How transcription factors (TFs) down-regulate gene expression remains ill-understood, especially when they bind to multiple enhancers contacting the same gene promoter. In particular, it is not known whether they exert similar or significantly different molecular effects at these enhancers.TGFB2 gene by the TF Fra-1 in Fra-1-overexpressing cancer cells, as Fra-1 binds to multiple enhancers interacting with the TGFB2 promoter. We show that Fra-1 does not repress TGFB2 transcription via reducing RNA Pol II recruitment at the gene promoter but by decreasing the formation of its transcription-initiating form. This is associated with complex long-range chromatin interactions implicating multiple molecularly and functionally heterogeneous Fra-1-bound transcriptional enhancers distal to the TGFB2 transcriptional start site. In particular, the latter display differential requirements upon the presence and the activity of the lysine acetyltransferase p300/CBP. Furthermore, the final transcriptional output of the TGFB2 gene seems to depend on a balance between the positive and negative effects of Fra-1 at these enhancers.To address this issue, we used a particularly well-suited study model consisting of the down-regulation of the TGFB2 gene expression. This has consequences for our general understanding of the functioning of the ubiquitous transcriptional complex AP-1, of which Fra-1 is the most documented component for prooncogenic activities. In addition, it raises the general question of the heterogeneity of the molecular functions of TFs binding to different enhancers regulating the same gene.Our work unveils complex molecular mechanisms underlying the repressive actions of Fra-1 on The online version contains supplementary material available at 10.1186/s13578-023-01077-5. TGFB2 gene down-regulation by the Fra-1 TF in Fra-1-overexpressing cancer cells, as Fra-1 binds to the numerous enhancers contacting the TGFB2 gene promoter.Gene up-regulation by transcription factors (TFs) has amply been studied. In contrast, how TFs down-regulate gene expression remains largely ill-understood. This question is all the more complex to solve that most gene promoters are now known to interact with multiple enhancers –5 and thFOSL1 gene) is a component of the ubiquitous dimeric transcriptional complex AP-1. The latter is predominantly made up of combinations of members of the Fos-  and Jun  multigene families but is also contributed to a lesser extent by members of the ATF and MAF multigene families [Fra-1  from TSSs than with gene promoters [trans-activating certain, but not all, of its target genes. The fine mechanistical consequences of these cooperations were however not studied.Fra-1 is a bZIP TF, i.e. a TF harboring a basic domain (b) enabling binding to DNA, adjacent to a leucine zipper (ZIP) permitting dimerization with other TFs . To exerromoters , 36–41. romoters –44 or NFromoters , and a fromoters , 47, thePLAU (encoding the urokinase plasminogen activator) [HMGA1  [HMGA1 but not at that of PLAU where it, instead, promotes the formation of the transcription-elongating form of Pol II.To better understand the molecular mechanisms underlying Fra-1-mediated transcription regulation in TNBCs, we turned to the model cell line MDA-MB-231 , 47, 48 PLAU and HMGA1, belonged to the minor category of those for which Fra-1-bound AP-1/TRE- and/or CRE elements reside in the proximity of the genes' TSSs (i.e. a few kbs upstream or downstream) [TGFB2 gene  ). In consiveness ), as on e’s TSS  as compared to cells transfected with a control siRNA (siCTL) .Thus, our data indicate that Fra-1 is key for transcriptional down-regulation of TGFB2, we next asked whether it could affect Pol II recruitment at this gene. This was achieved by ChIP-qPCR assay of total Pol II at various places on the TGFB2 locus, as indicated in Fig. TGFB2. We then addressed whether Fra-1 could affect the formation of the transcription-initiating form of Pol II. The latter is phosphorylated on Serine 5 (Pol II-PSer5) of the heptad repeats of the carboxyl terminal domain (CTD) of Pol II catalytic subunit (RPB1) and is usually found enriched around TSSs and at the beginning of genes but little further on gene bodies [TGFB2 gene after RNAi-mediated depletion of Fra-1  , i.e. the cell type TNBCs are derived from  where the sequencing background is always very high due to the intrinsic design of the NG Capture-C technology (see details on technology in reference 55). One, residing at position—46 kb, lies at the TAD border and harbors a CTCF-binding site. The other, located at position + 32 kb, corresponds to a TGFB2 enhancer identified by the FANTOM5 consortium [To gain a better insight into how Fra-1 down-regulates  results . NG Capt results . It allo results . These d package . These Pnsortium . Moreovensortium , 57. Of TGFB2 TAD  was not affected upon RNAi-mediated knockdown of Fra-1, as assayed by Affymetrix array-based transcriptomic analysis , as they were not represented on the Affymetrix arrays we used , Pol II and CTCF, as well as with ATAC-seq data serving to identify open chromatin regions with potential regulatory roles  that bind both Fra-1 and p300/CBP, (iii) PIRs − 46, + 1041, + 1222 and + 1316 most probably bear elements only involved in the 3D structuring of the TGFB2 gene chromatin, as they show binding of CTCF but neither signs of open chromatin conformation nor of histone marks featuring active enhancers or promoters, (iv) PIR + 836, which is not bound by Fra-1, corresponds to an annotated promoter for both LYPLAL1-ID and LYPLAL1 genes. It might also carry an Epromoter for TGFB2, i.e. a gene promoter showing enhancer activity on another gene [TGFB2 than the latter, (vi) PIR + 314 contains 2 Fra-1-bound enhancers at + 314 kb and + 315 kb, (vii) PIR + 729 contains a CTCF-bound element residing in a closed chromatin region at position + 722 kb that is likely to constitute a structural element, as well as a Fra-1- and p300/CBP-bound enhancer at + 744 kb that most probably constitutes a TGFB2 enhancer and (viii) PIR + 1449 contains a Fra-1-bound enhancer showing a low p300/CBP signal that is located at + 1426 kb, as well as two domains binding CTCF at positions + 1460 kb and + 1468 kb that most probably correspond to chromatin-organizing elements. Finally, it must be noted that the CTCF-bound elements located within PIRs − 46 and + 1449 are located at the TGFB2 TAD borders and might be important for the individualization of this TAD.Our results Fig. : Data S3her gene , (v) PIRTGFB2 PIRs (see TableTGFB2 promoter interacts with 12 remote Fra-1-bound elements showing marks of active enhancers (and possibly an Epromoter) strongly supports, not only the idea of TGFB2 being a direct transcriptional target of Fra-1, but also that of a complex chromatin 3D organization/dynamics underlying TGFB2 transcriptional regulation. This notion is strengthened by the observation that a number of CTCF-binding elements lie between certain enhancers  show a strong heterogeneity in Fra-1-, p300/CBP-, H3K4me1-, H3K4me3-, H3K27ac- and Pol II ChIP-seq- and ATAC-seq signal intensities  in the vicinity of Fra-1-binding sites , the expression of which most often correlates with both enhancer activity and transcription of target genes –61. To a31 cells , as the TGFB2 gene and whose transcription does not interfere with that of other transcription units, the analysis of the + 32 kb enhancer deserved a specific analysis to distinguish between production of the + 32 kb eRNAs and that of the TGFB2 pre-mRNA due to its intronic localization. Thus, + 32 kb eRNA production was deduced from the difference in RNAs amounts between position + 32 kb and positions + 21.6 kb and + 55.8 kb, which correspond to 2 sites within intron 2 harboring no regulatory elements  and situated at a distance sufficient to eliminate possible overlaps between pre-mRNA- and eRNA synthesis  located in the proximity 90–400 bp) of the Fra-1-binding sites  we identified in our previous work in the whole genome of MDA-MB-231 cells based on the analysis of histone modifications . The data presented in Fig. TGFB2 enhancers  by Fra-1 and most probably relies on reconfiguration of the macromolecular complexes responsible for TGFB2 expression rather than on changes in enhancer accessibility (see Discussion).We next asked whether increased eRNA production upon RNAi-mediated knockdown of Fra-1 could be associated with local changes in chromatin opening at Fra-1-bound TGFB2 transcription. p300 and CBP are often studied together due to their close structural and functional relationships and in this case termed p300/CBP [TGFB2 mRNA  a strong reduction in TGFB2 mRNA level, indicating that p300/CBP KAT activity is required for TGFB2 expression  where they significantly increased  p300/CBP recruitment at only one fourth of them.Next, we tested whether Fra-1 could operate via altering p300/CBP recruitment at the TGFB2 expression involves p300/CBP enzymatic activity for the regulation of TGFB2 enhancers.Finally, we addressed whether the Fra-1-induced limitation of TGFB2 expression in the presence of Fra-1 as shown in Fig. TGFB2 upon Fra-1 down-regulation. To this aim, MDA-MB-231 cells were transfected with either siFra-1 or siCTL for 72 h with A485 being added 56 h after the beginning of the transfection to inhibit p300/CBP enzymatic activity . Addition of the drug abrogated the increase in TGFB2 mRNA steady-state level triggered by Fra-1 depletion .For 7 enhancers  where eRNA production was increased upon simple Fra-1 down-regulation  [Our work indicates that promoter  and thatTD Ser2)  in MDA-MTGFB2 promoter interacts with 10 PIRs that contain at least one Fra-1-bound enhancer. Moreover, we also report that the formation of the chromatin loops between the TGFB2 promoter and its enhancers is not modulated by Fra-1. It is therefore most likely that Fra-1 exploits pre-exiting chromatin loops to prevent the formation of the transcription-initiating form of Pol II and exert its repressive effects. Importantly, our data indicate that the decrease in phosphorylation of Pol II CTD Ser5 is not linked to a decrease in CDK7 recruitment that would be mediated by Fra-1. Coherently with this observation, recent proteomic studies conducted in another TNBC cell line (BT549) [Here, we have shown that the  (BT549)  and in h (BT549)  did not TGFB2 expression, our run-on- and pre-mRNA level assays suggest that there could also exist a post-transcriptional one. This possibility is all the more to be taken into consideration that Fra-1 has been shown to regulate the expression of various miRNAs that may affect the stability of other RNAs in other cell contexts [Besides the transcriptional contribution of Fra-1 to the down-regulation of contexts , 72.TGFB2 gene regulation depends on complex chromatin 3D organization and/or dynamics within the TGFB2 TAD. Thus, the TGFB2 promoter interacts with 15 PIRs scattered from one to the other extremity of this TAD with most of them being located far away from the TGFB2 gene's TSS (i.e. from 116 kb up to 1450 kb for 13 of them). Moreover, amongst the PIRs, (i) 8 harbor Fra-1-bound active enhancers, (ii) 4 harbor CTCF-bound candidate chromatin 3D structure-organizing domains, (iii) 2 harbor at least one CTCF-bound candidate chromatin 3D structure-organizing domain plus a Fra-1-bound active enhancer, and (iv) one bears a possible Epromoter. Finally, the PIRs are not clustered according to their types  but are rather tangled, especially in the most distal moiety of the TGFB2 TAD. In this context, it is interesting to note that several genes are located between the TGFB2 PIRs or overlap