome.

FOXK1 associates with SIN3A and MAX to repress HSV-1 replication and antagonizes activating FOX proteins
To understand the repressive mechanisms of FOXK1, we re-used the proteomic data to identify proteins proximal to FOXK1-TurboID relative to FOXF1-TurboID and unfused TurboID, resulting in 85 proteins (Fig. 7a), among which MAX, KIN, GTF2A1, SIN3A and ARID4B are implicated in gene regulation (NCBI database). Among siRNAs for these genes, each of two siRNAs against MAX and one of two siRNAs against SIN3A increased ICP0-null HSV-1 yields in Neuro-2a cells overexpressing FOXK1 (Supplementary Table 1, Fig. 7b, Supplementary Fig. 11a). In FOXK1 KO cells, both SIN3A siRNAs eliminated virus suppression by transfected FOXK1 and both MAX siRNAs attenuated the suppression (Fig. 7c) suggesting that FOXK1 work with MAX and SIN3A to suppress HSV-1 replication. Because SIN3A is a scaffolding protein known to recruit histone deacetylases to chromatin, we further investigated the role of histone deacetylation. Interestingly, treatment with a histone deacetylase inhibitor trichostatin A (TSA), a chemical that can induce reactivation in neuronal culture models, not only increased viral yields substantially, but also weakened the suppressive effects of FOXK1 (Fig. 7d) suggesting that histone deacetylation is involved in FOXK1 function. Since the effects of MAX siRNAs were remarkably substantial, we also tried single guide RNAs (sgRNAs). All three sgRNAs effectively reduced MAX expression (Supplementary Fig. 11b) and caused ~2-log increases in viral yields (Fig. 7e), which further indicates that MAX strongly suppresses HSV-1 replication. Although we failed to co-immunoprecipitate FOXK1 with MAX or SIN3A (Supplementary Fig. 9), Western blotting of biotin-captured FOXK1 proximal proteins labeled using FOXK1-TurboID readily detected MAX and SIN3A (Fig. 7f) confirming their proximity to FOXK1. We note that previous studies reported FOXK1-SIN3A interactions in yeast two-hybrid and co-immunoprecipitation assays. It is possible that these protein-protein interactions are transient, weak and/or indirect, so that they did not persist during our co-immunoprecipitation assay.

Given that FOXF1 associates with co-activators while FOXK1 is at least proximal to co-repressors and may function with them to repress HSV-1 replication, we wondered whether activating and repressive FOX proteins are antagonistic. Indeed, during co-transfection, FOXK1 abolished the strong effects of simultaneously expressed FOXA1 and E1, and greatly reduced the effects of FOXC1 and F1 on ICP0-null HSV-1 replication (Fig. 7g). Antagonism of FOXA1 was also achieved by FOXK2, but not by FOXP1, FOXH1, or the DBDs of FOXC1 or A1 (Fig. 7h) suggesting that non-DBD domains are required for the antagonism. Therefore, FOXK proteins work with repressive cofactors to antagonize activating FOX proteins.

Methods
Cells
Vero (catalogue no. CCL-81), 293T (catalogue no. CRL-3216), U2OS (catalogue no. HTB-96), HFF (catalogue no. CRL-1634), and Neuro-2a (catalogue no. CCL-131) cells were obtained from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% newborn calf serum (for Vero cells) or fetal bovine serum (for other cells) at 37 °C in 5% CO2. Mouse TG neurons were isolated and cultured according to a reported protocol with slight modifications. Briefly, 6-week-old male Institute for Cancer Research (ICR) mice (Shanghai Laboratory Animal Center) were anesthetized with isoflurane (RWD Life Science) for 1 min and transcardially perfused with 10 ml of phosphate buffered saline (PBS). TG were dissected and digested for 1 h at 37 °C in a collagenase/dispase II solution (Sigma-Aldrich, C9891 and D4693). Neurons were isolated in an OptiPrep gradient (Sigma-Aldrich, D1556), counted and seeded onto 10-mm coverslips coated with poly-D-lysine (Sangon Biotech, catalog number: E607014-0002) and laminin (Sigma-Aldrich, catalog number: L2020) in Neurobasal-A medium (Thermo Fisher Scientific, catalog number: 10888022). Approximately 5000 neurons were plated on each coverslip. One hour after seeding, the coverslips with neurons were transferred to a 48-well plate and cultured in Neurobasal-A medium supplemented with 2% N21 (Bio-Techne, AR008), 50 ng/mL β-neuronal growth factor (Novoprotein, C060), 50 ng/mL glial-derived neurotrophic factor (Novoprotein, C060), C226) and 1 mg/mL mitomycin C (MedChemExpress, hay-13316) for two to three days before use. Neurogenin 3 inducible human iPSCs (iNGN3 iPSCs) kindly provided by George Church of Harvard Medical School were differentiated into neurons for the experiment in Fig. 2a as described previously, or for the experiment in Supplementary Fig. 3a using another previously described method as follows: Briefly, iNGN3 iPSCs were seeded in Matrigel (STEMCELL)-coated 6-well plates (Corning or Costar, 1×106 cells) and NGN3 was induced using doxycycline (Dox, 1 µg/mL)-containing mTeSR media with supplement (STEMCELL) for 3 days. The medium was replaced with mTeSR/N2 media (mTeSR+supplement: Neurobasal plus+N2 supplement (Thermo Fisher Scientific) = 1:1) containing five inhibitors (5i; LDN193189 (APExBIO, 0.1 µM), SB431542 (APExBIO, 10 µM), DAPT (APExBIO, 10 µM), SU5402 (APExBIO, 10 µM), and CHIR99021 (APExBIO, 5 µM) for 5 days with daily replacement. After 5i treatment, the cells were re-plated to poly-L-ornithine (50-100 µg/mL, Sigma)/laminin (10 µg/mL, Gibco) coated plates and further differentiated in 4G media [4G: BDNF, GDNF, β-NGF, and NT-3 (all 10 ng/mL, PeproTech), ascorbic acid (0.2 mM) in Neurobasal plus (supplemented with B27 plus, Thermo Fisher Scientific): DMEM/F12 (Thermo Fisher Scientific) = (1:1)]. The cells were incubated with 5-fluoro-2’-deoxyuridine (20 µM, FUdR, RPI) for 7 days to eliminate undifferentiated dividing cells and maintained in 4G media thereafter.

Viruses
HSV-1 strain KOS, HSV-2 strain 186 and ICP0-null mutant (7134) HSV-1 derived from KOS were propagated and assayed as described previously. Basically, wild-type HSV-1 and HSV-2 were propagated and titrated in Vero cells, and ICP0-null HSV-1 was propagated and titrated in U2OS cells. PRV and BoHV-1 were generous gifts from Zhengfei Liu at Huazhong Agricultural University. BoHV-1 was originally from Shafiqul Chowdhury of Louisiana State University. PRV was propagated and titrated in PK15 cells, which were kindly provided by Yaowei Huang of Zhejiang University. BoHV-1 was propagated and titrated in MDBK cells kindly provided by Liqian Zhu at Hebei University. VZV bacterial artificial chromosome (BAC) DNA was a kind gift from Hua Zhu of Rutgers University and purified as described previously. Recombinant HSV1FOXC1, HSV1FOXC1delAD and HSV1FOXC1delADb viruses were constructed in our laboratory, as described previously. For infection of cell culture, the viruses were added to cells at the indicated MOIs, incubated for 1 h at 37 °C with gentle shaking every 15 min for adsorption. Then the cells were washed once with PBS and the supernatants were replaced with fresh media before further incubation at 37 °C. Viral titers were determined by regular plaque assays. For TCID50 assays of BoHV-1, Neuro-2a cells were seeded in 96-well plates and infected when the cells reached 60% confluency. BoHV-1 was diluted in a 10-fold serial dilution using serum-free DMEM medium, with eight replicates per dilution gradient. The diluted BoHV-1 was added to Neuro-2a cells. After 1 hour of infection, the culture medium was supplemented with a final concentration of 2.5% fetal bovine serum. Cytopathic effects were observed daily. At 7 dpi, cells were fixed and stained with crystal violet, where non-viable cells remained unstained. TCID50 values were calculated using the Reed–Muench method. All viral infection procedures were performed in a biosafety level 2 laboratory.

