And P63-Responsive Enhancer Regulates IFN-Κ Expression In

A TGF- −β and p63-Responsive Enhancer Regulates IFN- κ Expression in Human Keratinocytes

This information is current as Katrin Klein, Christina Habiger, Thomas Iftner and Frank of September 25, 2021. Stubenrauch J Immunol published online 14 February 2020 http://www.jimmunol.org/content/early/2020/02/13/jimmun ol.1901178 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 14, 2020, doi:10.4049/jimmunol.1901178 The Journal of Immunology

A TGF-b– and p63-Responsive Enhancer Regulates IFN-k Expression in Human Keratinocytes

Katrin Klein, Christina Habiger, Thomas Iftner, and Frank Stubenrauch

Type I IFNs have antiviral and immune-modulating activities. IFN-a/-b have very low basal expression levels but are strongly induced upon activation of pattern recognition receptors. In contrast, IFN-k is constitutively expressed in uninfected keratinocytes and responds only weakly to pattern recognition receptor activation. IFN-k expression has been implicated in the pathogenesis of inﬂammatory skin diseases and in limiting human papillomavirus replication in human keratinocytes. We have identiﬁed an enhancer ∼5 kb upstream of the IFNK gene driving its expression in keratinocytes. The enhancer consists of binding sites for the transcription factors jun-B, SMAD3/4, AP-2a/g, and p63, of which the latter two are key regulators of keratinocyte biology. The jun-B and SMAD3/4 elements confer activation by the TGF-b pathway. Furthermore, inhibition of ERK1/2 kinases activates

IFN-k expression. Our study provides a framework for the cell type–speciﬁc, constitutive expression of IFN-k and its modulation Downloaded from by signal transduction pathways in human keratinocytes. The Journal of Immunology, 2020, 204: 000–000.

nterferons are secreted cytokines that bind to membrane re- activated by virus infection or PRR signaling (5–10). IFN-ε is ceptors and initiate signal transduction cascades that result in constitutively expressed in the reproductive organs and can be I the increased transcription of several hundred IFN-stimulated induced by estrogen but not by PRR signaling or overexpression genes (ISG) (1, 2). ISG proteins inhibit a variety of steps during the of IRF3 or 7 (6). IFN-k is constitutively expressed in normal http://www.jimmunol.org/ infection and replication of many viruses, modulate the immune human keratinocytes (NHK), peripheral blood monocytes, and response, and have antitumor activities (1, 2). IFNs are classified monocyte-derived dendritic cells (7, 11). LaFleur et al. (7) according to the membrane receptor used: type I IFN bind to the reported a 3- to 4-fold induction of IFNK upon infection with IFNAR1/2 receptor, type II IFN bind to the IFNGR1/2 receptor, encephalomyocarditis virus 5 h postinfection, which increased and type III IFN bind to the IFNLR1/IL-10RB receptor (1, 2). The up to 10-fold at 15 h postinfection. Stimulation of NHK with type I IFN family in humans consists of more than 10 subtypes of poly(I:C), an activator of RIG-I, MDA5, and TLR3 pathways, for IFNA and single IFNB1,-E,-K, and -W1 genes. Type I IFN-a and 6 h resulted in only a 1.5-fold induction of IFNK, whereas IFNB1 -b and type III IFN-l are present at very low levels in many cell and -L1 were activated 1500- and 3000-fold, respectively (8). types but are strongly induced upon the activation of pattern Similarly, poly(dA:dT), which activates several cytoplasmic DNA by guest on September 25, 2021 recognition receptors (PRR), such as TLR and RIG-I–like recep- sensors and RIG-I, induced IFNK 3- to 4-fold in the human tors, and DNA sensors such as IFI16, cGAS, and RNA polymerase keratinocyte NIKS cell line, whereas IFNA,-B1,and-L1 were III (1, 2). PRR signaling cascades activate IFN regulatory factor activated 110-, 15,000- and 40,000-fold, respectively (12, 13). (IRF) 3 and 7, NF-kB family members p50 and p65, and a het- Semiquantitative analyses also suggested a strong induction of erodimeric complex of ATF-2 and Jun (1, 2). Different combi- IFN-b, but not of IFN-k, by