certain of them but are not regulated by Fra-1, which suggests the existence of regulation mechanisms capable of discriminating between them and TGFB2. While these observations point to a complex regulatory scheme in this particular region of the genome, they unfortunately do not allow to propose a precise overall functioning for it, as the Hi-C-, NG Capture-C- and ChIP-seq techniques used in our study give averaged frequencies in chromatin interactions or markings occurring in a whole cell population but do not provide any insight into their stability, dynamics, simultaneity or succession over time in individual cells. Further fine molecular studies will therefore be necessary to understand why Fra-1 regulates the TGFB2 gene but not the other genes residing within the TGFB2 TAD. Whatever these mechanisms might be, our NG Capture-C experiments indicate that they do not involve gross Fra-1-dependent chromatin rearrangements at the TGFB2 locus.Several of our data suggest that TGFB2 by binding exclusively to active enhancers that all bear at least one AP-1-binding site. We consider this observation important for two reasons. On one side, it excludes the possibility that Fra-1 exerts its repressive effect on TGFB2 expression via binding to silencer elements [TGFB2 transcription. On the other side, it strongly supports the idea that Fra-1 attenuates the activity of multiple enhancers collaborating to TGFB2 expression through direct binding to them. As already mentioned, this negative effect of Fra-1 cannot be explained by induction of major changes in the chromatin 3D organization of the TGFB2 locus and our ATAC-seq data indicate that they do not involve marked changes in local chromatin accessibility either.Our data indicate that Fra-1 represses elements  distinctTGFB2 enhancers or limits the activity of the latter via other mechanisms. The first possibility is challenging to address, as silencers still constitute particularly ill-understood genomic entities [trans-activating- and -trans-repressing activities, the contributions of which may vary according to the cell and/or signaling context [Further work will therefore have to establish whether Fra-1 binds to discrete autonomous silencer elements embedded within Fra-1-bound entities , and the context .TGFB2 enhancers, but also suggests that the final TGFB2 transcriptional output may result from a balance between repressive, neutral and positive actions of Fra-1 at these 3 categories of enhancers, respectively. Thirdly, our data also strongly point to heterogeneity amongst the Fra-1-bound TGFB2 enhancers with respect to their dependence upon p300/CBP presence and KAT activity for eRNA production, as well as to different implications of Fra-1 at these regulatory elements. Thus, we show that the activity of one of the 12 Fra-1-bound enhancers is independent of the presence of p300/CBP even though p300/CBP binds to it. Moreover, among the 11 p300/CBP-dependent enhancers, only 8 of them require the KAT activity, suggesting that p300/CBP may only play a structural/organizational role at the remaining other 3 elements. The latter possibility was not unexpected, as enzymatic activity-independent scaffolding properties have regularly been reported for p300/CBP [TGFB2 enhancers in a general context where the biochemical, epigenetic, operational and functional definition of enhancers is a matter of intense debate [Interestingly, several of our observations suggest that the role of Fra-1 at the regulatory elements it binds to is likely pleiotropic. Firstly, the various Fra-1-bound active enhancers show strong molecular heterogeneity, as indicated by marked differences in Fra-1-, p300/CBP-, H3K4me1-, H3K4me3-, H3K27ac- and Pol II ChIP-seq- and ATAC-seq signal intensities. Secondly, assessing enhancer activity on the basis of their ability to produce eRNAs showed that, among the 12 Fra-1-bound candidate active enhancers, 9 of them have their activity repressed by Fra-1, 2 were unaffected by Fra-1 and 1 was even stimulated by Fra-1. This, not only provides additional support to the idea of functional heterogeneity for the Fra-1-bound p300/CBP –68. FinaTGFB2 enhancers in MDA-MB-231 cells deserves additional comments. It has been reported that the RNAs transcribed proximally to p300/CBP-binding sites in chromatin (at the forefront of which are eRNAs) stimulate the KAT activity of p300/CBP, leading to enhanced acetylation of histones and, thereby, increased transcription of target genes [TGFB2 enhancers with KAT activity being necessary for only 8 of them suggests that this model is not universal and also raises the question of the molecular role of p300/CBP in the activity of these enhancers. In particular, for the 8 enhancers where KAT activity is required for eRNA production, it will be interesting to identify which of the many possible p300/CBP non-histone substrates [TGFB2 enhancer activity and gene transcription. Additionally, it will also be interesting to elucidate how Fra-1 limits eRNA production at 2 of these enhancers where it reduces the recruitment of p300/CBP, as well as at those where it decreases KAT activity. In the latter case, indirect mechanisms will have to be considered as, even though Fra-1 and p300 have been shown co-immunoprecipitable in cell transfection experiments in one study [The role of p300/CBP at the et genes . Our obsbstrates –68, 75 ine study , the prone study , 70.TGFB2 enhancers function collectively through the formation of transcriptional hubs whose importance is increasingly documented in the literature [TGFB2 promoter and the various chromatin regions it can interact with in a whole cell population. However, it gives no indication on the number of PIRs that are in contact with other PIRs and/or with the TGFB2 promoter in each cell at a given time, as well as on whether these interactions are stochastic or sequentially ordered. Moreover, if our eRNA assays suggest that all Fra-1-bound TGFB2 enhancers actually possess some transactivation potential, they do not indicate whether certain of them are more potent than others at transactivating the TGFB2 gene (in the presence or in the absence of Fra-1). Future work will therefore have to characterize the individual contributions of each one of these regulatory elements to TGFB2 transcriptional regulation. In particular, it will have to decipher their collective versus redundant functioning, as well as to establish whether they come into play concurrently or dependently on structural and/or sequential constrains.Another question raised by our work is whether the Fra-1-bound terature , 76, incterature , 39. As TGFB2 enhancers could converge to reduce transcription initiation at the TGFB2 promoter. There is accumulating evidence that liquid–liquid phase separation-induced biomolecular condensates incorporating gene promoters and enhancers are essential for transcription to occur and that gene regulation may involve controlling their assembly/dissolution [TGFB2 enhancers would perturb the crowding of the other TFs at these enhancers and, thereby, would lead to rearrangements within these condensates. As RNAs [TGFB2 enhancers would also affect the organization of TGFB2 transcriptional condensates. Interestingly, it has recently been proposed that, rather than tight physical contacts between promoters and their cognate enhancers, sufficient proximity would be sufficient for molecular communication to occur between these gene regulatory elements [TGFB2 in MDA-MB-231 cells, it would be important to identify the molecules trafficking from its enhancers to its promoter within transcriptional condensates and to elucidate whether and how Fra-1 alters their movements.A next important issue concerns how the diverse mechanisms used by Fra-1 at the various Fra-1-bound solution , 78. As  As RNAs , 78, and As RNAs , produceelements . In thisTGFB2. This issue is, in fact, complex and we cannot exclude that multiple partners come into play at the various TFGB2 candidate enhancers for various reasons: (i) even though proteomic studies have shown that Fra-1 can heterodimerize with the 3 members of the Jun family in TNBC cells [TGFB2 upon simple RNAi-mediated down-regulation of Fra-1 indicates that Fra-1 plays a crucial role in the concerned AP-1 dimers. This observation is consistent with the fact that Fra-1 was described to exert a prominent part, not only in the transcriptional activity of the AP-1 dimers where it is involved, but also in AP-1-binding site selection [As Fra-1 does not bind to DNA as an homodimer , 8, anotBC cells , it mustBC cells , 8, (ii)BC cells , (iii) ABC cells  and (iv)election .TGFB2 expression outcome results from an equilibrium between enhancers negatively or positively regulated by Fra-1. This highlights the important notion that the molecular actions of Fra-1 bound to multiple enhancers for down-regulation of the same gene can be heterogeneous. As most gene promoters interact with several enhancers [TGFB2 may also apply to other Fra-1-down-regulated genes, as well as to other TFs capable of binding several enhancers of the same gene. Among the latter, the other Fos and Jun components of the ubiquitous AP-1 transcription complex must be considered in priority, as recent genome-wide transcriptomic and genomic investigations have shown that several Fos and Jun family proteins can nearly equivalently up- or down-regulate gene expression in the same cellular context [We report here that Fra-1 can down-regulate one of its target genes via binding to multiple enhancers showing different molecular and functional features. Interestingly, some of its actions are even antagonistic since the final nhancers , we feel context . Future 2-containing atmosphere. They were routinely tested for the absence of mycoplasma contamination.MDA-MB-231 cells were obtained from the ATCC. Cells were cultured at 37 °C in DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin (100 µg/ml each) in a humidified, 5% COsiRNAs were transfected at a final concentration of 5 nM for 72 h using INTERFERin (Polyplus) according to the supplier’s specifications. For RNAi-mediated depletion of Fra-1, a pool of 3 siRNAs (siFra-1) was used as described in Tolza et al.  to proviThe anti-Fra-1- (sc-376148X and sc-28310X),—Pol II- (sc-55492X) and -GAPDH (sc-25778) antibodies were from Santa Cruz Biotechnology, as well as control IgG (sc-2025). The anti-p300-  -CBP  and -CDK7 (#2916) antibodies were from Cell Signaling Technology. The anti-Pol II-PSer5 antibody  was from Biolegend. The mouse anti-IgM antibody (04–6800) was from Invitrogen. The anti-H3K36me3 antibody (61101) was from Active Motif and the anti-H3 antibody (05–928) from Millipore. The anti-H3K27ac (Ab4729) and -p300/CBP antibodies (Ab14984) were from Abcam. The anti-rabbit (sc-2313) and anti-mouse (sc-2954) horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology.Immunoblotting experiments were performed as described previously  and protCellular p300/CBP HAT activity was inhibited by adjusting the cell culture medium to 3 nM of A485  using a stock solution prepared in DMSO at a concentration of 5 mM. In control samples, an equivalent volume of DMSO was added to the culture medium.Transcriptional run-ons, RNA extraction, reverse transcription and qPCR were performed as described previously . For RT-TGFB2 gene are given in Additional file ChIP-qPCR were performed as described previously . The seq2 3 mM and Igepal CA-630 0.1%). Lysed cells were centrifuged at 500 g at 4 °C for 10 min and the pellets were then resuspended in 100 µl of the Transposase reaction mix  to which 5 µl Tn5 transposase  and 45 µl of Nuclease-free water were added before an incubation of 30 min at 37 °C. DNA was then purified using MinElute Qiagen columns (Qiagen cat. n° 28004) according to the manufacturer’s instructions. At this stage, libraries were indexed using the Phusion® High-Fidelity DNA Polymerase (NEB cat. n° M0530). Sequencing was performed using NovaSeq 6000 (Illumina) in paired-end (50 nt). Reads were aligned to the hg19 Homo sapiens genome using Bowtie2 and, then, processed using the R package PASHA [ATAC-seq was performed according to Buenrostro et al. . Brieflyge PASHA . The thr6 MDA-MB-231 cells were transfected with either siCTL or siFra-1. 