Plasmids
All Fox genes were cloned into the Flag-HA-pcDNA3.1- (here referred to as pcDNA) vector. The coding sequences were amplified from cDNA derived from 293T or Neuro-2a cells by PCR as previously described. We also removed the Flag tag in the pcDNA vector to generate the pcDNA-HA vector, into which we cloned the genes encoding FOXF1, FOXK1, FOXF1Δ200–300, FOXA1, FOXC1, and FOXE1 for co-immunoprecipitation assays. All mutations were introduced into the plasmids using a Q5 Site-Directed Mutagenesis Kit (NEB #E0554). The coding sequences of Foxk1 and Foxf1 were cloned into pTRIPZ, a Dox-inducible lentiviral expression vector (Addgene #206981) to construct pTRIPZ-FOXF1 and pTRIPZ-FOXK1 plasmids. To construct the FOXK1 and FOXK2 shRNA plasmids, we selected the target sequences 5’-GGAGCCTCACTTCTATCTTCG-3’ and 5’-CCAGAGCTCAAGCGAGTTAAA-3’. The sequences were annealed and inserted into the pLKO.1 vector (Addgene #128073). To construct different lentiviral plasmids, the coding regions of Foxa1, Foxc1, Foxe1, Foxf1 and Foxk1 genes were cloned into pLVX-EF1α-IRES-mCherry (Addgene) vector using BamHI and XbaI restriction sites. To construct MAX-knockout sgRNA plasmids, we designed the target sequence using the Synthego online tool (https://design.synthego.com/#/) (Supplementary Table 2). The complementary oligonucleotides were annealed and subsequently cloned into the LentiCRISPRv2 vector (Addgene #98290) via the BsmBI restriction site. Primer sequences used for cloning are listed in the Supplementary Table 2.

Transfection
Plasmids, VZV BAC DNA or siRNAs were transfected into cells using a Lipomaster 3000 transfection reagent (Vazyme Biotech Co., TL301-01) according to the manufacturer’s instructions. For small scale transfection, cells were seeded in 24-well plates at a density of 1.5x105 cells per well and transfected six hours later with 100–200 ng of plasmids or 30 pmol of siRNA per well, as specified in figure legends. For co-transfection of VZV BAC DNA and plasmids, 100 ng of VZV BAC DNA and 150 ng of a plasmid were added to each well of a 24-well plate. For immunoprecipitation, ~107 cells in a 100-mm plate were transfected with 4 μg of plasmid per plate, or 4 μg+4 μg of plasmid for co-transfection. siRNAs were synthesized and purchased from Qingke Biological Company, and their sequences, as well as knockdown efficiencies calculated from target mRNA levels quantified by RT-qPCR, are provided in Supplementary Table 1.

Western blot analysis
Cells were lysed in an SDS-PAGE sample loading buffer: 10 mM Tris-HCl (pH 8.0), 50 mM dithiothreitol (DTT), 1% SDS, 10% Glycerol, 0.008% bromophenol blue. Proteins were resolved on 10–12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore #IPVH00010) before being probed with the following antibodies at the following dilutions. Primary antibodies: anti-Flag M2 antibody (1:2000, sigma #F1804); anti-HA antibody (1:2000, Sangon Biotech #D110004-0100); anti-actin antibody (1:10000, ABclonal #AC026); anti-H3 antibody (1:2000, Solarbio #K200033M); anti-FOXK1 antibody (1:1000, invitrogen PA5-36257); anti-FOXK2 antibody (1:1000, ABclonal #A14245); anti-CREBBP antibody (1:1000, Cell Signaling Technology #7389s); anti-H3K27ac antibody (1:2000, Abcam #ab4729); anti-ARID1A antibody (1:2000, Proteintech #83733-3-RR); anti-PML antibody (1:500, Merck #05-718); anti-MAX antibody (1:2000, Proteintech, #10426-1-AP, for Fig. 7f); anti-MAX antibody (1:2000, Abcam #ab199489, for other experiments); anti-SIN3A antibody (1:1000, Proteintech #67197-1-Ig); anti-FOXC1 antibody (1:1000, Abcam #ab227977); anti-FOXA1 antibody (1:1000, Proteintech #20411-1-AP); anti-FOXF1 antibody (1:1000, Abcam #ab168383); anti-ICP0 antibody (1:1000, Santa Cruz #sc-53070). Secondary antibodies: HRP-conjugated goat anti-mouse (1:4000, Southern Biotech #1030-05) and goat anti-rabbit antibodies (1:4000, Southern Biotech #4030-05).

Lentivirus production
Four μg of lentiviral transfer plasmid, 3.8 μg of psPAX2 plasmid (Addgene), and 1.9 μg of pMD2.G plasmid (Addgene) were transfected into 293T cells in a 100 mm dish using ExFect transfection reagent (Vazyme Biotech Co., T101-02). At 16 h post-transfection, the culture medium was replaced with fresh medium. At 48 h post-transfection, the supernatant containing lentivirus was collected and stored at 4 °C, after which fresh medium was replenished. At ~92 h post-transfection, a second batch of supernatant was collected and combined with the first batch. The pooled supernatant was centrifuged at 400×g for 5 min to remove cell debris and filtered through a 0.45 μm syringe filter (JetBio-Filtration #FPV-403-030) to further clarify the solution. Lentiviral particles were concentrated by ultracentrifugation using a 50 Ti rotor (Beckman Coulter) at 110,000 xg for 90 min. The concentrated lentiviral particles were re-suspended in the medium suitable for the next step and stored at -80 °C for long-term preservation.

Construction of N2AFOXF1, N2AFOXK1 and N2A-FOXK1KO cells
Lentivirus expressing FOXK1 or FOXF1 derived from pTRIPZ-FOXF1 or pTRIPZ-FOXK1 plasmid was transduced into Neuro-2a cells in the presence of 8 μg/ml polybrene (Sigma). Twenty-four h later, the supernatant was replaced with fresh medium containing 2 μg/ml puromycin. Puromycin-resistant cells were selected over a period of 10–14 days, during which time the medium was changed every 48 h. Stable expression of FOXK1 and FOXF1 was confirmed by Western blotting. To induce the expression of FOXK1 or FOXF1, cells were treated with 1 μg/ml doxycycline hydrochloride (Solarbio #D8960) for at least 24 h. N2A-FOXK1KO cells were constructed by CRISPR-Cas9 technology as described previously. Briefly, sgRNA was designed based on the mouse Foxk1 gene sequence with the aid of the online resource https://zlab.bio/guide-design-resources. The selected target sequence was 5’-GCCGAAGUCGGCGAGGACAG-3’. The sgRNA sequence was cloned into the PX459 vector (Addgene). Neuro-2a cells were seeded in a 24-well plate and transfected with the sgRNA expressing plasmid. At 24 h post-transfection, the supernatant was replaced with fresh medium, and cells were transfected again with the same plasmid. Twenty-four h later, the supernatant was replaced with fresh medium containing 2 μg/ml puromycin. At 48 h after adding puromycin, cells were trypsinized and seeded in 96-well plates with a density of 0.5 cells per well in the presence of 1 μg/ml puromycin to expand single colonies. When cells grew to confluency, they were trypsinized and transferred to larger wells or plates for further propagation in the presence of 1 μg/ml puromycin. Successful knockout of FOXK1 was confirmed by Western blotting.

RNA-seq analysis of iPSC-derived neurons
Twenty-one days after seeding at 1x105 cells/well on Poly-L-ornithine/laminin coated plates, iNGN3 iPSC-derived neurons were mock-infected or infected with HSV-1 strain KOS at MOIs of 10 or 1 at 37 °C for 2 h. The inocula were removed, the cells were washed with DMEM/F12, and fresh prewarmed 4G medium was added. At 10 hpi, for each sample, RNA was harvested from four wells of cultured neurons using an RNeasy Plus Mini Kit (Qiagen Catalog #74134). The culture medium was removed, and 100 μl of lysis buffer provided in the kit was added to each well. Genomic and viral DNA were eliminated by treatment with the gDNA Eliminator Spin Columns supplied in the kit followed by RNA extraction according to the manufacturer’s protocol. Extracted RNA samples were quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, USA), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Catalog #E7760S) following the manufacturer’s instructions. Oligo(dT) enrichment was performed as the first step of library preparation using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Catalog #E7490S), followed by fragmentation and first and second-strand cDNA synthesis. After adapter ligation and index addition followed by PCR, libraries were analyzed for length and concentration using the Agilent Bioanalyzer and qPCR, and 200 bp Paired-End sequencing was performed using a NextSeq High-Output—Illumina HiSeq instrument by the BPF NGS Genomics Core Facility, Harvard Medical School. The human hg38 reference genome was first used for aligning fastq reads using STAR (version 2.7.11a). Transcript abundances were then quantified using Salmon (version 1.10.2), and scaled by library size using tximport in the R package (version 1.26.1), with each gene then normalized to transcripts per million (TPM) counts. The expression values of selected Fox genes were then selected and visualized using ComplexHeatmap in the R package (version 2.14.0). Raw data have been uploaded to the NCBI GEO repository under access number GSE299792.

qPCR and RT-qPCR
To quantify HSV-1 genomes, total DNA was purified using a FastPure Cell/Tissue DNA Isolation Minikit (Vazyme Biotech Co., #DC102-01) and a segment of the HSV-1 TK gene was assayed. Purified viral genomes of known concentrations as described previously were serially diluted to generate standard curves for determination of genome copy numbers. Viral genome levels were normalized to mouse Adipsin gene levels. To quantify mRNAs, total RNA was purified using a FastPure Cell/Tissue total RNA isolation kit V2 (Vazyme Biotech Co., #RC112-01). For co-purification of DNA and RNA, a DNA/RNA co-extraction kit (TIANGEN, #A0722A) was used. RNA was reverse transcribed using a HiScript II Q RT SuperMix for qPCR (+gDNA wiper) kit and oligo(dT) primer according to the manufacturer’s instructions (Vazyme Biotech Co., #R233-01). Serially diluted total RNA was used to generate standard curves for calculation of relative quantities. For quantifying Fox mRNAs in TG, absolute mRNA copy numbers were estimated by cDNA copy numbers, assuming that reverse transcription was efficient. For standard curves, regular PCR was first performed using the same pair of primers as for qPCR. The PCR product was purified using a HiPure Gel DNA Mini kit (Magen #D2111-03) and quantified by a spectrophotometer before being serially diluted and used as standards. GAPDH mRNA was used as an internal control for normalizing mRNA levels. For both DNA and RNA quantification, PCR reactions were prepared using a ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., #Q711-02) and performed on a QuantStudio 3 real-time PCR system instrument (Applied Biosystems). qPCR primer sequences are listed in Supplementary Table 3.