Sendai virus infection in human nations of these transcription factors (TF) are required for the keratinocytes expressing the human papillomavirus (HPV) 16 E7 induction of IFNB1 (IRF3, p50/p65, ATF-2/Jun), IFNA (IRF7), protein (9). Furthermore, inhibitors of the epidermal growth factor and IFNL (IRF3, IRF7, p50/p65) (3, 4). (EGF) receptor (EGFR) and of MEK activate IFN-k and ISG In contrast to IFN-a,-b, and -l, IFN-ε and -k are expressed in expression in NHK (14, 15). a cell type–dependent manner and are not (or only very weakly) Increased expression of IFN-k has been implicated in the pathology of cutaneous lupus erythematosus and allergic contact dermatitis, whereas decreased expression has been observed in Institute for Medical Virology and Epidemiology of Viral Diseases, University psoriasis, atopic dermatitis, and high-risk (HR) HPV–infected Hospital Tuebingen, University of Tuebingen, D72076 Tuebingen, Germany lesions (9, 12, 16, 17). HR-HPV have also been shown to diminish ORCIDs: 0000-0002-5929-7078 (T.I.); 0000-0002-6685-299X (F.S.). IFN-k expression in cultivated keratinocytes (8, 9, 13, 18, 19). Received for publication September 26, 2019. Accepted for publication January 19, 2020. IFN-k repression is mainly caused by the HR-HPV E6 protein and involves an increase in DNA methylation at CpG islands in the This work was supported by a grant from the Wilhelm-Sander-Stiftung (2007.007.3 to F.S.). IFNK promoter (9, 18). Transcriptome analyses upon inducible Address correspondence and reprint requests to Prof. Frank Stubenrauch, University expression of IFN-k in HPV31-positive keratinocytes have Hospital Tuebingen, Elfriede-Aulhorn-Straße 6, 72076 Tuebingen, Germany. E-mail revealed that 71% of IFN-k target genes are known ISG, indi- address: [email protected] cating that its ability to regulate gene expression is similar to The online version of this article contains supplemental material. IFN-a and -b (20). IFN-k has anti-HPV activity, and the ISG Abbreviations used in this article: BS, binding site; ChIP, chromatin immunoprecip- Sp100 and IFI16 have been shown to limit HR-HPV replication itation; EGF, epidermal growth factor; EGFR, EGF receptor; FLuc, firefly luciferase; GLuc, Gaussia luciferase; HPV, human papillomavirus; HR, high-risk; HS, hyper- (20–23). Reconstitution of IFN-k expression in HR-HPV–positive sensitive; ISG, IFN-stimulated gene; mt, mutated; NHK, normal human keratinocyte; cells might therefore have therapeutic potential, and in line with PRR, pattern recognition receptor; qPCR, quantitative PCR; siRNA, small interfering this, a recent report suggested that TGF-b1 specifically induces RNA; TF, transcription factor; TFBS, TF binding site; wt, wild-type. IFN-k by demethylation of the IFNK promoter in HPV16-positive Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 cells but not in normal keratinocytes (18).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901178 2 REGULATION OF IFN-k EXPRESSION IN KERATINOCYTES

The cell type–specific expression of IFN-k and its deregulated poly(dA:dT) or poly(dI:dC)] and incubated at room temperature for 5 min. expression in pathologic conditions prompted us to investigate its Then, double-stranded 59-DY681–labeled oligonucleotides (40 fmol) transcriptional regulation. We have identified an enhancer element were added and incubated for 15 min at room temperature. Complexes were separated in 3 or 5% polyacrylamide-0.53 Tris-borate-EDTA gels. that controls IFNK expression in human keratinocytes. IFNK ex- Fluorescent signals were recorded with an Odyssey Fc Infrared Imaging pression requires binding sites (BS) for AP-2, SMAD3/4, jun-B, System (LI-COR Biosciences). and p63 proteins in this enhancer. AP-2 and p63 proteins are Compound treatments highly expressed in keratinocytes and control important aspects of keratinocyte biology and thus most likely contribute to the Cells were seeded in six-well plates and treated the next day with the keratinocyte-specific expression of IFN-k. SMAD3/4 proteins can respective substances or vehicle controls