72 h later, cells were fixed at 25 °C for 7 min using 1% paraformaldehyde (Euromedex). Fixation was stopped by addition of glycine 2.5 mM. Cells were then resuspended in 1 ml of Cell Lysis Buffer  and let on ice for 10 min. Nuclei were recovered by mild centrifugation at 4 °C and lysed in 250 µl of Nuclei Lysis Buffer  at 4 °C for 2 h. They were then sonicated at 4 °C using 10–15 cycles (up to clarification) of the Bioruptor Pico device from Diagenode. After centrifugation at 13,000 g at 4 °C for 15 min, samples were split into two 100 µl aliquots. One of the aliquots, which constituted the total DNA reference, was decrosslinked at 65 °C for 6 h after successive RNAse A and Proteinase K treatments and DNA was purified by phenol/chloroform extraction following the procedures described in ref. [versus total DNA amounts, were also performed as described in ref. [TGFB2 locus are given in Additional file Formaldehyde-assisted isolation of regulatory elements (FAIRE) was performed as described by Rodriguez-Gil et al. . Briefly in ref. . The oth in ref. . PrimersNG Capture-C experiments and peak calling analyses are described in detail in Bejjani et al. . Peak caData were reported as the means ± SEM and analyzed by unpaired two-tailed Student’s t-test or ANOVA depending on the number of samples being compared  with the GraphPad Prism5 software. P-values were considered as significant when *, ≤ 0.05 and highly significant when **, ≤ 0.01; ***, ≤ 0.005; ****, ≤ 0.001).Additional file 1: Data S1. Transcriptional repression of TGFB2 by Fra-1. The experiments presented in (A), (B) and (C) are the same as those presented in Figure 1B, -C and -D, except that other amplicons were used. The sequences of the oligonucleotides used in RT-qPCR assays are given in Additional file RPS26 gene and ChIP-qPCR. The upper panel shows the amplicon positions used in ChIP-qPCR experiments, indicated in kb from the TSS. The middle panel shows ChIP-qPCR analysis of total Pol II on the RPS26 gene (n=4) and the lower panel shows ChIP-qPCR analysis of Pol II-PSer5 on the RPS26 gene (n=4). All values were normalized to that of amplicon − 0.8 kb under control condition arbitrarily set to 1. The arrows indicate the RPS26 TSS. All experiments were carried out using MDA-MB-231 cells transfected with either siFra-1 (green boxes) or siCTL (violet boxes) for 72 hours. (E) Expression levels of the genes located in the TGFB2 TAD upon Fra-1 down-regulation. The transcriptome regulated by Fra-1 was formerly identified using Affymetrix GeneChip Human Gene 2.0 ST arrays [versus siCTL conditions showed that the mRNA steady-state levels of RRP15, LYPLAL1 and LINC02869 were not affected upon Fra-1 down-regulation. Expression of LYPLAL1-DT, LYPLAL1-AS1, ZC3H11B and LINC01710 could not be analyzed (NA), as the corresponding probes were absent from the Affymetrix arrays used.T arrays . Gene exAdditional file 2: Data S2. Positions of AP-1 motifs under the Fra-1 ChIP-seq peaks at the Fra-1-bound TGFB2 enhancers. The positions of the Fra-1-bound enhancers are given with respect to the TGFB2 TSS. AP-1 motif positions are given both with respect to the TGFFB2 TSS and in the reference genome Hg19. The sequences of the AP-1- and AP-1-related motifs are indicated in black and blue, respectively.Additional file 3: Data S3. Molecular features of the TGFB2 PIRs. ChIP-seq data for H3K4me3, H3K27ac, H3K4me1, Pol II, CTCF, p300/CBP, Fra-1, as well as ATAC-seq data, were used to, not only classify the 15 TGFB2 PIRs defined by NG Capture-C in MDA-MB-231 cells, but also show their molecular heterogeneity. The first category contains the PIRs harboring only a Fra-1-bound candidate active enhancer . These are all characterized by overlapping Fra-1- and ATAC-seq signals and absence of CTCF and H3K4me3 signals. However, they show some heterogeneity in H3K4me1, H3K27ac, Pol II, and p300/CBP signal intensities. The second category contains PIRs marked by CTCF but not by Fra-1 . At variance with Fra-1-bound active enhancers, CTCF-bound elements are not associated with ATAC-seq signals. The third category corresponds to a PIR (PIR +836) that is not bound by Fra-1 but bears an active gene promoter  with possible enhancer function for TGFB2 (Epromoter). The fourth category contains PIRs (PIR +116 and +314) that carry two Fra-1-bound active enhancers specified by overlapping Fra-1- and ATAC-seq signals. Like in the first category of PIRs, the candidate enhancers are not marked by CTCF and H3K4me3 and show some heterogeneity in H3K4me1, H3K27ac, Pol II, and p300/CBP signals. Finally, the fifth category (PIRs +729 and +1449) contains PIRs bearing one Fra-1-bound active enhancer, as well as at least one CTCF-binding elements.Additional file 4: Data S4. Bidirectional transcription at the TGFB2 locus in MDA-MB-231 cells. Nascent RNA production was assessed by others in MDA-MB-231 cells using Gro-seq [TGFB2 locus NG Capture-C data (lower panel), as well as Fra-1 ChIP-seq data obtained in Bejjani et al. [TGFB2 gene transcription and orange ones antisense (-) transcription. Gro-seq . The pubi et al. . Purple Additional file 5: Data S5. assay of eRNAs at the +32 Fra-1-bound PIR. (A) Molecular features of intron 2 from position 218,536,777 to 218,577,587. The Fra-1-binding site located at +32 kb resides in a region of open chromatin  also recruiting p300/CBP and marked by H3K4me1 and H3K27ac but not by CTCF. In contrast, there is no sign of open chromatin at positions +21.6 kb and +55.8 kb, as well as of marks specifying active enhancers. The amplicons used to assay eRNA versus TGFB2 pre-mRNA at position +32 kb are indicated by black boxes. (B) RT-qPCR assay of eRNA versus TGFB2 pre-mRNA at position +32 kb in MDA-MB-231 cells. RNA levels at positions +21.6-, 32- and 55.8 kb from the TGFB2 TSS were quantified by RT-qPCR. RNA amplified at position +21.6- and +55.8 kb correspond to pre-mRNA, whereas RNA amplified at position +32 kb corresponds to pre-mRNA+eRNA. The data are the results of 6 independent experiments. Relative RNA abundances at the 3 positions were calculated by the ∆∆CT method, amplicon +32 being taken as the reference. Primer sequences for qPCR amplification are given in Additional file Additional file 6: Data S6. ATAC-seq signal ratios obtained in the absence and presence of Fra-1 at the Fra-1-bound TGFB2 enhancers. Three independent ATAC-seq experiments were carried out. (A) The ratios of ATAC-seq signal intensities in siFra-1 versus siCTL conditions are given for the 12 Fra-1-bound enhancers, as well as for the TGFB2 promoter. The start and end positions of the ATAC-seq peaks are given as well as the peak positions relative to the TGFB2 TSS. (B) The ratios of ATAC-seq signal intensities in siFra-1 versus siCTL conditions are given for two peaks (peak A on chromosome 16 and peak B on chromosome 15) for which the ATAC-seq signals were found increased or decreased (FC≥±1.5), respectively. The start and end position of peaks are given in the table. (C) The ratios of p300/CBP signal intensities in siFra-1 versus siCTL conditions at the Fra-1-bound TGFB2 enhancers showing a FC ≥ ±1.5 are indicated. ChIP-seq data obtained in MDA-MB-231 cells were previously presented in Bejjani et al. [TGFB2 TSS, as well as the start and end positions of the p300/CBP peaks are given.i et al. . The peaAdditional file 7. Additional Tables.


Irx5−/− mice have delayed anagen onset, with increased DNA damage and diminished HFSC proliferation. Open chromatin regions form near cell cycle progression and DNA damage repair genes in Irx5−/− HFSCs. DNA damage repair factor BRCA1 is an IRX5 downstream target. Inhibition of FGF kinase signaling partially rescues the anagen delay in Irx5−/− mice, suggesting that the Irx5−/− HFSC quiescent phenotype is partly due to failure to suppress Fgf18 expression. Interfollicular epidermal stem cells also show decreased proliferation and increased DNA damage in Irx5−/−mice. Consistent with a role for IRX5 as a promoter of DNA damage repair, we find that IRX genes are upregulated in many cancer types and that there is a correlation between IRX5 and BRCA1 expression in breast cancer.The molecular mechanisms allowing hair follicles to periodically activate their stem cells (HFSCs) are incompletely characterized. Here, we identify the transcription factor IRX5 as a promoter of HFSC activation.  •IRX5 promotes cell cycle progression and DNA damage repair through epigenetic regulation•Irx5−/− mice display delayed anagen due to suppression of Fgf-mediated quiescence•High IRX5 and BRCA1 gene expression is correlated in breast-invasive carcinoma In this article, Chen and colleagues show that IRX5 is an essential transcription factor for hair follicle stem cell activation. Drosophila, Iroquois (IRX) homeodomain transcription factors function early in development to differentiate the dorsal head and mesothorax but also remain active in later stages of development to subdivide these territories  and interfollicular epidermal stem cells (EpiSCs) also accumulate mutations from environmental insults, including ultraviolet radiation. Unsurprisingly, HFSCs and EpiSCs have evolved robust DNA damage repair mechanisms that make them resistant to DNA damage , 2013. LBRCA1 in hair follicle growth are unknown.One factor implicated in DNA damage repair in HFSCs is BRCA1, a tumor suppressor with roles in double-stranded DNA break resection, DNA repair, chromatin remodeling, and cell cycle checkpoint regulation . In the Brca1 and other DNA repair genes as targets of IRX5, consistent with the hair follicle phenotype of Brca1−/− mice , we identified IRX binding sites in the gene regulatory regions of the pro-differentiation Grainyhead like transcription factor 3 (GRHL3), suggesting that IRX could regulate epidermal differentiation . To charntiation A.Figure We then used siRNA knockdown and RNA sequencing (RNA-seq) to characterize the IRX2-, IRX4-, and IRX5-regulated genes in NHEKs. Principal component analysis (PCA) of these data B shows tComparing NHEK-Ds to NHEK-Ps, we identified 289 differentially expressed genes (DEGs) , which clustered into eight unique expression patterns among proliferating control and proliferating siIRX knockdown  and Irx5+/+ (n = 2) mice into single cell suspension. Through RNA-seq, we identified 478 upregulated and 1,513 downregulated genes in the Irx5−/− epidermis  and the Irx5−/− (n = 4) epidermis contained, respectively, 8% and 2% EdU+ cells (p = 0.0002) , whereas 5% of keratinocytes in the Irx5−/− epidermis (n = 3) were γH2AX+ (p = 0.0001)  K-1M. Alspidermis N and 1O; 0.0001) P, sugges 0.0001) A–S3C and 0.0001) D–S3F staIrx5−/− mice at second telogen  showed that Irx5 is expressed in the bulge region and adjacent secondary hair germ at P20 (early anagen)  B and 2C. anagen) C, when Ir matrix B and S4A P5 skin B  and Irx5+/+ (n = 5) littermates at P20 and monitored hair regrowth as an indicator of new growth phase (anagen) initiation  in fur coverage compared with Irx5+/+ littermates. Irx5−/− mice eventually entered anagen on P37, 