Mouse procedures
All mouse housing and experimental procedures were in compliance with national ethical guidelines and approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang University with an approval code of ZJU20210251. Mouse infections were performed in an animal biosafety level 2 laboratory. Mice were housed at ambient temperature (~23 °C) with low humidity in an air-conditioned room with 12 h light-dark cycles. All experiments used male mice because female AvilCre+/− mice were reported to exhibit Cre expression in oocytes according the information from the manufacturer (https://www.jax.org/strain/032536). ICR mice were obtained from the Shanghai Laboratory Animal Center. Genetically modified mice based on the C57BL/6J strain were generated as described below. For HSV-1 infection, six-week mice were anesthetized with a solution containing 4 mg/ml sodium pentobarbital and 500 µg/ml xylazine hydrochloride, followed by inoculation of WT HSV-1 strain KOS on scarified corneas. The viral dose was 2×105 PFU/eye for ICR mice and 2×106 PFU/eye for the C57BL/6J strain. For tear film viral titer determination, mice were anesthetized using 3% isoflurane (RWD Life Science) in oxygen at a flow rate of 0.5 ml/min with a V1 Table Top anesthesia machine (Colonial Medical Supply), and ocular swabs were collected using cotton-tipped applicators. For analysis of infectious viral titers in TG, mice were euthanized by cervical dislocation and TG were harvested and then homogenized in cell culture medium, subjected to three freeze/thaw cycles before titration by plaque assays. For quantification of DNA or RNA, TG was harvested in the same way and then homogenized in the lysis buffer for DNA or RNA purification before qPCR or RT-qPCR analysis as described above. For explant-induced reactivation, TG were excised and placed in a 12-well plate containing 1 ml of culture medium at 37 °C. Supernatants were collected and replenished daily. The collected supernatants were added to Vero cells to assess the presence of infectious virus by cytopathic effects.

Construction of sensory neuron-specific FOXK1 knockout mice
C57BL/6JGpt-Foxk1em1Cflox (Foxk1f/f) mice with the Foxk1 gene flanked by loxP sites were obtained from GemPharmatech (Strain No. T014811). AvilCre+/− mice (JAX Laboratory Strain No. 032536) that express Cre specifically in sensory neurons, also in the C57BL/6J background, were kindly provided by Liang Wang at Zhejiang University School of Medicine. The mice were housed and bred in the isolation barrier facility of the Laboratory Animal Center at Zhejiang University. Foxk1f/f mice were crossed with AvilCre+/− mice to generate AvilCre+/−Foxk1f/f mice. For offspring genotyping, toe samples were collected, lysed in 200 µL of 50 mM NaOH at 99 °C, and then neutralized with 20 µL of 1 M Tris-HCl (pH 6.8). The lysates were centrifuged at 13,800×g for 5 min, and the supernatants were used for PCR to identify genotypes. PCR primer sequences for genotyping are listed in Supplementary Table 4.

HSV-1 latency and reactivation in primary mouse TG neurons
To establish HSV-1 latent infection, two days after isolation and culture of TG neurons, neurons were treated with 100 μM ACV. On the next day, neurons were infected with a genetically engineered HSV-1 expressing GFP (64-GFP) at an MOI of 20 in the presence of 100 μM ACV. The supernatant was replaced with fresh medium containing 100 μM ACV at 2 and 4 dpi. At 6 dpi, ACV was removed and replaced with regular medium for neuronal culture. At two days after ACV removal, neurons were transduced with lentiviruses at a dose of 1.5x108 genome copies per well in the presence of 2 μg/ml polybrene (Sigma-Aldrich). Lentiviruses were removed by medium replacement on the next day. Supernatants were collected every two days and assayed for infectious virus by evaluating cytopathic effects in Vero cells.

ChIP-seq
Neuro-2a cells were cultured in 100-mm dishes, transfected with 4 μg of pcDNA or pFOXF1 for 42 h, and then infected with ICP0-null HSV-1 for 5 h. Cells were crosslinked with 1% formaldehyde for 10 min and neutralized with 0.125 M glycine for 5 min, followed by two washes with 5 ml of ice-chilled PBS. Cells were collected into 15 ml conical tubes using 5 ml of cold PBS and centrifuged at 400×g for 5 min at 4 °C. The resulting cell pellets were lysed in 300 µl of a lysis buffer (0.4% SDS, 10 mM of EDTA, 50 mM of Tris-HCl, pH 8.0) for 30 min before being transferred to 1.5 ml Eppendorf tubes. The lysates were sonicated on ice using a SCIENTZ-IID ultrasonic homogenizer for six cycles of 1 min each, with a 30-sec on and 30-sec off interval, at a power output of 90 watts. A 10 µl aliquot of the sonicated lysate was used for analyzing DNA size distribution. This aliquot was incubated with 110 µl of water containing 0.2 M NaCl at 65 °C for 3–6 h to reverse crosslinking, followed by treatment with 0.2 mg/ml RNase A (Transgenic Biotechnology #GE101-01) for 1 h at 37 °C, then with 0.5 mg/ml proteinase K (Transgenic Biotechnology #GE201-01) for 1 h at 55 °C. DNA was purified using a HiPure Gel DNA Mini Kit (Magen #D2111-03). DNA size distribution was analyzed by agarose gel electrophoresis. This DNA sample also served as an input control. After an optimal DNA fragment size range of 100–500 bp was achieved, immunoprecipitation reaction was carried out by incubating 40 µl of FLAG affinity gel (Merck Millipore #F2426) and 280 µl of the sonicated cell lysate in 1 ml of ChIP dilution buffer [20 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA (pH 8), 1% Nonidet P-40, 0.1% sodium deoxycholate, and 0.01% SDS], supplemented with protease inhibitors (1 tablet per 50 ml, Roche #K562) with rotation for 3 h at 4 °C. The gel was washed sequentially with an ice-cold low-salt wash buffer, high-salt wash buffer, lithium chloride wash buffer, and TE buffer as described previously. The cross-linked DNA-protein complexes were competitively eluted with a 3X Flag peptide (Beyotime #P9801). Crosslinking reversal and DNA purification were performed as described above. The purified input and immunoprecipitated DNA samples were submitted to Novogene for ChIP-seq high-throughput sequencing, followed by data analysis using protocols described previously. Briefly, clean reads were aligned to the mm10+KOS no repeat genome using Bowtie2 (version 2.3.4.3) with default parameters. Then the virus and mouse reads were split. DeepTools (version 3.5.1) bamCoverage was used to calculate read coverages. PlotProfiles and ComputeMatrix were utilized to plot signals around the TSS. The peak calling algorithm MACS2 (version 2.2.7.1) was used to identify significant peaks representing DNA binding sites for mouse and virus separately. The peaks were annotated to known genomic features using ChIPseeker (version 1.32.1). Homer2 (version 4.11.1) findMotifsGenome.pl was used to identify enriched known and de novo motifs. Integrative Genomics Viewer (version 2.16.2) was used to visualize read coverages. Raw data have been uploaded to the NCBI GEO repository under GEO access number GSE285910.

ChIP-qPCR
293T or Neuro-2a cells in 100-mm plates were infected or transfected then infected under conditions described in figure legends. The procedures for crosslinking, sonication and DNA purification were the same as described above for ChIP-seq. For analysis of DNA associated with FOXF1 and FOXK1 in Neuro-2a cells, the same procedure was also followed for immunoprecipitation. For analysis of DNA associated with histone H3 and its modifications, each immunoprecipitation reaction was conducted by incubation of 20-60 µl of the sonicated cell lysate, 20 µl of Dynabeads Protein A (Invitrogen #10002D) and 2 µg of antibody in 1 ml of ChIP dilution buffer with rotation overnight at 4 °C. For analysis of DNA associated with endogenous FOXF1 and FOXP1 in 293T cells, each immunoprecipitation reaction was carried out by incubating a mixture consisting of 100 μL of the sonicated cell lysate, 880 μL of ChIP dilution buffer, supplemented with protease inhibitor (1 tablet per 50 mL, Roche, cat. no. 4693132001), 20 μL of Dynabeads Protein A (Invitrogen, cat. no. 10001), and 3 μg of anti FOXF1 or anti-FOXP1 antibody or normal rabbit IgG, with rotation at 4 °C overnight. A-485 was from MCE (#HY-107455). The following antibodies were used: anti-histone H3 (Abcam #ab1791); anti-H3K27ac (Abcam #ab4729); anti-H3K18ac (Abcam #ab1191); anti-H3K9me3 (Abcam #ab8898); normal rabbit IgG (Merck Millipore #12-370); anti-FOXF1 (Abcam #ab168383); anti-FOXP1 (Abcam #ab134055). After washing, crosslinked DNA-protein complexes were eluted by incubation with 120 µl of elution buffer (1% SDS, 0.1 M NaHCO3) at 65 °C for 10 min before crosslinking reversal and DNA purification as described above. Input and immunoprecipitated DNA samples were analyzed by qPCR using primers listed in Supplementary Table 3.