as listed in Supplemental Table I. be activated by the TGF-b signal transduction cascade (24), and After 24 h, RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and reverse-transcribed using consistent with this, the TGF-b pathway activates IFN-k expres- the QuantiTect Reverse Transcription Kit (Qiagen). Twenty-five nano- sion, which is dependent on both the SMAD3/4 and jun-B BS. grams of cDNA per well were used for quantitative RT-PCR using the Furthermore, inhibition of ERK1/2 kinases activates IFN-k ex- SYBR Green I Master Mix (Roche) and analyzed with the Light Cycler pression. Interestingly, the simultaneous activation by TGF-b1 480 Instrument (Roche). The primers are listed in Supplemental Table I. The efficiency of each primer pair was determined by serial dilution of and inhibition of ERK1/2 resulted in the highest levels of IFN-k in NHK-derived cDNA and melting curve analysis. The standard SYBR both normal and HR-HPV16–positive keratinocytes, which points green program of software version 1.5 was used for cDNA quantification, to possibilities to manipulate IFN-k expression in pathological the second derivate/max analysis was chosen to obtain crossing point settings. values, and melting curves were analyzed to ensure measurement of a single amplicon. Afterwards, crossing point values were converted to fold Downloaded from induction compared with vehicle control according to Pfaffl (27) and Materials and Methods PGK1 as the reference gene transcript. Cell culture Western blot analysis NHK were isolated from human foreskin after routine circumcision upon Cell extracts from TGF-b1– or ERKi-treated cells were obtained by lysing informed consent of patients, which was approved by the ethics committee cells in RIPA buffer (75 ml per well; 1% [v/v] Igepal CA-630, 1% [w/v] of the medical faculty of the University Tu¨bingen (6199/2018BO2) and sodium deoxycholate, 0.1% SDS [v/v], 150 mM NaCl, 10 mM sodium http://www.jimmunol.org/ performed according to the principles of the Declaration of Helsinki. NHK phosphate [pH 7.2], 2 mM EDTA, 50 mM sodium fluoride, cOmplete were cultured in keratinocyte serum-free medium supplemented with Protease Inhibitor mixture [Roche], PhosSTOP phosphatase inhibitors recombinant human EGF, bovine pituitary extract and gentamicin (Thermo [Roche]). For Nutlin-3a–treated cells, the flow-through from RNeasy Fisher Scientific). Keratinocyte cell lines carrying episomal HPV16 wild- columns was precipitated using acetone as described in the RNeasy Mini type (wt) genomes were maintained as previously described (25, 26). Handbook (Qiagen). The protein pellet was then resuspended in 60 mlof Plasmids Roti-Load (Carl Roth) and incubated at 95˚C for 5 min. Separated proteins were transferred onto 0.22-mm nitrocellulose membrane (Protran) in To generate the pGL3 25631 IFNK promoter construct (25631) sequences 10 mM CAPS and 10% (v/v) methanol (pH 10.3). Membranes were from 25631 to 21 relative to the ATG of the IFNK gene were amplified by blocked in 5% milk in either 0.05% PBS-Tween20 or 0.1% TBST. PCR from human genomic DNA and inserted by restriction enzyme Membranes were incubated over night with the primary Abs cloning into the promoterless pGL3-basic plasmid (Promega), which en- (Supplemental Table I) at 4˚C. Secondary fluorescence-labeled Abs (1: by guest on September 25, 2021 codes firefly luciferase (FLuc). To generate the 27502 construct, se- 15,000; LI-COR) were added for 1 h, and signals were detected using the quences from 27502 to 25631 were amplified by PCR from genomic Odyssey Fc Infrared Imaging System (LI-COR Biosciences). DNA and inserted by restriction enzyme cloning into the 25631 construct. Additional IFNK promoter plasmids are derived from the 25631 con- Small interfering RNA knockdown experiments struct by deleting or mutating sequence stretches by overlap extension Cells were seeded in six-well plates the day before transfection. Small PCR– and/or restriction enzyme–based cloning strategies. All inserts interfering RNA (siRNA) (90 pmol per well; Supplemental Table