7 days later than Irx5+/+ littermates. Furthermore, hair growth in Irx5−/− mice initiated in an abnormal pattern, in a region in the center that propagated laterally. Typically, as in Irx5+/+ littermate mice, first anagen starts in the shoulder region and propagates in a cranial-caudal direction , Irx5−/− follicles displayed a thin epithelial column characteristic of anagen initiation. At P32, Irx5+/+ follicles displayed regression of the epithelial column, indicative of catagen, whereas Irx5−/− skin displayed a dramatically thickened dermis containing the lower bulbs of hair follicles, suggesting the follicles had reached a delayed mid-anagen. At P46, Irx5+/+ follicles exhibited telogen morphology, while Irx5−/− follicles were still in late anagen. At P56, Irx5−/− follicles eventually entered telogen.Histology confirmed anagen initiation delay in −/− mice F. By P24Irx5−/− hair follicles, we quantified proliferation by means of 2-h EdU pulses (Irx5−/− hair germ (p = 0.0579) and bulge (p = 0.0418) compared with Irx5+/+ (Irx5−/− hair bulb cells continued to display significantly decreased proliferation (p < 0.0001), with 1% of Irx5−/− hair bulb cells staining EdU+ compared with 30% of Irx5+/+ cells  and P20 (early anagen in WT), and we subjected them to bulk RNA-seq analysis  and Irx5+/+ (n = 3) HFSCs identified 2,259 downregulated and 630 upregulated genes in Irx5−/− HFSCs (Irx5−/− (n = 4) and Irx5+/+ (n = 4) HFSCs identified 1,668 downregulated and 1,464 upregulated genes in Irx5−/− HFSCs  sequencing on fluorescence-activated cell sorting-isolated HFSCs from the back skins of Irx5−/− (n = 2) and Irx5+/+ (n = 2) mice at P20. We identified on average 20,970 peaks in Irx5+/+ and 32,692 peaks in Irx5−/− HFSCs that were proportionally similar in their locations in introns, exons, and promoters   D–5J. BETresponse E, while response H. FurtheA repair F, while function I.Irx5−/− unique open chromatin regions   or 1% DMSO saline (n = 9) every two days from P20 to P46. Irx5+/+ littermates were treated with 1% DMSO saline (n = 7) as additional controls. AZD4547 partially rescued hair growth with a 4-day acceleration of back fur recovery in the AZD4547-treated Irx5−/− group compared with the DMSO-treated Irx5−/− group  (Irx5−/− mice.To determine whether upregulation of FGF could mediate the delayed anagen initiation in  AZD4547 . Irx5−/− 0.0106) H and 6I. 0.0106) I. These Brca1 expression is downregulated in Irx5−/− hair follicles, we used RNA-FISH to analyze its expression at P20, P28, and P32  and P28 (p < 0.001)   B. By P32ollicles B. Analys 0.0001) B. Togeth and P20 D, these Brca1 expression is Irx5 dependent, we characterized the expression of IRX5 and the relationship between IRX5 and BRCA1 in tumorigenesis, using publicly available TCGA (The Cancer Genome Atlas) data. IRX5 is overexpressed in the majority of tumor types, although some tumor types have decreased IRX5 expression  and homologous recombination (HR). While there are no accounts of these DNA damage repair mechanisms in HFSCs, NER and HR are the preferential DNA damage repair pathways of proliferating adult stem cells similar to activated HFSCs  of al layer J. The reull mice  and an eull mice , is unknull mice , suggestFgf18 expression in Irx5−/− HFSCs  indicate that syndrome . This sysyndrome . Recentlsyndrome , a phenoIRX5 may suppress tumorigenesis, multiple studies have found IRX5 to be upregulated in cancers .Requests should be directed to corresponding author Bogi Andersen .− CD34+ CD49f+ HFSCs were isolated for bulk RNA-seq and ATAC-seq.P18 and P20 back skin was dissected and the epidermis isolated. Ly-6A/E2+ was added to the medium to induce differentiation. RNA lysate was collected 72 h after transfection.NHEKs were grown in keratinocyte serum free media supplemented with epidermal growth factor and bovine pituitary extract. 30 nM pooled siRNAs was transfected into semi-confluent monolayers. 12 hours after transfection, 1.8 mM CaLibrary preparation was done with Illumina TrueSeq library preparation kit, and single-end Illumina HiSeq 2500 sequencing was performed.Nuclei were incubated in 50 uL of Tn5 transposition buffer for 30 min at 37°C. Library preparation with Illumina TrueSeq library preparation kit and paired-end Illumina HiSeq 2500 sequencing (80 million reads per sample) was performed at the University of California, Irvine High Throughput Genomics Facility. Motif analysis was conducted with MEME Suit 5.5.0 using predicted IRX5 motif .For immunofluorescence localization of protein, fresh tissue samples were harvested and embedded in optimal cutting temparture (OCT) compound. For RNA-FISH, fresh frozen 10-μM-thick OCT sections were processed and stained using RNAscope Multiplex Fluorescent Detection Kit v1.Details on mouse work, cell culture, RNA and protein detection in tissues, RNA-seq, and ATAC-seq are provided in the J.K.C. and B.A. wrote the manuscript with input from all the authors. J.K.C., J.W., M.V.P., and B.A. designed and interpreted the experiments. J.K.C., L.N., Z.L., and M.T. conducted wet-lab experiments. J.K.C. and J.W. conducted dry-lab experiments. C.-C.H., M.V.P., and B.A. provided resources. B.A. was the overall supervisor.


Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection, but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) when transcription termination is disrupted by the viral ICP27 protein. This is accompanied by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperone FACT is not sufficient to induce dOCRs in ΔICP22 infection but increases dOCR induction in wild-type HSV-1 infection. Interestingly, this is accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone repositioning downstream of genes in the wake of Pol II in HSV-1 infection. Herpes simplex virus 1 (HSV-1) infection disrupts transcription termination by RNA Polymerase II. Here, Djakovic et al. identify the immediate-early protein ICP22 protein of HSV-1 to induce open chromatin downstream of genes upon read-through transcription involving the histone chaperone FACT. Herpes Simplex Virus type 1 (HSV-1) infection induces a profound shut-off of host gene expression by targeting multiple steps of RNA metabolism1. This curtails antiviral host responses and facilitates efficient, productive infection2. Disruption of transcription termination (DoTT) of cellular genes significantly contributes to HSV-1-induced host cell shut-off3. DoTT commonly results in read-through transcription extending for tens to hundreds of thousands of nucleotides beyond poly(A) sites. Read-through transcription originating from disrupted transcription termination often extends into downstream genes (denoted as “read-in” transcription for these genes)3. The viral immediate-early protein ICP27 plays a direct, bimodal role in HSV-1-induced DoTT4. On the one hand, ICP27 interacts with and disrupts the essential mRNA 3′-end processing factor CPSF, thereby inducing the assembly of a dead-end 3′ processing complex that is unable to cleave mRNA 3′ ends. On the other hand, ICP27 acts as a sequence-dependent activator of mRNA 3′ processing by binding to viral (and some host) transcripts thereby restoring CPSF activity and 3′ mRNA cleavage. Interestingly, poly(A) read-through transcription is accompanied by a dramatic increase in chromatin accessibility downstream of the affected poly(A) sites5. In uninfected cells, open chromatin is predominantly observed around gene promoters as well as in the gene bodies of highly expressed genes, but also in intergenic regions6. During HSV-1 infection, chromatin accessibility selectively increases downstream of genes with strong read-through transcription indicative of impaired histone repositioning in the wake of RNA polymerase II (Pol II) downstream of affected poly(A) sites. Disrupted transcription termination with Pol II transcription extending far downstream of gene 3′ ends (DoGs) is also observed upon cellular stress responses8, influenza A virus infection10 and cancer11. However, downstream open chromatin regions (dOCRs) do not arise upon salt or heat stress5, indicating that transcription read-through is either not sufficiently strong for dOCR induction in stress responses or that additional viral factors are involved.Productive 12). The histone-chaperoning activity of the FACT complex facilitates both the removal and reassembly of histones in the wake of actively transcribing Pol II. Distinct regions of FACT interact with both the H2A-H2B dimer and the (H3-H4)2 tetramer and can promote displacement of H2A-H2B from nucleosomes13. In particular, FACT mediates both histone eviction and repositioning at the early stages of transcription elongation14. SPT6 directly interacts with histones and assembles nucleosomes in vitro15. This activity is required for the maintenance of a chromatin structure that prevents improper usage of cryptic promoter elements, suggesting that SPT6 also reassembles nucleosomes in the wake of Pol II16. Interestingly, the viral ICP22 protein interacts with the FACT complex and relocalizes it to viral replication compartments (RCs)17. ICP22 (encoded by the HSV-1 US1 gene) is an important, but non-essential, multifunctional viral 63-kDa polypeptide conserved in Alphaherpesviruses that is expressed with immediate early kinetics. HSV-1 mutants lacking ICP22 do not form plaques in skin and lung fibroblasts but replicate in Vero or BHK cells18. ICP22 features a structurally-conserved core domain flanked by poorly-conserved, intrinsically disordered regions that are extensively phosphorylated by the viral pUL13 protein kinase and, to a lesser extent, by the pUS3 protein kinase20. ICP22 is also phosphorylated by yet unidentified cellular kinases and nucleotidylated by casein kinase II22. It is thus very likely that ICP22 exerts multiple different functions throughout productive virus infection that are governed by post-translational modifications. Accordingly, ICP22 has been shown to interact with a variety of different cellular proteins including cyclin-dependent kinase 9 25, cell division cycle 25C (CDC25C)26 as well as FACT27. Notably, ICP22 facilitates the recruitment of FACT and SPT6 to RCs and to the transcribing Pol II complex27. Very early during productive infection, ICP22 induces the formation of virus-induced chaperone-enriched (VICE) domains, which may aid the proper folding of newly synthesized viral proteins28. It may by itself also function as a virally encoded co-chaperone (J-protein/Hsp40) by interacting with heat shock protein 70 (Hsc70) to prevent aggregation of misfolded proteins29. During productive infection, ICP22 and the viral pUL13 protein kinase mediate an intermediate phosphorylation of the carboxyl terminal domain (CTD) of Pol II, termed Pol IIi12. ICP22 also alters the activity of the cell cycle component CDK1 to enhance viral late (L) gene expression13. This effect of ICP22 on key cell cycle regulators may explain the cell type-dependent growth defect of ICP22 null mutants30. Finally, it also plays an important role in HSV-1 nuclear egress by interacting with the viral nuclear egress complex (pUL31 and pUL34 proteins)31.During the process of transcription, Pol II removes and subsequently repositions histones to facilitate efficient transcription elongation while maintaining chromatin architecture. The two transcription elongation factors and histone chaperons SPT6 and FACT (comprised of SSRP1 and SPT16) play a key role in this process  read-through transcription induced by ectopic expression of ICP27. Read-through transcription and induction of dOCRs downstream of genes were associated with a notable depletion of histones downstream of gene 