ATAC-seq
Neuro-2a cells were transfected with pFOXF1 for 42 h and infected with ICP0-null HSV-1 for 3 h at an MOI of 1. Cells were trypsinized, suspended in PBS and counted. A total of 5×104 cells were lysed by an ice-cold lysis buffer, consisting of 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.1% (vol/vol) Igepal CA-630. DNA enzymatic reaction was performed using a TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech Co., #TD501). VAHTS DNA Clean Beads (Vazyme Biotech Co., #N411-01) were used for purification of fragmented DNA, which was then used for library construction using a TruePrepTM Index Kit V2 for Illumina (Vazyme Biotech Co., TD202). The DNA library was submitted to Novogene for high-throughput sequencing. Data processing and analysis were conducted as previously described. Raw data have been uploaded to the NCBI GEO repository under access number GSE285909.

Immunofluorescence assays
Cells were fixed in 4% paraformaldehyde for 10 min, washed with PBS for 5 min, permeabilized with 0.1% Triton X-100 for 10 min, and then washed three times with PBS for 5 min each. The cells were then incubated with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Primary antibodies were applied overnight in 1% BSA at 4 °C. After three washes with PBS for 10 min each, cells were incubated with secondary antibodies in 1% BSA for 1 h at room temperature. Nuclei were stained with DAPI solution (Solarbio #C0060) for 10 min, followed by three additional washes with PBS. Coverslips were sealed with an anti-fade mounting medium (Beyotime #P0126). The following antibodies and dilutions were used: anti-ATRX antibody (1:500, Abcam #ab97508); anti-PML antibody (1:500, Merck #05-718); anti-Flag M2 antibody (1:2000, Sigma #F1804); anti-Flag antibody (1:500, MBL #PM020); anti-ICP4 antibody (1:1000, Abcam #ab6514); anti-FOXC1 antibody (1:1000, Abcam #ab227977); goat anti-rabbit IgG Alexa Fluor 488 (1:500, Thermo Fisher #A11008); goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 (1:1000, Thermo Fisher #A11004); donkey anti-goat IgG Alexa Fluor 405 (1:1000, Abcam #ab175664). Images were captured using an Olympus IX81-FV1000 microscope at ×100 magnification.

Co-Immunoprecipitation
Cells were transfected and infected as described in figure legends before being lysed in a 1-ml lysis buffer containing 50 mM HEPES-KOH (pH 7.4), 1% Triton X-100, 150 mM NaCl, 10% glycerol, and 2 mM EDTA, supplemented with Complete EDTA-free protease inhibitors (Roche, one tablet per 50 ml) and 1.5 μl of Benzonase (enzyme activity 250-300 unit/μl; Yeasen Bio #20156ES50) at 4 °C for 1 h. Lysates were incubated with anti-Flag antibody-conjugated magnetic beads (Thermo Fisher #A36797) or anti-HA antibody-conjugated magnetic beads (Thermo Fisher #88837) at 4 °C for 2 h. For enrichment of endogenous proteins, lysates were incubated with Dynabeads Protein A (Invitrogen #10002D) together with either a specific antibody or normal rabbit IgG (Millipore #12-370) as a negative control. The beads were washed three times for 10 min each with lysis buffer at 4 °C and eluted using an SDS-PAGE sample loading buffer. The eluates were analyzed by Western blots.

Proximity labeling and sample preparation for mass spectrometry (MS)
Proximity labeling was conducted according to a published protocol. Briefly, the TurboID gene was cloned and fused to the N-terminus of the FOXK1, FOXF1, or FOXF1Δ200–380 gene with a linker. Unfused TurboID served as a negative control. The primer sequences are provided in Supplementary Table 2. Plasmids pTurboID, pFOXF1-TurboID, pFOXF1Δ200–380-TurboID, and pFOXK1-TurboID were transfected into Neuro-2a cells at a concentration of 10 μg per 150-mm dish. At 42 h post-transfection, cells were infected with ICP0-null HSV-1 for 6 h at an MOI of 5, followed by treatment with 50 μM biotin for 10 min. Biotin treatment was terminated on ice, and cells were washed five times with cold PBS. Cells were lysed using RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (wt/vol) SDS, 0.5% (wt/vol) sodium deoxycholate, 1% (vol/vol) Triton X-100)] for 1 h, and the lysate was incubated with streptavidin magnetic beads (Invitrogen #65002) overnight at 4 °C. Beads were washed sequentially twice with RIPA buffer (1 ml, 2 min each), once with 1 M KCl (1 ml, 2 min), once with 0.1 M Na2CO3 (1 ml, ~10 s), once with 2 M urea in 10 mM Tris-HCl (pH 8.0) (1 ml, ~10 s), twice with RIPA buffer (1 ml, 2 min each), once with 200 μl of 50 mM Tris-HCl (pH 7.5), twice with 2 M urea in 50 mM Tris-HCl (pH 7.5, 200 μl, ~10 s). The final supernatant was removed, and beads were incubated with 80 μl of 2 M urea in 50 mM Tris-HCl (pH 7.5) containing 1 mM DTT and 0.4 μg trypsin at 25 °C for 1 h with vigorous shaking. The supernatant was collected and transferred to fresh tubes. Streptavidin beads were eluted twice more with 60 μl of 2 M urea in 50 mM Tris-HCl (pH 7.5), and all supernatants were combined. The released proteins were reduced by 5 mM DTT at 25 °C for 30 min and alkylated by 15 mM iodoacetamide in dark for 45 min. Additional 0.5 µg trypsin was added for overnight digestion at 25 °C. Peptides were desalted by µElute Oasis HLB plate (186001828BA, Waters) before LC-MS/MS analysis.

LC-MS/MS analysis of proximity labeling samples
Peptides (500 ng) were dissolved in 2% ACN 0.1% FA and loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptides were separated by a 120-min non-linear ACN gradient on a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) at a flow rate of 300 nL/min on a U3000 nano UPLC system. Eluted peptides were analyzed by a Fusion Lumos Tribrid Orbitrap mass spectrometer (Thermo Fisher Scientific). The data-independent analysis (DIA) included a full MS survey scan from 400 to 900 Th at the resolution of 240,000 FWHM (at m/z 200 Th) with automatic gain control (AGC) set to 1e6, followed by 63 MS2 spectra with isolation windows of 8 Th that covers precursors from 400-900 Th in consecutive order. Precursors co-isolated in each window were fragmented by higher-energy collision dissociation (HCD) with normalized collision energy set to 30%. All MS2 spectra were acquired with 30,000 FWHM resolution with AGC set to 2e5. All data were recorded by Xcalibur software (V4.2.28.14, Thermo Scientific). The acquired .RAW files were loaded into DIA-NN (version 1.9) for spectra-library-free search against the all SwissProt protein sequences of mouse and HSV-1 proteomes. Trypsin with up to two missed cleavages was set. Carbamidomethyl (C) was set as fixed modification. Oxidation (M) and acetyl (protein N-term) were set as variable modifications. Mass tolerance of 7 ppm and 15 ppm were set for MS and MS2 spectra, respectively. Protein level 1% FDR was set to filter the result. Potential contaminant proteins were identified by DIA-NN build-in database and were removed from subsequent analysis. For quantitative analysis, missing values were filled with a MS intensity of 1000 (noise level), and all intensity values were log2-transformed. Proteins enriched in the TurboID-labeled samples were then selected using t tests (P value < 0.05) in Excel and required a fold-change of at least 2 (corresponding to an average intergroup difference of ≥ 1 on the log2 scale).