I) was and manipulated sequences were verified by DNA sequencing (Eurofins transfected with Lipofectamine RNAiMAX (Thermo Fisher Scientific) Genomics). The sequences of all plasmids used in this work are available according to the manufacturer’s instructions. Cells were harvested 48 h upon request. after transfection and subjected to quantitative PCR (qPCR) analysis as Luciferase reporter assays described above. Proteins from the flow-through were acetone-precipitated following the protocol in the RNeasy Mini Handbook (Qiagen). After- Cells were seeded in 24-well plates and transfected the next day using wards, proteins were subjected to Western blot analysis as described the FuGene HD Transfection Reagent (Promega). Cells were transfected above. If detection was not feasible from acetone-precipitated proteins, with 300 ng of the different FLuc reporter constructs and 0.5 ng of pCMV– nuclear extracts (as described in the EMSA section) were prepared. Gaussia luciferase (GLuc) (New England Biolabs) per well in duplicates. GLuc activity was assessed 48 h after transfection using the Gaussia Juice Chromatin immunoprecipitation Big Kit (PJK Biotech). FLuc activity was determined after the addition of For chromatin immunoprecipitation (ChIP) assays, NHK were seeded on a lysis buffer (100 mM potassium phosphate buffer [pH 7.8], 1% [v/v] 15-cm dishes and grown to 80% confluency. Cells were treated for 4 h with Triton-X 100, 1 mM DTT). Relative luminescent units were measured 20 ng/ml TGF-b1 and then processed according to the manual of the using a Lumat LB 9507 Luminometer (Berthold Technologies). Simple ChIP Enzymatic Kit (Magnetic Beads; Cell Signaling Technology). EMSA Immunoprecipitation was performed for 4 h at 4˚C with the Abs listed in Supplemental Table I. qPCR analysis using primers listed in Supplemental Nuclear extracts were prepared by resuspending cell pellets in cold lysis Table I was performed as described above. buffer (10 mM HEPES [pH 7.9], 300 mM saccharose, 50 mM NaCl, 0.5% [v/v] Igepal CA-630, 1 mM EDTA, cOmplete Protease Inhibitor Mixture [Roche], PhosSTOP phosphatase inhibitors [Roche], 1 mM DTT). Nuclei Results were extracted using an elution buffer (10 mM HEPES [pH 7.9], 20% [v/v] Identification of a putative NHK-specific regulatory element in glycerol, 500 mM NaCl, 0.5 mM EDTA, cOmplete Protease Inhibitor the IFNK locus Mixture [Roche], PhosSTOP phosphatase inhibitors [Roche], 1 mM DTT). Debris was removed by centrifugation (20,000 3 g, 5 min, 4˚C) and su- To identify putative regulatory regions, the human IFNK locus was pernatant was transferred to precooled tubes and stored at 280˚C until analyzed using the Encyclopedia of DNA Elements database. We usage. The oligonucleotides used (Thermo Fisher Scientific or Biomers) compared data from NHK with GM12878 (an EBV-transformed can be found in Supplemental Table I. Nuclear extracts were mixed with 4-pmol unlabeled double-stranded oligonucleotides and Ab (Supplemental lymphoblastoid cell line), H1-hESC (H1 human embryonic stem Table I), where indicated, in a binding buffer [20 mM HEPES (pH 7.9), cell line), K562 (human immortalized myelogenous leukemia cell 0.5 mM EDTA (pH 8), 5% (v/v) glycerol, 1 mM DTT and 50 mg/ml line), HSMM (human skeletal muscle myoblasts), HUVECs, and The Journal of Immunology 3 normal human lung fibroblasts. Only in NHK, significant tran- to 40%, deletion of HS 2 reduced activity to 16%, and deletion of scription of the two exons of IFNK occurred, consistent with HS 3 reduced activity to 37% (Fig. 1C). In contrast to this, the previously published data [Fig. 1A, (7)]. In contrast, the first exon deletion of the region between HS 1 and HS 2 resulted in a 5-fold of the MOB3B gene, which is located ∼3 kb downstream of IFNK induction of reporter activity (Fig. 1C). This indicated that each and is transcribed in antisense orientation, is transcribed in all HS region contributes to transcriptional activity. analyzed cell types (Fig. 1A). We then analyzed the occurrence of Five putative transcription factor binding sites