3’ends. Efficient knock-down of the histone chaperone FACT not only enhanced dOCR induction but also increased chromatin accessibility within gene bodies in an ICP22-dependent manner. It thereby alleviated the selective, DoTT-dependent increase in chromatin accessibility downstream of genes. We propose a model in which functional inhibition of FACT by the viral ICP22 protein and allosteric changes in Pol II composition downstream of the poly(A) signal result in selectively impaired histone repositioning in the wake of Pol II downstream of genes.Here, we show that the viral ICP22 protein is necessary to induce dOCRs in HSV-1 infection, we first assessed whether viral genome replication and, thus, viral late gene expression was required for the induction of dOCRs. We infected primary human fibroblasts (HFFFs) with HSV-1 strain 17 for 8 h or strain F for 8 and 12 h in the presence or absence of the viral DNA polymerase inhibitor phosphonoacetic acid (PAA) and performed ATAC-seq (n = 2). The length of dOCRs was quantified for 4162 protein-coding and lincRNA genes that exhibited no read-in transcription originating from the read-through transcription of an upstream gene. These genes were identified in our previous study comparing transcriptional regulation in HSV-1 strain 17 and Δvhs infection32. We excluded genes with read-in transcription as read-in transcription can result in dOCRs from an upstream gene extending into downstream genes  responsible for the induction of dOCRs during HSV-1 infection are not limited to induction of downstream open chromatin but also include other changes, e.g., changes at promoters that are associated with alterations in Pol II promoter occupancy. Here, we only focus on the induction of open chromatin downstream of genes during HSV-1 infection. PAA treatment of HSV-1-infected cells was particularly useful for the investigation of dOCRs as it resulted in a much higher percentage of cellular reads, i.e., reads aligning to the human genome, in the ATAC-seq data due to the inhibition of viral DNA replication . Second, the detection of dOCRs was likely enhanced by PAA due to technical reasons. RCs at 8 h p.i. comprise much uncompacted viral DNA, which attracts and sequesters DNA binding proteins non-specifically33. Thus, the transposase used for ATAC-seq likely preferentially incorporates sequencing adapters into viral DNA over cellular dOCRs as the latter is  still more compacted. When PAA is added to the infected cells, the transposase is not sequestered in RCs and can better sample the loosely packaged dOCRs. Since PAA also prevents viral late gene expression, we conclude that neither viral late gene expression nor sequestration of a cellular factor to viral RCs is required for dOCR induction.It should be noted that changes in chromatin accessibility during HSV-1 strains and ± PAA treatment.To assess the role of individual viral genes regarding the induction of dOCRs, we first aimed to identify a subset of cellular genes that showed strong and consistent dOCR induction upon infection with both virus strains. To this end, we performed a hierarchical clustering analysis of dOCR lengths in mock, WT, and WT + PAA infection Fig. . The cut3. We analyzed (i) the percentage of read-through transcription  and (ii) the absolute extent of transcriptional read-through activity . While this showed statistically significant increases in the percentage of read-through transcription for Clusters 2 and 5 compared to all other analyzed genes  at least for some time-points, the differences were nevertheless small (fold-change of medians < 1.35). Please note that by definition, read-through in mock infection was set to zero and thus not shown. Cluster 2  showed the highest median percentage of read-through transcription of all clusters  than in all other clusters . Cluster 5 thus comprises the most strongly expressed cellular genes with the highest absolute levels of downstream transcription due to DoTT. Clusters 2 and 6 also showed slightly elevated downstream transcriptional activity compared to clusters without apparent induction of dOCRs, but this was only significant for Cluster 2 . Accordingly, the absolute extent of transcriptional activity downstream of the respective genes rather than the percentage of read-through transcription determines the extent of dOCR induction. This also explains why genes with a high percentage of read-through transcription but relatively low gene expression (Cluster 2), as well as genes with a moderate percentage of read-through transcription but higher gene expression (Cluster 6), exhibit some induction of dOCRs. Strikingly, induction of open chromatin predominantly occurred downstream of affected gene 3′ ends, although transcription of the respective gene bodies was at least as high as downstream transcription.To investigate the cause of these differences in the induction of dOCRs between the different gene clusters, we made use of our previously published 4sU-seq time-course of the first 8 h of WT strain 17 infectioners Fig. . In conters Fig. . This waHSV-1 infection, in particular upon PAA treatment. In the absence of both read-through and dOCR induction in mock infection, no correlation was observed. While the correlation in HSV-1 infection was not perfect, it nevertheless confirmed a general trend with longer dOCRs observed for genes with higher downstream transcriptional activity, which were enriched for Cluster 5 genes  sites. Furthermore, inhibition of viral DNA replication by PAA enhances the induction of dOCRs due to the reduced shut-off of host transcription.As transcription downstream of genes is very low prior to infection, median downstream FPKM in mock infection is 3- (without PAA) to 13-fold (with PAA) lower than in WT infection, even in total RNA . This included null mutants of the immediate early genes ICP0, ICP22 (R325), and ICP27, as well as of the virion host shut-off protein (vhs), which is expressed late during infection but is delivered to infected cells by the incoming virions. Due to the attenuated progression of ΔICP0 and ΔICP22 infections, ATAC-seq was performed at 12 h p.i. for these mutants. For WT and the other mutants, infection was performed at 8 h p.i. For ΔICP22 infection, we also included PAA treatment as we hypothesized that the loss of serine 2 phosphorylation of RNA polymerase II (Ser-2P) during HSV-1 infection might play a role in the induction of dOCRs. This loss of Ser-2P results from the combined effects of both an ICP22-dependent and an unknown ICP22-independent, viral late gene-dependent mechanism34. To analyze the induction of dOCRs in the single-gene deletion mutants, we first focused on genes in Cluster 5. Infection with ΔICP0 and Δvhs showed induction of dOCRs at levels comparable to WT strain 17  . This confirmed the presence of extensive DoTT and strong downstream transcriptional activity in ΔICP22 infection in the dOCR regions observed in WT infection  Fig.  as well lls Fig. . In contlls Fig. . We concHSV-1-induced read-through, ΔICP27 infection still induced dOCRs in Cluster 5 genes, albeit to a lesser extent  albeit to a lesser extent than observed in WT infection . Omni-ATAC-seq represents a recent improvement in the ATAC-seq protocol published during the course of this study, which improves signal-to-background ratios and reduces the amount of contaminating mitochondrial reads37. As expected, Dox-induced ICP22 expression in the absence of ICP27-induced read-through transcription and Dox-induced ICP27 expression alone did not induce dOCRs  that express either ICP22 (T-HF-ICP22 cells) in isolation (T-HF-ICP22 cells) or in combination with ICP27 (T-HF-ICP22/ICP27 cells) upon doxycycline (Dox) exposure . It is noteworthy, however, that this was also observed for genes without dOCR induction, although not as pronounced. In contrast, genes without dOCR induction showed a small but significant increase in H3K36me3 downstream of the TTS. While we also observed some relative enrichment around promoters for H3, H3K27me3, and H3K36me3, this was not specific to Cluster 5 genes and was likely associated with the global loss of host transcriptional activity and reduction of promoter-proximal Pol II pausing in HSV-1 infection39.We hypothesized that the induction of dOCRs was due to impaired histone repositioning in the wake of Pol II read-through transcription into downstream genomic regions. To test this hypothesis, we first analyzed genome-wide occupancy of histone H3 as well as the two major histone marks associated with heterochromatic regions H3K27me3) or active transcription (H3K36me3) in uninfected and WT strain 17-infected  cells by ChIPmentation , gene bodies excluding promoter regions (TSS + 1.5 kb to TTS) and downstream regions (TTS to TTS + 25 kb) for all protein-coding and lincRNA genes. Consistent with the metagene analyses, we observed small but highly significant increases in H3, H3K27me3, and H3K36me3 at gene promoters  compared to gene body and downstream regions . However, as no significant differences in gene promoters were observed between our 9 clusters, the respective changes are thus unlikely to be linked to dOCRs. Interestingly, however, downstream regions of Cluster 5 genes, but not of other clusters, tended to show a small reduction in H3K36me3 (median log2 fold-change −0.05) but not H3 or H3K27me3. This reduction was statistically significant compared to all protein-coding and lincRNA genes or all other genes included in our analysis . In summary, we found no differences in the distribution of H3 or H3K27me3 following HSV-1 infection that could be linked to dOCR induction. However, small differences in H3K36me3 were observed downstream of genes with strong dOCR induction.To confirm the results from the metagene analyses, we performed a genome-wide differential analysis of H3, H3K27me3, and H3K36me3 in 33, we repeated the ChIPmentation experiments upon PAA treatment . This time, we also analyzed histones H1 and H4 in addition to H3, H3K27me3, and H3K36me3. Quality of the histone ChIPmentation data was again confirmed by metagene analyses on all protein-coding genes showing the expected occupancy profiles with a strong depletion of H1, H3, and H4 at transcription start sites (TSSs) compared to gene bodies and, in particular, downstream regions  for some of these positions, it was observed consistently for all three histones. Furthermore, no such reduction was observed for genes without induction of dOCRs  and to a lesser degree for H3 and H4 downstream of Cluster 6 genes , a loss in histone occupancy would likely be masked by the cells not transcribing the respective genes anymore. Thus, to prevent the virus-induced sequestration of Pol II to RCs and alleviate the reduction in host transcriptional activityHSV-1 infection, even with PAA treatment. For both histone modification marks, a reduction was observed for Cluster 5 genes selectively in the region from the TTS or upstream of the TTS to around 25 kb downstream of the TTS  at 8 h p.i. Fig. . We used12. During the course of this study, both histone chaperones were shown to be recruited into viral RCs by the viral ICP22 protein27, which suggests some kind of functional modulation. Since PAA treatment increased dOCRs and ICP22/ICP27 expression in isolation were sufficient for dOCR induction, this excludes sequestration of ICP22-binding factors to RCs as a mechanism of dOCR induction. However, since functional inhibition of FACT or SPT6 on host cell chromatin by ICP22 could still explain the induction of dOCRs, we investigated whether depletion of either of the two factors would restore the induction of dOCRs