Statistical analyses
Statistical analyses were performed using Prism version 10.0 (GraphPad Software). All measurements were taken from distinct samples. All multiple comparisons have been corrected before adjusted P values are presented. For comparisons between two groups, unpaired two-tailed t tests were used. For comparisons among three or more groups, one-way ANOVA with Dunnett’s multiple comparisons tests were employed. For multi-condition group comparisons, two-way ANOVA with Sidak’s multiple comparisons tests were used. To compare reactivation curves, log-rank (Mantel-Cox) tests were used. Data are expressed as mean ± SD.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript “Counteracting FOX proteins epigenetically control the herpesvirus lytic-latent balance” by Xiang et al. describes the role of FOX pioneer transcription factors in HSV-1 replication. The family of FOX transcription factors consists of several members in humans that are known for their role in embryonic development. FOX proteins consist of a forkhead box (winged helix) that mediates binding to DNA. The authors convincingly show that some family members have either an antiviral or proviral role in HSV-1 replication, which is an important finding per se. The manuscript is well-written and well-structured in general. There are too many jumps from one FOX protein to another between the individual images which is somehow confusing. In my opinion, the manuscript would have benefited from focusing on maybe two FOX proteins, one promoting viral replication and one hampering viral replication. The data about how FOX proteins influence viral replication in vitro and in vivo are convincing. However, the mechanistic studies would benefit from additional experiments, in particular in regard to ChIP / ChIP-seq. experiments. Most experiments were performed with overexpressed proteins, which is not the best setting to study transcription factor binding to DNA.\nMajor points:\n- Throughout the manuscript the authors use a ICP0-null HSV-1 virus based on an initial experiment in Figure 1 where they show that both ICP0-null virus and wildtype virus behave similar. However, it would be crucial to repeat some of the key experiments also with a wildtype virus to exclude effects that are due to the absence of ICP0.\n- Along this line, ICP0 is a ubiquitin E3-ligase. Could the authors please show a western blot +/- infection with wt / delta ICP0 virus in cells that have endogenous expression of most of the FOX proteins (e.g. BJ fibroblasts and HepG2). This would show how virus infection affects physiological FOX protein levels and whether ICP0 is able to degrade some of the FOX proteins.\n- The finding that FOXF1 is supposed to have a different binding motive in the viral genome compared to the host genome is quite unusual. In general, I am critical of ChIP assays in the context of protein overexpression because overexpression often leads to artifacts. Transcription factors possess the ability to bind to DNA and the more transcription factor present, the more binding to DNA. The authors should perform ChIP assays in infected cells (HSV-1 wt / delta ICP0) with endogenous FOXF1 expression with antibody Abcam; ab168383 from Cancer Discov (2018) 8 (2): 234–251. https://doi.org/10.1158/2159-8290.CD-17-0468.\n- In addition, the authors must include a suitable transfection control protein in their ChIP-seq. assay and ChIP assays in Figure 4 and 6. Desirable would be to use another FOX transcription factor from Figure 1 that did not affect HSV-1 replication like FOXO1.\n- The authors should also show selected regions from the host genome in order to compare FOXF1 binding to the human genome upon FOXF1 overexpression with more physiological settings e.g. from Cancer Discov (2018) 8 (2): 234–251. A list with all binding sites in the host and viral genomes in the supplement would also help.\n- For Figure 5C, does FOXF1 expression also induce changes in Histone H3 / Histone modifications at binding sites in cellular chromatin?\n- The authors show dispersal of PML-nuclear bodies by FOXF1. That would mean that cell lines like HeLa, 293T and BJ fibroblasts that express FOXF1 (Fig. 2A) should not have PML bodies, which is obviously not the case. In addition, ICP0 is highly efficient in degrading PML-nuclear bodies. This would mean that disruption of PML bodies by FOXF1 has no physiological relevance in the context of wildtype infections because it is anyway rapidly degraded upon infection. Could the authors please comment.\n- Minor point:\nThe study would benefit from IF experiments analyzing the presence of FOX proteins in viral replication compartments e.g. by co-staining with ICP8.\n- In some experiments (e.g. in Figures 4 F,H,I,J) the authors used a low MOI of 0.2 and virus was titrated after 48h. Would the effect be the same with higher MOI of 1 after 24h?", "reviewer_2": "In this manuscript, the authors investigate the role of FOX family transcription factors in regulating the lytic-latent balance of herpesviruses, with the primary focus on herpes simplex virus type 1 (HSV-1). The study identifies specific activating (FOX A, C, E, and F family members) and repressive (FOX K1 and K2) FOX proteins and investigates their mechanisms of action through a range of sequencing and molecular assays. While the majority of the work is in mouse cells, some of the activity could be recapitulated in human cells to varying degrees between cell lines, indicating a general but cell type-dependent effect. RNA-seq indicates that repressive FOXK proteins are widely expressed while the activating proteins are more abundant in non-neuronal cells, but expression of activating FOX proteins increases in neurons after infection or cellular stress. Focusing on latency, reducing the basal levels of repressive FOXK1 and 2 in trigeminal ganglia of infected mice with siRNAs was sufficient to induce reactivation along transduction of activating FOX proteins. Furthermore, FOX proteins were found to bind the viral genome extensively, even at sites lacking the RAAACA motif seen for host genes. The functional activating/repressive activity was found through screening of FOX mutants to depend on backbone interactions rather than the recognition of this motif. Using timepoints and chemical inhibitors of viral DNA replication, the role of FOX proteins was shown to occur early in the viral lifecycle. Interestingly, expression of FOXF1 even dispersed silencing PML bodies, mimicking one of the major roles of the viral ICP0 protein. Through a series of IP assays and proximity labeling assays, the authors identify activating FOX proteins interacting with chromatin remodelers, namely CBP/P300, and that this leads to increased H3 acetylation and opening of viral chromatin. On the other hand, repressive FOXK1 interacts with MAX and SIN3A to provide the repressive role through the opposite effect.\nOverall, the manuscript provides a thorough investigation of FOX proteins and their roles in viral replication through of a number of techniques and settings. It links binding of these proteins to the viral genome to functional roles in chromatin formation of viral DNA and outcomes on viral gene expression and titers, however it would be helpful to have the ChIP and ATAC-Seq done on latently infected neurons before and after induction of FOX members. The techniques are well described and the findings presented well, and the findings should be on interest to researchers investigating neurotrophic DNA viruses. I have a few comments for consideration:\nMajor Comments\nSome clarification on the design of the siRNAs used for knockdown experiments is required, or more direct evidence of their functionality such as Westerns when antibodies are available. According to Table S1, the siRNA sequences are perfectly complementary and lack the two-nucleotide 3' overhangs essential for loading into the RNA-induced silencing complex (RISC).\nThe authors indicate that BoHV-1 inefficiently infects N2A neurons, and use an MOI of 5 MDBK units. Could the authors provide a relative MOI for N2A cells based on TCID50 assays and immunofluorescence?\nRegarding FOX ChIP-Seq peaks around transcription start sites, could this analysis be extended to metagene analyses of host and viral genes? On this note, does the RTAAACA motif exist in other parts of the viral genome that did not give rise to peaks in ChIP?\nWithout a marker for infection, it is difficult to claim that the cells in Fig. S6B are both infected and transfected. There is only one cell, in the FOXF1+WT HSV panel at the bottom right, that shows a dispersal of DAPI signal due to viral replication compartments.\nMinor Comments\nLine 40: change to \"an ubiquitous\"\nPerhaps the authors could elaborate more on the HSV1FOXC1 virus mutants so readers get an overview without having to reference the earlier study\nLine 443: perhaps the authors mean PLO, which is a more common abbreviation for poly-L-ornithine\nLine 649: place a space in the middle of \"pH8.0\" for consistency\nThe supplemental figures do not need \"supplemental data\" in the title\nThe ChIP and ATAC-seq are already publicly available, but I was unable to locate the proteomics data with the given accession number", "reviewer_3": "In their manuscript, Xiang et al. investigate the role of FOX proteins — a family of transcription factors – in HSV-1 replication. The authors use a variety of approaches and sophisticated methods to decipher the functions of FOX proteins in controlling HSV-1 latency, including proximity labelling experiments to identify potential FOXF1- and FOXK1-interacting proteins with roles in gene regulation.\nAt the editor’s request, this review focuses exclusively on the proteomic experiments presented in the manuscript. Overall, the proteomic studies are well designed, appear to have been conducted by experienced proteomics researchers, and are analysed using appropriate cut-off criteria. Please see my specific comments below.\nMajor comments\n1. The authors state that their proteomic data have been deposited to the ProteomeXchange repository under the dataset identifier PXD059232, and that the dataset should be publicly available. However, I was unable to locate the dataset using this identifier, or by searching relevant keywords or author names. I urge the authors to verify the accuracy of the dataset ID and ensure that the data are publicly accessible at the time of review. If the dataset is still private, reviewer credentials should be provided to allow proper evaluation of the proteomic data.\nMinor comments\n1. I commend the authors for clearly distinguishing between proteins that are proximal to the bait protein and those that directly interact with it — a distinction that is often overlooked in proximity labelling studies and in many manuscripts I have reviewed previously.