located in the epigenetic marks associated with active enhancer elements in the IFNK enhancer contribute to promoter activity IFNK locus (28). This revealed that two regions outside the IFNK gene are enriched for histone 3 lysine 27 acetylation marks To identify candidate TF in HS 1–3 the JASPAR CORE [H3K27Ac; Fig. 1A, (28)]. In contrast to other cell types, which vertebrata database of TF binding profiles was used (http:// do not express IFNK, NHK display an increased H3K27Ac signal jaspar.genereg.net). This analysis identified putative FOXA1 (posi- in a region located upstream of the IFNK transcription start site tion 24794/24784) and AP-2a/g BS (position 24696/24681) in between 25800 and 23300 relative to the IFNK start codon HS 1, HOX family member (position 24204/24194), AP-1 family (Fig. 1A). The second H3K27Ac-enriched region is located im- (position 24165/24158) and an inverted tandem repeat SMAD3/4 mediately upstream of exon 1 of the MOB3B gene and is present BS (position 24149/24139) in HS 2, and two p53/p63 BS (position in all analyzed cell types and thus might be a regulatory region 24015/23996 and 23941/23922) in HS 3 (Supplemental Fig. 1). controlling MOB3B expression (Fig. 1A). Furthermore, only in To analyze the contribution of these putative BS, point mutations NHK does transcription occur throughout the H3K27Ac-enriched were created in the context of the 25631 construct. The mutated region upstream of the IFNK gene, which may represent enhancer- (mt)_FOX displayed an activity similar to the wt 25631 construct Downloaded from associated RNAs (29, 30). Additionally, DNase I–hypersensitive (Fig. 1C). In contrast, mt_AP-2 reduced activity to 10%, mt_HOX (HS) sites, which identify accessible chromatin, are present only reduced activity to 12%, mt_AP-1 reduced activity to 18%, in NHK in both areas with enriched H3K27Ac marks and at the mt_SMAD3/4 reduced activity to 30%, mt_p63 reduced activity beginning of the first exon of IFNK. Although there are three to to 50%, and mt_p63#2 reduced activity to 19% of the wt 25631 four HS regions in the H3K27ac-enriched region downstream of construct (Fig. 1C).

IFNK in all analyzed cell types, three HS regions in the H3K27ac- To determine if nuclear proteins from keratinocytes bind to these http://www.jimmunol.org/ enriched region upstream of IFNK are only present in NHK, and sequences, EMSAs were carried out. The putative AP-2 BS formed one of those is unique to NHK (Fig. 1A). Cell type–specific several complexes, of which only one was competed away by a DNase I–HS sites are often enriched in enhancer elements that 100-fold excess of unlabeled wt oligonucleotide but not by mt control cell type–specific gene expression (31). In summary, the oligonucleotide (Fig. 2A). The addition of Abs to AP-2a or AP-2g NHK-specific presence of HS sites, enrichment of H3K27ac, and resulted in the appearance of supershifts, which suggests that the presence of putative enhancer-associated RNAs in the region be- AP2 sequence can be bound by both AP-2a and AP-2g (Fig. 2B). tween 25800 and 23300 strongly suggests that this region may Despite repeated attempts, we were not able to detect specific represent an NHK-specific enhancer responsible for the tran- binding of nuclear proteins to the putative HOX BS (data not scription of IFNK. shown). Therefore, it is unclear if the effect of the mt HOX BS on by guest on September 25, 2021 promoter activity is due to the loss of binding of a TF. Using the Deletion analysis of the 59-upstream region of IFNK gene putative AP-1 BS, two specific complexes were observed and were indicates that the H3K27ac-enriched DNase I–HS regions competed by the wt (but not the mt) oligonucleotides, suggesting represent an enhancer for IFNK expression that they contribute to transcriptional activity (Fig. 2A). The pu- To test this hypothesis, a fragment from 27502 bp relative to the tative SMAD3/4 BS gave rise to one specific complex (Fig. 2A). ATG of the IFNK gene up to the ATG of IFNK was cloned into the For the p63#2 BS, the binding of two complexes