upon infection with an ICP22-null mutant. For this purpose, we generated T-HF cells with Dox-inducible artificial miRNA-mediated knockdown of SSRP1 and SPT640. For both cellular proteins, efficient knockdown was achieved with two different miRNAs after 3 days of Dox treatment (1 µg/ml) . Omni-ATAC-seq was performed on cells from the same experiment as utilized for the Western blots in Supplementary Fig. HSV-1, depletion of FACT significantly increased dOCR induction  but no other clusters . Interestingly, however, when analyzing the 74 genes with a ≥2-fold decrease in chromatin accessibility upon FACT depletion in WT infection  but no other cluster. We hypothesize that Cluster 5 genes were preferentially affected by FACT depletion as these represent the most highly expressed genes  recognition of cryptic poly(A) sites in the respective gene bodies. Knockdown of FACT in mock- and ΔICP22-infected cells also resulted in a modest increase in chromatin accessibility within gene bodies for 202 and 164 genes, respectively, and a decrease for 51 and 96 genes, respectively . However, in contrast to WT infection, genes with a decrease in chromatin accessibility within gene bodies upon FACT knock-down in mock and ΔICP22 infection showed no significant enrichment in any cluster. FACT-depletion-induced chromatin accessibility increases in mock- and ΔICP22-infected cells were also less prominent than observed in WT infection. We conclude that transcription of the highly expressed Cluster 5 genes may generally be more susceptible to FACT depletion and, thus, dOCR induction. In summary, FACT knockdown not only enhanced the induction of dOCRs in WT HSV-1 infection but also alleviated its restriction to regions downstream of genes with read-through transcription. These data directly implicate FACT in the ICP22-mediated impairment of histone repositioning in the wake of Pol II.The two histone chaperones FACT (SPT16/SSRP1) and SPT6 play a key role in the re-assembly of nucleosomes after the passing of Pol IIion Fig. . Down-saion Fig. . Analysiely Fig. . Genes wHSV-1 infection and various stressors trigger extensive transcription downstream of genes, but dOCRs only arise in HSV-1 infection. Our study explains this difference by demonstrating that the viral ICP22 protein is required for the induction of dOCRs in HSV-1 infection. Importantly, dOCR formation critically depended on transcriptional activity downstream of genes as dOCRs were only induced in HSV-1 infection for genes with strong downstream transcription, while ICP22 expression alone, which does not induce read-through, did not induce dOCRs. The absolute level of transcriptional activity downstream of a given gene is determined by the transcriptional activity of the gene itself and the extent of failure in terminating transcription at the gene’s 3′end, i.e., the extent of read-through. This explains why genes in Cluster 5 and, to a lesser extent, genes in Cluster 2 and 6 but not any of the other clusters identified in our analysis showed prominent dOCR induction upon HSV-1 infection. For the six clusters without dOCR induction, absolute levels of transcriptional activity downstream of genes likely did not reach sufficiently high levels to induce dOCRs since they were neither particularly highly expressed nor exhibited particularly high read-through. Low transcriptional activity downstream of the respective genes during the last few hours prior to ATAC-seq analysis in a large percentage of cells thus probably explains the absence of dOCRs.Both PAA treatment substantially increased the number of ATAC-seq reads mapping to the cellular genome by reducing the contribution of viral reads. However, it also increased the extent of dOCR induction due to PAA-mediated preservation of Pol II on host chromatin and, consequently, host transcriptional activity within and downstream of genes. Thus, PAA treatment is ideal for comparative analysis of dOCR induction between different virus mutants or strains, as it also prevents additional secondary effects on the progression of productive infection.HSV-1 infection, which increased again upon PAA treatment. As Pol II transcription extends beyond the poly(A) site before termination, some open chromatin can also be observed prior to infection downstream but in the proximity of the poly(A) site, in particular for highly expressed genes. Due to the virus-induced loss of host transcriptional activity38, this downstream transcription is reduced for genes without read-through transcription, leading to a further reduction of these short dOCRs. PAA treatment prevents the extensive loss of host transcriptional activity and thus restores these short dOCRs. We would like to stress that the nine identified clusters are not set in stone but rather serve as a tool to identify the viral genes that may or may not be involved in dOCR formation. While the susceptibility of a gene’s poly(A) site to disruption of transcription termination by HSV-1 is likely an inherent feature, dOCR induction also depends on the expression level of the respective gene in the cells under study. This will vary between different cell types and conditions.The association between dOCRs and downstream transcriptional activity also explains why some of the clusters without dOCR induction, e.g., Clusters 1 and 3, showed a reduction in the already relatively short dOCR length during HSV-1 null mutants. Both the selective analysis of genes with strong dOCR induction in human fibroblasts, i.e., Cluster 5, as well as a global regression analysis across all 4162 cellular genes analyzed in our study, revealed that ICP22 but not ICP0, ICP27, or vhs are required for dOCR induction. Furthermore, Dox-induced expression of ICP22 in combination with ICP27 confirmed that ICP22 is sufficient for dOCR induction upon disruption of transcription termination by ICP27. Notably, ICP27 alone did not induce dOCRs despite inducing transcription downstream of genes. Finally, ICP22 alone did not induce dOCRs in the absence of ICP27-mediated read-through transcription. Interestingly, infection with an ICP27-null mutant still induced a reduced but nevertheless significant induction of dOCRs. This is consistent with the reduced but nevertheless detectable levels of transcription downstream of genes in infection with an ICP27-null mutant3, which likely represents a stress response. This suggests that cellular stress responses, and thus DoG transcription42, may be sufficient to induce dOCRs in the presence of ICP22. It will be interesting to see whether ICP22 also triggers dOCRs in other models of impaired transcription termination.We ruled out that other viral factors are required for dOCR induction by analyzing chromatin structure in PAA-treated infected cells, which reduces or abolishes the expression of late genes, and by using several HSV-1 strains with KOS1.1 (KT887224.1) showing only six amino acid substitutions to the identical strain F (GU734771.1) and strain 17 (NC_001806.2) ICP22 sequences. The ICP22 mutants, which we employed in this study, either lack the ICP22 protein completely (KOS1.1 and strain 17) or just its C-terminal 220 aa . This implies that the N-terminus of ICP22, which is important for VICE domain formation43, is not sufficient for the induction of dOCRs. The N-terminus of ICP22 harbors five of the six mutations in KOS1.1. The respective mutations may nevertheless contribute to the reduced dOCR induction of this strain compared to strain F and 17. The dependency of dOCR induction on strong downstream transcriptional activity observed in our data supports a model in which the extent of dOCR induction by different strains is determined by the absolute extent of downstream transcriptional activity. The latter is defined by the extent of ICP27-induced disruption of transcription termination, i.e., the percentage of transcripts showing read-through and the overall extent of virus-induced transcriptional shut-off. In summary, the observed strain-specific differences in the extent of dOCRs likely reflect differences in the extent of downstream transcription. These differences result from the cumulative effects of sequence variations between the different strains in viral proteins other than ICP22 and ICP27.All three strains included in our analysis  induced dOCRs, although to different extents. dOCR induction was most pronounced for strain 17, followed by strain F and KOS 1.1  present in all α-herpesvirus U13 and H3–H4 tetramers45 to unwrap the nucleosomal DNA. However, SSRP1 was recently shown to also bind to the linker histone H1 as a homodimer and to mediate eviction of H1, suggesting an SPT16-independent function of SSRP146. How ICP22 induces dOCRs by interacting with SSRP1 and SPT16 thus remains unclear. Nevertheless, our results support a model in which ICP22 impairs histone repositioning in the wake of Pol II downstream of genes. It is important to note that the loss of histone occupancy downstream of genes with dOCR induction was only detectable when viral genome replication was inhibited by PAA. Our findings thus stress the importance of studying the function of viral immediate early gene products on the host transcriptional machinery in the absence of the HSV-1-induced shut-down of host transcription.ChIPmentation for major histones and common histone marks revealed a selective decrease in histone occupancy within the first ≈25 kb downstream of genes with dOCR induction. This is consistent with the increased chromatin accessibility observed in ATAC-seq experiments and was dependent on the presence of ICP22. The concordant loss of total H1, H3, and H4, as well as of both the activating and inhibitory histone marks H3K36me3 and H3K27me, respectively, indicates that impaired histone repositioning is not restricted to histones with specific histone marks but affects histone repositioning in general. The SSRP1–SPT16 heterodimer (FACT) is well described to bind both H2A–H2B dimers12). This is facilitated by Pol II-associated histone chaperones, including SPT6 and FACT. During the course of this study, ICP22 was found to directly interact with FACT and recruit both FACT and SPT6 to the viral RCs17. However, induction of dOCRs upon ectopic expression of both ICP22 and ICP27, as well as our findings involving PAA treatment, imply that deprivation of cellular factors by sequestration to large viral RCs is not responsible for dOCR induction. Interestingly, the knockdown of FACT but not SPT6 significantly increased dOCR induction in WT HSV-1 infection. The requirement for FACT, but not for SPT6, in nucleosome reassembly, may be explained by a model in which SPT6-mediated histone chaperoning can be compensated by FACT, which can chaperone all four core histones onto DNA. In contrast, loss of FACT activity cannot be compensated by SPT6, which only chaperones histones H3 and H412. Importantly, FACT knockdown also resulted in a small but nevertheless significant increase in chromatin accessibility within gene bodies of a few hundred genes in uninfected and ΔICP22-infected cells. This was substantially increased upon WT infection and often reached similar levels as observed downstream of the respective genes. Of note, a slight increase in chromatin accessibility within gene bodies was also observable for many of these genes in WT HSV-1 infection without FACT depletion, thereby demonstrating that the increased chromatin accessibility observed in HSV-1 infection is not completely restricted to genomic regions downstream of genes. Interestingly, affected genes, as well as genes with a reduction of chromatin accessibility upon FACT depletion in WT infection, were