\n2. The authors should provide more detailed information on how the proteomic data were processed, including steps such as the handling of missing values, removal of potential contaminants, normalization procedures, and any filtering criteria applied. Additionally, specify which software tools or statistical packages were used for the analysis.\n3. The authors may consider validating the nuclear localisation of the TurboID-tagged FOX constructs using IF to confirm their proper subcellular distribution, and include these data in Fig. S8.\n4. Fig. 6: It would be helpful to include a Venn diagram in to highlight the 11 proteins enriched by more than 2 log2 in all pairwise comparisons. This addition would improve the overall flow of the figure and help readers more easily understand the rationale behind validating the interaction with CBP in panels C–E, as well as the selection of genes shown in panel F for silencing experiments.\n5. Fig. 7A: Would MAX, KIN, GTF2A1, SIN3A, and ARID4B still be considered hits if a more stringent cut-off of log₂ fold-change > 2 were applied, as was done for the FOXF1 dataset? This may be particularly relevant given that MAX and SIN3A were not successfully validated in the co-IP assays, which—beyond the possibility of a temporal or indirect interaction, as the authors suggest—could also indicate that the interactions are generally weaker. Along these lines, were the authors able to validate, via co-IP, the interaction between FOXK1 and any other protein that showed a similar level of enrichment as MAX or SIN3A? Generally, increasing the amount of protein input in co-IP assays can improve the likelihood of successful detection, which might be worth considering if validation has been challenging."}, "author_response": "We thank the reviewers for taking time to review this manuscript and providing many helpful comments. We have addressed all points raised by the reviewers and included new data from the requested experiments. We believe that these revisions have greatly improved the manuscript. The point-by-point responses are written under the corresponding comments in bold letters.\nReviewer #1 (Remarks to the Author):\nThe manuscript “Counteracting FOX proteins epigenetically control the herpesvirus lytic-latent balance” by Xiang et al. describes the role of FOX pioneer transcription factors in HSV-1 replication. The family of FOX transcription factors consists of several members in humans that are known for their role in embryonic development. FOX proteins consist of a forkhead box (winged helix) that mediates binding to DNA. The authors convincingly show that some family members have either an antiviral or proviral role in HSV-1 replication, which is an important finding per se. The manuscript is well-written and well-structured in general. There are too many jumps from one FOX protein to another between the individual images which is somehow confusing. In my opinion, the manuscript would have benefited from focusing on maybe two FOX proteins, one promoting viral replication and one hampering viral replication. The data about how FOX proteins influence viral replication in vitro and in vivo are convincing. However, the mechanistic studies would benefit from additional experiments, in particular in regard to ChIP / ChIP-seq. experiments. Most experiments were performed with overexpressed proteins, which is not the best setting to study transcription factor binding to DNA.\nResponse: We thank the reviewer for the positive comments as well as the constructive suggestions for improvement. Regarding the comments regarding the jumps from one FOX protein to another, we apologize for the confusion. Focusing on two FOX proteins, FOXF1 and FOXK1, was mainly what we did in mechanistic studies. Some results about other FOX proteins were included only for confirmation. To clarify, we have added this sentence at the beginning of mechanistic investigation: “The following studies focused mainly on FOXF1 and FOXK1 as examples of an activator and repressor, respectively, of viral replication and reactivation.” We have also added the names of the FOX proteins on some figure panels with indications regarding whether they are activating or repressive in some places. Regarding the additional experiments, we agree that overexpressed proteins can sometimes be problematic and address this issue below under the relevant major points.\nMajor points:\n- Throughout the manuscript the authors use a ICP0-null HSV-1 virus based on an initial experiment in Figure 1 where they show that both ICP0-null virus and wildtype virus behave similar. However, it would be crucial to repeat some of the key experiments also with a wildtype virus to exclude effects that are due to the absence of ICP0.\nResponse: We understand the concern. Our rationale for using an ICP0-null virus was that FOX proteins have greater effects on it than on wild-type (WT) virus suggesting that ICP0 interferes with FOX protein functions, making it harder to dissect the mechanisms. We note that in our original submission most of our functional studies were conducted using WT virus (Figs 1-3), and that absence of ICP0 is biologically relevant during latency and the initial stages of reactivation. Nevertheless, we repeated experiments regarding the effects of knocking down CBP/P300 on FOXF1 functions with WT virus and have added the results in Fig. S10B. Moreover, the new ChIP experiments in 293T cells (see below and Fig. 4A) were also conducted using both WT and ICP0-null viruses.\n- Along this line, ICP0 is a ubiquitin E3-ligase. Could the authors please show a western blot +/- infection with wt / delta ICP0 virus in cells that have endogenous expression of most of the FOX proteins (e.g. BJ fibroblasts and HepG2). This would show how virus infection affects physiological FOX protein levels and whether ICP0 is able to degrade some of the FOX proteins.\nResponse: We have done this in 293T cells, which have substantial endogenous expression of most of the FOX proteins we have studied. The results have been added to Fig. S1B. The FOX protein levels were largely unaffected by ICP0. We speculate that ICP0 interference with FOX protein functions may be related to the ability of both ICP0 and FOX proteins to regulate viral chromatin.\n- The finding that FOXF1 is supposed to have a different binding motive in the viral genome compared to the host genome is quite unusual. In general, I am critical of ChIP assays in the context of protein overexpression because overexpression often leads to artifacts. Transcription factors possess the ability to bind to DNA and the more transcription factor present, the more binding to DNA. The authors should perform ChIP assays in infected cells (HSV-1 wt / delta ICP0) with endogenous FOXF1 expression with antibody Abcam; ab168383 from Cancer Discov (2018) 8 (2): 234–251. https://doi.org/10.1158/2159-8290.CD-17-0468.\nResponse: We do not think our data suggest that FOXF1 has different binding motifs in the viral genome relative to the host genome and we did not state that. The two de novo motifs in Fig. 4C look similar and both conform to the RTAAACA consensus sequence. The minor difference may be because the viral genome is too small to allow accurate motif determination. We share the reviewer’s concerns about experiments using overexpression. Our original thought was to study this in neuronal cells where FOXF1 is poorly expressed so we used overexpression to mimic the condition where FOXF1 expression is induced due to stresses. We thank the reviewer for providing information about the antibody. We have used the antibody for ChIP-qPCR in 293T cells and have demonstrated FOXF1 association with all the viral sites as well as two host sites tested. Two of the viral sites tested (in UL22 and UL50 genes) conform to the motifs in Fig. 4C and two of the viral sites (in ICP8 and ICP27 promoters) do not conform to it supporting our finding that FOXF1 associates with viral DNA in both sequence-specific and nonspecific manners. These new data are in Fig. 4A. This indeed strengthens the argument that FOXF1 can associate with viral DNA broadly in physiological conditions.\n- In addition, the authors must include a suitable transfection control protein in their ChIP-seq. assay and ChIP assays in Figure 4 and 6. Desirable would be to use another FOX transcription factor from Figure 1 that did not affect HSV-1 replication like FOXO1.\nResponse: Following the reviewer’s suggestion, we used FOXP1 (for which we found a good commercially available antibody) as a transfection control for the added experiment of FOXF1 ChIP in 293T cells (Fig. 4A). However, we detected FOXP1 association with both the host and viral DNA sites (FOXF1 association is still higher than FOXP1, but they are both much higher than the IgG negative control). This is not surprising because FOX proteins have similar binding motifs. This raises the interesting possibility that FOX proteins generally associate with the viral genome but such association is insufficient to confer regulation of viral genes.\nWith regard to Fig. 6I, it was performed using a cell line with inducible FOXF1 expression, not by transfection. Nevertheless, we added an experiment which examined the effects of transfected FOXF1 on H3K27ac/H3, where both transfected FOXP1 and the empty vector were used as negative controls (Fig. 5D, right panel). The results recapitulated the results using inducible cell lines (Fig. 5C) and also showed similar H3K27ac/H3 values between FOXP1 and the empty vector-transfected cells suggesting that the results in Fig. 6I from inducible cell lines should be valid.\n- The authors should also show selected regions from the host genome in order to compare FOXF1 binding to the human genome upon FOXF1 overexpression with more physiological settings e.g. from Cancer Discov (2018) 8 (2): 234–251. A list with all binding sites in the host and viral genomes in the supplement would also help.\nResponse: The positions of viral and host peaks and their corresponding genes are listed in Supplementary Data File 1. For host sites, since they are too numerous, we selected the top 1000. Among them are many binding sites in the mouse genome reported previously (Data source: Cistrome Data Browser, GSM2062274), such as Sfi1, Bcl10, Mpzl1, Fos, Tex14, Ablim1, Fcho2, Ints3, Pitpnc1, Ncor2, etc. The experiments in the recommended Cancer Discov article used human cells, but we used mouse cells, so the sequences are not directly comparable. Nevertheless, we detected peaks on Etv1 and Gpr20 genes, which were identified as FOXF1 targets also in the published study.\n- For Figure 5C, does FOXF1 expression also induce changes in Histone H3 / Histone modifications at binding sites in cellular chromatin?\nResponse: We analyzed sites in cellular Fos and Etv1 genes, which are high-ranking host sites in our ChIP-seq data. We see that on the Etv1 site, both H3K18ac/H3 and H3K27ac/H3 ratios were increased by induced FOXF1 expression, with the same trend observed with the Fos site. However, H3 and H3K9me3/H3 on these sites were not reduced like the viral sites. We conclude that inducing histone acetylation may be a FOXF1 function applicable to both viral and host genes. The added data are in Fig. S6B.