significantly enriched for Cluster 5 genes, indicating that these highly expressed genes are most dependent on FACT. Our findings indicate that increased chromatin accessibility both within and downstream of genes results from a common mechanism, namely functional impairment of FACT by the combined effects of FACT knockdown and the viral ICP22 protein. More direct proof of the involvement of FACT in increased chromatin accessibility would come from FACT chromatin immunoprecipitation (ChIP-seq or ChIPmentation) experiments. However, we were unable to obtain data of sufficient quality for FACT despite multiple attempts. We nevertheless would like to propose a model in which the viral ICP22 protein, via its interaction with FACT, interferes with the function of FACT to reposition histones in the wake of Pol II. The FACT complex is thought to associate indirectly with Pol II by either HP1 or the PAF1 complex bridging the interaction (reviewed in48). Furthermore, chromatin remodelers can interact with histones and FACT and likely promote FACT association with chromatin. It is thus probably not surprising that the resistance of FACT to viral inhibition by ICP22 changes during transcription and drops downstream of genes where allosteric changes in Pol II result in a loss of transcription elongation factors from Pol II before transcription termination. The selective induction of dOCRs in HSV-1 infection thereby supports the allosteric model of transcription termination, which proposes allosteric changes in Pol II composition at the end of genes49. In the presence of both efficient FACT knockdown and impairment of FACT function in Pol II transcription by the viral ICP22 protein (possibly by interfering with the recruitment of FACT to Pol II), these differences are alleviated, often resulting in concordant increases in chromatin accessibility both within and downstream of genes for genes with strong dOCR induction.During Pol II transcription, nucleosomes are first destabilized and momentarily removed to be reassembled once again at the same position in the wake of Pol II (reviewed in27. Manipulation of other cellular factors by ICP22 may thus contribute to dOCR induction by ICP22.Multiple different functions have been attributed to ICP22. One of the most striking is the loss of serine 2 phosphorylation (Ser-2P) of the Pol II CTD, which governs the recruitment of other cellular proteins to Pol II. We cannot exclude that viral interference with Ser-2P and resulting changes in Pol II composition, in addition to the direct interaction of ICP22 with FACT, contribute to the observed effects. Finally, a recent study identified many other cellular factors involved in transcription elongation, including P-TEFb and additional CTD kinases that interact with ICP2217. Due to the multiple functions of ICP22, the relative contribution of FACT manipulation will be difficult to decipher. It is important to note that FACT has also been shown to play an important role during the early steps of transcription. In Drosophila, it alleviates transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor)50. The ICP22-mediated induction of dOCRs may thus only represent a bystander effect of more important viral interference with key mechanisms of upstream transcription elongation. However, it may also help to increase the pool of free histones52 to aid the chromatinization of incoming viral genomes at early times of infection when both ICP22 and ICP27 are expressed. In summary, our findings highlight HSV-1 as an exciting model to study fundamental aspects of the transcriptional machinery in human cells.At present, we can only speculate about the functional importance of this viral interference with histone repositioning. Interestingly, efficient knockdown of neither SPT6 nor FACT did significantly impair viral protein expression during productive infection, arguing against an important role of the two factors in viral transcription. Importantly, both FACT and SPT6 represent important transcription elongation factors. Viral interference with their activities as well as their recruitment to viral RCs, is thus likely to contribute to the selective virus-induced shut-off of host transcription by interfering with transcription elongation53, Baby Hamster Kidney cell line , Human Bone Osteosarcoma Epithelial Cells , ICP27-complementing Vero 2-2 cell line (kindly provided by Prof. Dr. Beate Sodeik) and Human embryonic kidney 293 T cells  were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% (v/v) Fetal Bovine Serum , 1× MEM Non-Essential Amino Acids (ThermoFisher #11140050) and 1% penicillin/streptomycin. All cells were incubated at 37 °C in a 5% (v/v) CO2-enriched incubator. HFFFs were utilized from passages 11 to 17 for all high-throughput experiments.Human Fetal Foreskin Fibroblasts , Telomerase-Immortalized Human Foreskin Fibroblasts (T-HF)HSV-1 strain 17, BAC-derived HSV-1 strain 17+Lox (kindly provided by Beate Sodeik)54, wild-type HSV-1 strain F, and mutant viruses R325 , wild-type KOS1.1 (kindly provided by Steven Rice)56, vhs-inactivated mutant , ICP27-null mutant  and ICP0-null mutant . Virus stocks were produced in BHK cells as described, except for the viruses mentioned below. Stocks of the ICP27-null mutant were produced on complementing Vero 2–2 cells60 and ICP0-null mutant in U2OS cells. All produced viruses were Ficoll-gradient purified. For all experiments, media were collected immediately prior to inoculation (conditioned media). Cells were infected for 1 h using a multiplicity of infection (MOI) of 10 in fresh media 24 h after the last split. Subsequently, the inoculum was removed, and warm, conditioned media was applied back to the cells. The time at which inoculum was replaced with growth media was marked as the 0 h time point. To block viral DNA replication, phosphonoacetic acid  was added in conditioned media to cultured cells after the inoculum was removed. The number of biological replicates that were performed for each experiment is indicated in the results section.This study was performed using wild-type (WT) 40, cloned into a doxycycline-inducible lentiviral vector (see below), and utilized to generate T-HF cells that enable efficient, dox-inducible knock-down of the respective host proteins. Primer sequences are listed in Supplementary Data Artificial miRNAs (amiRNAs) against SSRP1 and SPT6 were selected as describedUL54 ORF under control of the doxycycline-inducible pTRE-Tight promoter were produced by cloning the corresponding ORF from the HSV-1 genome (strain 17) via intermediate vectors into pW-TH3. The pW-TH3 vector was derived from pCW57.1 by sequential insertion of a synthetic multi-cloning site (prW64/65) and three stop codons (prW110/111) between the NheI and AgeI restriction sites. pCW57.1 was a gift from David Root . pW-TH7 (3xFlag-V5-NT1) was created by amplifying the N-terminal part of NT1 from the V5-NT1 vector61 by using primers prW196/197 and inserted back between the BamHI and EcoRI of the same V5-NT1 vector. The 3xFlag-V5-NT1 ORF was excised with EcoRI and XbaI and inserted between the EcoRI and NheI sites of pW-TH3 (now designated pW-TH9). The UL54 ORF was amplified from the HSV-1 genome by PCR using primers prW365/366. The PCR product was digested with BamHI and BglII and inserted into BamHI cut pW-TH9 (now designated pW-TH57). To generate the doxycycline-inducible vector with HA-tagged US1 ORF (designated as LDJ5), the vector YC1 was used as the backbone. YC1 was generated to carry the blasticidin resistance gene instead of puromycin by restriction digestion of the pW-TH3 vector with XbaI and AgeI and insertion of an hPGK.blast construct (purchased from GeneArt) via infusion cloning. The US1 ORF was amplified from the HSV-1 genome by PCR using primers prW1656/1657, and the extracted band was cloned via infusion cloning into YC1, linearized by restriction digest with MluI and NheI. Primer sequences used for generating HA-ICP22 and V5-ICP27 cell lines are listed in Supplementary Data HA-ICP22 and HA-ICP22 + V5-ICP27 cells were generated as follows. Lentiviral vectors encoding N-terminally 3xFLAG and V5-tagged (tandem tag) To generate V5-ICP27 and HA-ICP22 doxycycline-inducible cell lines, HEK-293T cells were transfected with pW-TH57 and LDJ5, respectively. Transduced T-HF cells were kept in selection with 5 µg/ml puromycin and 5 µg/ml blasticidin, respectively. To generate a V5-ICP27 + HA-ICP22 doxycycline-inducible cell line, V5-ICP27 cells were lentivirally transduced with LDJ5. Transduced T-HF cells were kept in selection with both 5 µg/ml puromycin and 5 µg/ml blasticidin. Expression of proteins was induced by 5 μg/ml doxycycline for 48 h.Samples were harvested at the indicated time points by removal of growth media, followed by 1× wash with phosphate-buffered saline  and lysis in 1× Laemmli buffer containing 5% (v/v) β-mercaptoethanol. Samples were sonicated and heated for 5 min at 95 °C before loading onto a Novex WedgeWell 4–20% Tris-Glycine Gel (ThermoFisher #XP04200BOX). Proteins were transferred to 0.2 µm nitrocellulose membranes (Sigma-Aldrich #GE10600001), blocked for 1 h at room temperature (RT) in 1x PBS with 0.2% Tween (PBS-T) containing 5% (w/v) milk (Carl Roth #T145.3), and probed using anti-V5 , anti-HA clone 11 , anti-FLAG  anti-α-Tubulin , anti-β-Actin clone C-4 , anti-GAPDH , anti-SPT6 , anti-SSRP1 , anti-Spt16 clone 8D2 , anti-RNA Pol II 1F4B6 , anti-ICP8 clone 11E2 , anti-gD clone DL6  overnight at 4 °C at the indicated dilution. Before the addition of each antibody, blots were washed with 3× PBS-T. After incubation with either anti-rabbit–horseradish peroxidase , anti-mouse–HRP , anti-rat-HRP , or IRDye 680RD goat-anti-rabbit IgG  and IRDye 800CW donkey anti-mouse IgG secondary antibody , bands were visualized using the LI-COR Odyssey FC Imaging System. PAGERuler Plus was used as a ladder. Source images are supplied without and with a ladder and as ‘ladder only’ scans.5 HA-ICP22 and HA-ICP22 + V5-ICP27 cells were plated in 12 well-dishes with the addition of 5 μg/mL of doxycycline (Merck #AMBH2D6FB132). At 48 h post-induction, cells were fixed with 4% formaldehyde (PFA) in PBS for 15 min at RT, washed three times in PBS, and either stored at 4 °C overnight in PBS or processed immediately as follows. Cells were permeabilized in 0.5% Triton X-100 in PBS for 5–10 min and blocked in a blocking buffer  for 1 h at RT. Anti-HA antibody clone F-7  or anti-V5 antibody  were incubated in 10% FBS and 1× PBS for 1 h at RT. Control imaging was performed in parallel for each ATAC-seq or ChIP experiment. Briefly, cells were seeded at the same density as for the specific assay and infected the next day with an MOI of 10 for 1 h. Cells were fixed at 6 hpi with 4% PFA and processed as described. Anti-ICP4 antibody  was incubated for 1 h to detect ICP4. For all assays, the secondary anti-mouse IgG, Alexa Fluor 488  or anti-rabbit IgG, Alexa Flour 568 , were incubated in 10% FBS in 1× PBS for 1 h at RT with 0.5 μg/mL 4′,6-diamidino-2-phenylindole (DAPI). All steps were followed by three 5 min washes in 1× PBS, after which the images were taken on a Leica DMi8 fluorescence microscope. Images were exported as tif files with 10 µm scale bars on black background.Totally, 105 cells per condition62. An improved ATAC-seq protocol, Omni-ATAC-seq, was performed according to the original protocol starting with 1 × 105 cells per condition36. For each experiment, biological duplicates were carried out. Sequencing libraries for ATAC-seq samples were prepared as specified using the Nextera DNA Library prep kit (Illumina #15028212) or using NEBNext Ultra II master mix in