\n- The authors show dispersal of PML-nuclear bodies by FOXF1. That would mean that cell lines like HeLa, 293T and BJ fibroblasts that express FOXF1 (Fig. 2A) should not have PML bodies, which is obviously not the case. In addition, ICP0 is highly efficient in degrading PML-nuclear bodies. This would mean that disruption of PML bodies by FOXF1 has no physiological relevance in the context of wildtype infections because it is anyway rapidly degraded upon infection. Could the authors please comment.\nResponse: The reviewer’s argument about the PML-NB makes sense, but the argument about ICP0 makes sense only with regard to acute infection. During latent infection as well as the initial stage of reactivation, there is little or no ICP0 expression, so FOXF1 can regulate PML bodies in these stages of infection. Regardless, we admit that it is possible that we introduced FOXF1 to levels that were artificially high. Therefore, we downplay this part by deleting the corresponding supplemental data. We have kept the panel in the main text because we still think FOXF1 dispersal of PML bodies is an interesting phenomenon that deserves revealing, and it may be physiologically relevant under stress conditions where certain Fox genes are strongly induced according to our data in Fig. 2. We also added the following sentence as a caution in the text: “Although these results may be an artifact of FOXF1 overexpression, we note that…”.\n- Minor point:\nThe study would benefit from IF experiments analyzing the presence of FOX proteins in viral replication compartments e.g. by co-staining with ICP8.\nResponse: This has been done and we see partial colocalization. The additional results are in Fig. S4A. We used an ICP4 antibody to stain replication compartments, which in our hands works well.\n- In some experiments (e.g. in Figures 4 F,H,I,J) the authors used a low MOI of 0.2 and virus was titrated after 48h. Would the effect be the same with higher MOI of 1 after 24h?\nResponse: We understand the reason to check effects at higher MOIs and shorter times. We did the experiment using the W122A mutants as a representative (see below). The result is the same as MOI of 0.2 and 48 h. We believe the other mutants also behave similar between the two conditions. We do not see a need to put this in the manuscript given the already crowded figures.\nICP0-null virus，MOI=1,24hpi\n0.6593\n5 <0.0001\n)L\nm 4\n/U\nF\nP\ng\n(013\no\nL\n2\npcDNA\npF\nO\nO\nX\nX\nF\nF\npF\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors investigate the role of FOX family transcription factors in regulating the lytic-latent balance of herpesviruses, with the primary focus on herpes simplex virus type 1 (HSV-1). The study identifies specific activating (FOX A, C, E, and F family members) and repressive (FOX K1 and K2) FOX proteins and investigates their mechanisms of action through a range of sequencing and molecular assays. While the majority of the work is in mouse cells, some of the activity could be recapitulated in human cells to varying degrees between cell lines, indicating a general but cell type-dependent effect. RNA-seq indicates that repressive FOXK proteins are widely expressed while the activating proteins are more abundant in non-neuronal cells, but expression of activating FOX proteins increases in neurons after infection or cellular stress. Focusing on latency, reducing the basal levels of repressive FOXK1 and 2 in trigeminal ganglia of infected mice with siRNAs was sufficient to induce reactivation along transduction of activating FOX proteins. Furthermore, FOX proteins were found to bind the viral genome extensively, even at sites lacking the RAAACA motif seen for host genes. The functional activating/repressive activity was found through screening of FOX mutants to depend on backbone interactions rather than the recognition of this motif. Using timepoints and chemical inhibitors of viral DNA replication, the role of FOX proteins was shown to occur early in the viral lifecycle. Interestingly, expression of FOXF1 even dispersed silencing PML bodies, mimicking one of the major roles of the viral ICP0 protein. Through a series of IP assays and proximity labeling assays, the authors identify activating FOX proteins interacting with chromatin remodelers, namely CBP/P300, and that this leads to increased H3 acetylation and opening of viral chromatin. On the other hand, repressive FOXK1 interacts with MAX and SIN3A to provide the repressive role through the opposite effect.\nOverall, the manuscript provides a thorough investigation of FOX proteins and their roles in viral replication through of a number of techniques and settings. It links binding of these proteins to the viral genome to functional roles in chromatin formation of viral DNA and outcomes on viral gene expression and titers, however it would be helpful to have the ChIP and ATAC-Seq done on latently infected neurons before and after induction of FOX members. The techniques are well described and the findings presented well, and the findings should be on interest to researchers investigating neurotrophic DNA viruses. I have a few comments for consideration:\nResponse: We agree that it would be best to have the ChIP and ATAC-seq done on latently infected neurons. However, this is technically challenging for us. Neurons are trivial to handle and transgene expression in them is often not very high. Therefore, we used models in primary neurons or iPSC-derived neurons mainly for functional validation, and used the Neuro-2a cell line for mechanistic investigation after showing that effects of FOX proteins in Neuro-2a cells and neurons were consistent.\nMajor Comments\nSome clarification on the design of the siRNAs used for knockdown experiments is required, or more direct evidence of their functionality such as Westerns when antibodies are available. According to Table S1, the siRNA sequences are perfectly complementary and lack the two-nucleotide 3' overhangs essential for loading into the RNA-induced silencing complex (RISC).\nResponse: We apologize for the negligence. In fact, siRNAs all have the two nucleotide 3’ overhangs. They have been added to Table S1. We also added Western blot validation of MAX siRNAs (Fig. S11A).\nThe authors indicate that BoHV-1 inefficiently infects N2A neurons, and use an MOI of 5 MDBK units. Could the authors provide a relative MOI for N2A cells based on TCID50 assays and immunofluorescence?\nResponse: We have conducted the TCID50 assay in Neuro-2a cells. Surprisingly, the titers of the BoHV-1 stock, which was determined to be 3.6 x 108 PFU/ml in bovine MDBK cells were determined to be 4 x 108 TCID50/ml in Neuro-2a cells indicating that Neuro-2a cells are equally susceptible to BoHV-1. However, we noticed that development of cytopathic effects in Neuro-2a cells took 7 days, which were much slower than in MDBK cells (~3 days), suggesting that the virus does not replicate efficiently after entering Neuro-2a cells. Therefore, we revised that part like this: “TCID assays of BoHV-1 stocks in Neuro-2a cells showed slow 50 development of cytopathic effects but TCID titers similar to PFU titers in bovine kidney 50 MDBK cells indicating efficient entry but inefficient replication. Regardless, sufficient BoHV- 1 yields were obtained in Neuro-2a cells by using a high MOI (Fig. S2C)”\nRegarding FOX ChIP-Seq peaks around transcription start sites, could this analysis be extended to metagene analyses of host and viral genes? On this note, does the RTAAACA motif exist in other parts of the viral genome that did not give rise to peaks in ChIP?\nResponse: We have tabulated the binding sites of FOXF1 on viral and host genomes from our ChIP-seq results (Supplementary Data File 1). According to the locations of the RTAAACA motif on the viral genome (green) in Fig. 4B, all RTAAACA sequences have peaks associated with them. Originally, we focused only on the few highest peaks and missed this important point. The text has been revised: “all RTAAACA sequences in the viral genome have peaks associated with them. However, many other viral sequences also show peaks despite lacking this consensus sequence”.\nWithout a marker for infection, it is difficult to claim that the cells in Fig. S6B are both infected and transfected. There is only one cell, in the FOXF1+WT HSV panel at the bottom right, that shows a dispersal of DAPI signal due to viral replication compartments.\nResponse: The reviewer’s question is very reasonable. This experiment lacks a marker for infection. However, considering Reviewer 1’s concern, we have decided to downplay this part about the PML body and delete this supplemental figure.\nMinor Comments\nLine 40: change to \"an ubiquitous\"\nResponse: Revised as suggested.\nPerhaps the authors could elaborate more on the HSV1FOXC1 virus mutants so readers get an overview without having to reference the earlier study.\nResponse: The description has been added: “we first utilized previously constructed a recombinant HSV-1 expressing FOXC1 (HSV1FOXC1) and, as controls, two viruses, each of which expresses an inactive FOXC1 mutant with both N-terminal and C-terminal activating domains of FOXC1 deleted (HSV1FOXC1delAD and HSV1FOXC1delADb)”.\nLine 443: perhaps the authors mean PLO, which is a more common abbreviation for poly-L-ornithine\nResponse: Yes. We mean PLO. But on second thought, there is no need to add an abbreviation here, so we deleted the abbreviation.\nLine 649: place a space in the middle of \"pH8.0\" for consistency\nResponse: Revised as suggested.\nThe supplemental figures do not need \"supplemental data\" in the title\nResponse: Revised as suggested.\nThe ChIP and ATAC-seq are already publicly available, but I was unable to locate the proteomics data with the given accession number\nResponse: We apologize for the problem. There were some technical issues, which have been resolved. We have confirmed that the data is accessible by using the accession number PXD059232 in https://proteomecentral.proteomexchange.org.\nReviewer #3 (Remarks to the Author):\nIn their manuscript, Xiang et al. investigate the role of FOX proteins — a family of transcription factors – in HSV-1 replication. The authors use a variety of approaches and sophisticated methods to decipher the functions of FOX proteins in controlling HSV-1 latency, including proximity labelling experiments to identify potential FOXF1- and FOXK1-interacting proteins with roles in gene regulation.\nAt the editor’s request, this review focuses exclusively on the proteomic experiments presented in the manuscript. Overall, the proteomic studies are well designed, appear to have been conducted by experienced proteomics researchers, and are analysed using appropriate cut-off criteria. Please see my specific comments below.\nMajor comments\n1. The authors state that their proteomic data have been deposited to the ProteomeXchange repository under the dataset identifier PXD059232, and that the dataset should be publicly available. However, I was unable to locate the dataset using this identifier, or by searching relevant keywords or author names. I urge the authors to verify the accuracy of the dataset ID and ensure that the data are publicly accessible at the time of review. If the dataset is still private, reviewer credentials should be provided to allow proper evaluation of the proteomic data.\nResponse: We apologize for the problem. There were some technical issues, which have been resolved. We have confirmed that the data is accessible by using the accession number PXD059232 in https://proteomecentral.proteomexchange.org.\nMinor comments\n1. I commend the authors for clearly distinguishing between proteins that are proximal to the bait protein and those that directly interact with it — a distinction that is often overlooked in proximity labelling studies and in many manuscripts I have reviewed previously.\nResponse: We thank the reviewer for the encouragement.\n2. The authors should provide more detailed information on how the proteomic data were processed, including steps such as the handling of missing values, removal of potential contaminants, normalization procedures, and any filtering criteria applied. Additionally, specify which software tools or statistical packages were used for the analysis.\nResponse: We have rewritten the Methods section about mass spectrometry data analysis including these details: “The acquired .RAW files were loaded into DIA-NN (version 1.9) for spectra-library-free search against the all SwissProt protein sequences of mouse and HSV-1 proteomes. Trypsin with up to two missed cleavages was set. Carbamidomethyl (C) was set as fixed modification. Oxidation (M) and acetyl (protein N-term) were set as variable modifications. Mass tolerance of 7 ppm and 15 ppm were set for MS and MS2 spectra, respectively. Protein level 1% FDR was set to filter the result. Potential contaminant proteins were identified by DIA-NN build-in database and were removed from subsequent analysis. For quantitative analysis, missing values were filled with a MS intensity of 1000 (noise level), and all intensity values were log2-transformed. Proteins enriched in the TurboID samples were then selected using a t-test (P value < 0.05) in Excel and required a fold-change of at least 2 (corresponding to an average intergroup difference of ≥ 1 on the log2 scale).”\nHowever, we did not perform normalization in this case. For whole proteome experiments, where it is assumed that the expression levels of the vast majority of proteins remain unchanged, we usually perform median or global normalization. However, for experiments like proximity labeling or co-IP-MS, the differences of identified proteins and their intensities among different groups are very significant, so in such cases, we generally do not force global normalization，which can artificially reduce the inter-group differences, leading to false negative results.\n3. The authors may consider validating the nuclear localisation of the TurboID-tagged FOX constructs using IF to confirm their proper subcellular distribution, and include these data in Fig. S8.\nResponse: We have done this experiment and the data are now included in Fig. S8B. Basically, all TurboID-FOX fusion proteins are in the nucleus but unfused TurboID is in the cytoplasm.\n4. Fig. 6: It would be helpful to include a Venn diagram in to highlight the 11 proteins enriched by more than 2 log2 in all pairwise comparisons. This addition would improve the overall flow of the figure and help readers more easily understand the rationale behind validating the interaction with CBP in panels C–E, as well as the selection of genes shown in panel F for silencing experiments.\nResponse: The Venn diagram was already in Fig. S8C in the original manuscript. Now we have moved it to Fig. 6B and moved the original Fig. 6B (about KEGG analysis) to Fig. S8D.\n5. Fig. 7A: Would MAX, KIN, GTF2A1, SIN3A, and ARID4B still be considered hits if a more stringent cut-off of log₂ fold-change > 2 were applied, as was done for the FOXF1 dataset? This may be particularly relevant given that MAX and SIN3A were not successfully validated in the co-IP assays, which—beyond the possibility of a temporal or indirect interaction, as the authors suggest—could also indicate that the interactions are generally weaker. Along these lines, were the authors able to validate, via co-IP, the interaction between FOXK1 and any other protein that showed a similar level of enrichment as MAX or SIN3A? Generally, increasing the amount of protein input in co-IP assays can improve the likelihood of successful detection, which might be worth considering if validation has been challenging.\nResponse: Applying a cut-off of log₂ fold-change > 2 gave rise to MAX, KIN and GTF2A1 only. We decided to use log₂ fold-change > 1 because we think SIN3A is a very interesting candidate and its interaction with FOXK1 has been previously reported. We did not validate the interaction between FOXK1 and other proteins because they do not show effects on viral replication. To try to address this comment, we tried more experiments to validate the interaction between FOXK1 and SIN3A given that previous studies reported FOXK1-SIN3A interactions in yeast two-hybrid and co-immunoprecipitation assays. We have attempted increasing the input protein quantity in 293T (Fig A) and Neuro-2a cells (Fig B), but still could not detect the interactions. We also tried leaving out benzonase (we’ve always added it for detecting DNA-independent interactions), but the result was also negative (Fig C). Therefore, we believe that these interactions were transient, indirect or weak despite that close proximity. We do not see the need to include these additional negative data, but we have revised that sentence: “It is possible that these protein-protein interactions are transient, weak and/or indirect, so that they did not persist during our co-immunoprecipitation assay.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed almost all points raised by the reviewers. For my part, concerning the overexpression of FOX proteins and binding of FOX proteins to the host/virus genome the authors show additional controls and experiments with endogenous protein levels that confirmed previous experiments and strengthen their findings. I still don't understand the exact mechanism given different binding patterns in the host and viral genome and differences in the chromatin between viral and host genomes, e.g. that differences in chromatin accessibility are independent on FOX binding sites. However, the authors convincingly show FOXF1 dependent recruitment of the histone acetyltransferase p300/CBP to the viral genome, resulting in changes in chromatin accessibility and histone acetylation and conversely that the repressive effect of FOXK1 is also dependent on histone acetylation. In my opinion, the quality of the manuscript improved and I think that the paper is well-suited for publication in Nature Communications.", "reviewer_2": "The authors have performed a comprehensive revision of their manuscript, which investigates how FOX transcription factors (specifically FOXF1 and FOXK1) act as epigenetic regulators of the HSV-1 lytic-latent cycle. The revision has greatly strengthened the manuscript, particularly in its transition from broad observations across the FOX family to a more focused exploration of specific activator and repressor proteins. The addition of experiments using wild-type (WT) virus and endogenous protein assays significantly strengthens the biological relevance of the findings, such as ChIP-Seq of endogenous FOXF1 in 293T cells.\nThe authors have addressed my previous concerns with new data, data accessibility, figure/table editing, and text revisions. The study provides a significant advancement in our understanding of how the host epigenetic machinery is leveraged to control the herpesvirus life cycle. I have no additional comments or concerns except to point out that in the methods for immunofluorescence, the new text should have a space between \"antibody\" and the parentheses.", "reviewer_3": "All of my comments have been addressed and I believe the manuscript is now ready for publication."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe authors addressed almost all points raised by the reviewers. For my part, concerning the overexpression of FOX proteins and binding of FOX proteins to the host/virus genome the authors show additional controls and experiments with endogenous protein levels that confirmed previous experiments and strengthen their findings. I still don't understand the exact mechanism given different binding patterns in the host and viral genome and differences in the chromatin between viral and host genomes, e.g. that differences in chromatin accessibility are independent on FOX binding sites. However, the authors convincingly show FOXF1 dependent recruitment of the histone acetyltransferase p300/CBP to the viral genome, resulting in changes in chromatin accessibility and histone acetylation and conversely that the repressive effect of FOXK1 is also dependent on histone acetylation. In my opinion, the quality of the manuscript improved and I think that the paper is well-suited for publication in Nature Communications.\nResponse：Thanks for recognizing the quality of the manuscript. We will continue to work on the exact mechanism.\nReviewer #2 (Remarks to the Author):\nThe authors have performed a comprehensive revision of their manuscript, which investigates how FOX transcription factors (specifically FOXF1 and FOXK1) act as epigenetic regulators of the HSV-1 lytic-latent cycle. The revision has greatly strengthened the manuscript, particularly in its transition from broad observations across the FOX family to a more focused exploration of specific activator and repressor proteins. The addition of experiments using wild-type (WT) virus and endogenous protein assays significantly strengthens the biological relevance of the findings, such as ChIP-Seq of endogenous FOXF1 in 293T cells.\nThe authors have addressed my previous concerns with new data, data accessibility, figure/table editing, and text revisions. The study provides a significant advancement in our understanding of how the host epigenetic machinery is leveraged to control the herpesvirus life cycle. I have no additional comments or concerns except to point out that in the methods for immunofluorescence, the new text should have a space between \"antibody\" and the parentheses.\nResponse：Thanks for the positive comments. The space has been added.\nReviewer #3 (Remarks to the Author):\nAll of my comments have been addressed and I believe the manuscript is now ready for publication.\nResponse：Thanks for the positive comment."}]

Decision letter