combination with primers made by IDT based on Illumina primers with unique dual (UD) index adapters for both i_5 and i_7. Sequencing libraries for Omni-ATAC samples were prepared as 50 µL reactions containing: 12,5 µL DNA, 6,25 µL i_5_x and i_7_x (10 µM), and 25 µL 2× NEBNext Ultra II Q5 Master Mix (NEB #M0544). The number of cycles necessary for the library amplification was determined from the pre-amplification of transposed fragments using quantitative PCR (SYBR green). Both ATAC-seq and Omni-ATAC-seq libraries were quantified by Agilent Bioanalyzer for fragments between 150 and 1000 base pairs (bs) to quantify the level of contamination with large DNA fragments. Libraries were then pooled to the same final concentration (range 150–1000 bp), loaded onto a 1% pre-cast agarose gel, excised in the specified range, and sequenced by NextSeq 500 (Ilumina) at the Core Unit Systemmedizin, Würzburg, Germany (35 bp paired-end reads). All samples were sequenced at equimolar ratios.ATAC-seq was performed according to the original protocol starting with 1 × 105 and 4sU-seq data for Fig. 4.To confirm the presence of read-through transcription, on the day of the ATAC/Omni-ATAC-seq experiment, total RNA was collected. Biological duplicates were carried out. For total RNA, cells were collected in 500 μl TRI reagent, and total RNA was isolated with Directzol-RNA Microprep Kit (Zymo Research #R1050) according to manufacturer’s instructions. The following steps were performed by the Core Unit Systemmedizin, Würzburg, Germany. For the total RNA libraries, both cytoplasmic and mitochondrial rRNA species were depleted. No rRNA depletion was performed for 4sU-RNA samples as rRNA only contributes about 40–50% of reads in 4sU-RNA samples. Library preparation for sequencing was performed using the stranded TruSeq RNA-seq protocol . Libraries were sequenced on NextSeq 500 (Ilumina). 4sU-seq data for Fig. n = 2 for all conditions except for H3K36me3 in WT strain 17 without PAA (n = 3)). PAA (350 μg/mL) was added to the conditioned cell culture media that was supplied to the cells after the removal of the virus inoculum. At 8 p.i., cells were fixed by adding ChIP Cross-link Gold according to the manufacturer’s instructions (Diagenode #C01019027) and subsequently with 1% PBS-buffered formaldehyde. Cells were scraped in 1 mL of ice-cold 1× PBS containing protease inhibitor cocktail (1×) (Roche #11836153001) with an additional 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were pelleted at 500g for 20 min at 4 °C. Supernatant was aspirated, and cell pellets were frozen in liquid N2.Two days prior to infection, two million HFFF cells were seeded in 15 cm dishes. On the day of infection, cells had expanded to ~80% confluency. Cells were infected with the respective viruses as described in the results section  to 200 μL. Input and gel samples were preserved. Lysates were incubated with 1:100/IP of anti-H1 antibody (Invitrogen #PA5-30055), 1:50/IP of anti-H3 antibody , 1:50/IP of anti-H4 antibody , 1 μg/IP of anti-H3K27me3  and 1 μg/IP of anti-H3K36me3  on a rotator overnight at 4 °C. Dependent on the added amount of antibody, the amount of Protein A magnetic beads (ThermoFisher Scientific #10001D) was adjusted  and blocked overnight with 0.1% [w/v] bovine serum albumin in RIPA buffer. On the following day, beads were added to the IP samples for 2 h on a rotator at 4 °C to capture the antibody-bound fragments. The immunoprecipitated chromatin was subsequently washed twice with 150 μL each of ice-cold buffers RIPA-LS, RIPA-HS , RIPA-LiCl  and 10 mM Tris pH 8.0 containing protease inhibitors. Beads were washed once more with ice-cold 10 mM Tris pH 8.0, lacking inhibitors, and transferred into new tubes.Cell pellets were resuspended in 1.5 mL 0.25% [w/v] SDS sonication buffer  with 1× protease inhibitors and 1 mM additional PMSF and incubated on ice for 10 min. Cells were sonicated in fifteen 1 min intervals, 25% amplitude, with Branson Ultrasonics SonifierTM S-450 until most fragments were in the range of 200–700 bp as determined by agarose gel electrophoresis. Two million cells used for the preparation of the ChIPmentation libraries were diluted 1:1.5 with equilibration buffer  and spun at 14,000×2O to the final volume and incubated at 37 °C for 10 min in a thermocycler. Beads were mixed after 5 min by gentle pipetting. To inactivate the Tn5 enzyme, 150 μL of ice-cold RIPA-LS was added to the tagmentation reaction. Beads were washed twice with 150 μL of RIPA-LS and 1x Tris-EDTA and subjected to de-crosslinking by adding 100 µL ChIPmentation elution buffer , 1× Tris-EDTA (Sigma #T9285) and incubating for 1 h at 37 °C followed by overnight shaking at 65 °C. The next day, 4 mM EDTA and 200 μg/mL Proteinase K  were added, and samples were incubated for another 2 h at 45 °C with 1000 rpm shaking. The supernatant was transferred into a new tube, and another 100 μL of ChIPmentation elution buffer was added for another hour at 45 °C with 1000 rpm shaking. DNA was isolated with MinElute PCR Purification Kit (Qiagen #28004) and eluted in 21 μL of H2O.Beads were resuspended in 25 μL of the tagmentation reaction mix  containing 5 μL of 5× Tagmentation buffer, 1 μL of Tagment DNA enzyme, topped up with H2O and 13.75 μL ChIPmentation DNA. The Cq value obtained from the library quantification, rounded up to the nearest integer plus one additional cycle, was used to amplify the rest of the ChIPmentation DNA. Library qualities were verified by High Sensitivity DNA Analysis on the Bioanalyzer 2100 (Agilent) before performing sequencing on NextSeq 500 (paired-end 35 bp reads) at the Core Unit Systemmedizin, Würzburg, Germany (samples without PAA) or DNBSEQ-G400 2x100bp in BGI, Hong Kong, China (samples with PAA). All samples were sequenced at equimolar ratios.DNA for the final library was prepared with 25 μL NEBNext Ultra II Q5 Master Mix, 3.75 μL IDT custom primer i5_n_x (10 μM); 3.75 μl IDT custom primer i7_n_x (10 μM)  the human genome (GRCh37/hg19), (ii) human rRNA sequences and (iii) the HSV-1 genome  using ContextMap v2.7.964 . For the two repeat regions in the HSV-1 genome, only one copy each was retained, excluding nucleotides 1–9213 and 145,590–152,222.Quality control on sequencing reads was performed using fastQCHSV-1 genomes were determined with samtools66. Promoter/transcript body (PT) scores were determined with ATACseqQC67. For peak calling in ATAC-seq and ChIPmentation data, BAM files with mapped reads were converted to BED format using BEDTools68, and peaks were determined from these BED files using F-Seq with default parameters69. The fraction of reads in peaks (FRiP) was calculated with featureCounts70 using identified peaks as an annotation. Annotation of peaks relative to genes was performed using ChIPseeker71. For ATAC-seq data, peaks identified to be in dOCRs were also additionally assigned to the downstream category.Statistics on the numbers of mapped reads and reads mapped to human and 5. In brief, dOCRs were assigned to each gene in the following way. First, all OCRs overlapping with the 10 kb downstream of a gene were assigned to this gene. Second, OCRs starting at most 5 kb downstream of the so far most downstream OCR of a gene were also assigned to this gene. This was performed iteratively until no more OCRs could be assigned. Here, individual OCRs could be assigned to multiple genes. dOCR length of a gene was then calculated as the total genomic length downstream of this gene covered by OCRs assigned to the gene. Similarly, OCR length in gene bodies was calculated as the total genomic length of the gene bodies covered by OCRs.dOCR length for a gene was calculated from OCRs (= peaks in ATAC-seq data) as previously described70 and gene annotations from Ensembl (version 87 for GRCh37). For genes, all read pairs (= fragments) overlapping exonic regions on the corresponding strand by ≥25 bp were counted for the corresponding gene. For downstream regions, all fragments overlapping the 5 kb downstream of the gene 3’end were counted. Gene expression and downstream transcriptional activity were quantified in terms of fragments per kilobase of exons per million mapped reads (FPKM) and averaged between replicates. Only reads mapped to the human genome were counted for the total number of mapped reads for FPKM calculation. The percentage of read-through was calculated as previously described5. In brief: First, the percentage of transcription downstream of a gene was calculated separately for each replicate as the percentage of downstream transcription = 100 × (FPKM in 5 kb downstream of gene)/(gene FPKM). The percentage of downstream transcription was averaged between replicates, and the percentage of read-through was calculated as the percentage of downstream transcription in infected cells —the percentage of downstream transcription in uninfected or untreated cells. Negative values were set to 0.The number of read fragments per gene or in downstream regions was determined from the mapped RNA-seq or 4sU-seq reads in a strand-specific manner using featureCounts72 using the software developed for this previous publication. For each gene, the regions −3 kb to +3 kb of the TSS were divided into 250 bp bins, the regions −5 kb to +100 kb of the TTS into 500 bp bins, and the remainder of the gene body (+3 kb of TSS to −5 kb of TTS) into 100 bins of variable length in order to compare genes with different lengths. For each bin, the average coverage per genome position was calculated and normalized to a total sum of 1. Metagene curves for each replicate were created by averaging results for corresponding bins across all genes considered, and metagene plots show the average metagene curves across replicates. To determine the statistical significance of differences between average metagene curves for two conditions, paired Wilcoxon signed rank tests were performed for each bin, comparing normalized coverage values for each gene for this bin between the two conditions. P-values were adjusted for multiple testing with the Bonferroni method across all bins within each subfigure and are color-coded in the bottom track of subfigures: red = adj. P-value ≤ P-value ≤ P-value ≤ 0.05.Metagene analyses were performed as previously described73 on read counts determined with featureCounts for promoter regions (TSS ± 1.5 kb), gene bodies (TSS + 1.5 kb to TTS), and downstream regions (TTS to TTS + 25 kb) for all protein-coding and lincRNA genes in the Ensembl gene annotation. P-values were adjusted for multiple testing using the method by Benjamini and Hochberg. Distributions of log2 fold-changes for genes in individual clusters and specific types of genomic regions  were then compared either against all other protein-coding and lincRNA genes or against all other genes included in the dOCR analysis using two-sided Wilcoxon rank sum tests. P-values for Wilcoxon tests were adjusted for multiple testing with the Bonferroni method separately for each histone or histone modification mark and infection.Differential analysis was performed with edgeR74 using the Bioconductor package Gviz75 and the R packages gplots (for heatmaps) and ggplots . All statistical analyses were performed in R using the wilcox.test, fisher.test, and lm functions. Hierarchical clustering analysis was performed using the heatmap.2 function according to Euclidean distances and Ward’s clustering criterion. Boxplots were created with default parameters, and linear regression analysis in scatter plots was performed using the lm function.All figures apart from Supplementary Fig. Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting SummaryPeer Review File