could be ob- promoterless pGL3-basic plasmid. The resulting pGL3 IFNK served that were also competed by the wt (but not the mt) oli- promoter 27502 plasmid (27502) was transfected into NHK, gonucleotides, suggesting that both contribute to transcriptional which revealed that it displayed a 9-fold increase in luciferase activity (Fig. 2A). Taken together, the data suggest that the loss of activity than the parental plasmid and a 5-fold increase in activity promoter activity is due to the loss of binding of nuclear proteins compared with an IFNB promoter reporter construct (Fig. 1B), to the putative AP-2, AP-1, SMAD3/4, and the p63 BS in the confirming that this region contains regulatory elements driving IFNK enhancer. IFNK promoter activity. To identify cis-elements necessary for b constitutive activity, 59 deletions of the 27502 construct were The TGF- pathway activates, and the ERK1/2 pathway generated and tested. The pGL3 25631 IFNK promoter (25631) inhibits, endogenous IFNK transcription in NHK construct had an 11-fold increase in activity than the parental Members of the AP-1 family and SMAD3/4 can be regulated by plasmid and was used as the reference construct for all further signal transduction pathways. The AP-1 TF is a dimeric complex experiments (Fig. 1B). A 59 deletion up to 24820 did not sig- composed of members from different protein families (JUN, FOS, nificantly diminish promoter activity. However, starting at 24785, ATF, MAF) whose activities can be regulated by MAPK signaling the activity of the reporter constructs constantly declined, and the (32). The addition of 40 mM T-5224, a small molecule inhibitor of 24331 deletion displayed an activity identical to the empty vector FOS/JUN DNA-binding activity (33), for 24 h reduced IFNK (Fig. 1B). This indicates that critical elements for constitutive transcription to 34% (Fig. 3A). The JUN family members Jun, IFNK promoter activity are located between 24785 and 24331, jun-B, and jun-D can be phosphorylated by JNK, which enhances which correlates well with the center of the H3K27ac-enriched their transactivation ability (34). The addition of 4 mM JNK-IN-8 region and coincides with the 59 end of HS 1 at 24811. (an inhibitor of JNK1, 2, and 3) did not alter IFNK expression in To elucidate the contribution of the different HS regions for gene NHK (Fig. 3A). ERK1/2 kinases can phosphorylate different AP-1 activity, internal deletions of HS 1 (24811 to 24660), the region members and thereby modulate their transactivation (35). The between HS 1 and HS 2 (24661 to 24250), HS 2 (24251 to ERK1/2 pathway can be stimulated by tetradecanoylphorbol- 24100), and HS 3 (24046 to 23895) were created in the back- acetate (TPA), and ERK1/2 activity can be blocked by inhibi- ground of the 25631 plasmid. Deletion of HS 1 reduced activity tors such as SCH 772984 (labeled throughout the text as ERKi). 4 REGULATION OF IFN-k EXPRESSION IN KERATINOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Identiﬁcation of a regulatory element in the IFNK locus. (A) Modiﬁed screenshot of the Encyclopedia of DNA Elements (ENCODE) at the University of California, Santa Cruz genome browser highlighting a putative regulatory element upstream of the IFNK gene (red brace). Transcription-, H3K27Ac-, and DNase I–HS sites (gray boxes) are depicted for different cell lines, that either do not have detectable IFNK transcription (top) or for NHK (bottom), which express IFNK transcripts. (B and C) NHK were transfected with the indicated constructs, and luciferase activity was assayed 48 h after transfection. Luciferase activity is shown relative to the activity of the 25631 reporter construct (highlighted in light gray). FLuc activity was normalized to the expression of a cotransfected GLuc construct. Bars represent the average values, and error bars represent the SEM. The average values for individual constructs in (B) are based on n =23(24659), n =18(25381, 25131, 24881), n =17(21341), n =16(24168), n =15(27502), n =13 (24743, 24578, 23540, 22890), n =11(24679, 24504), n =9(22224), n =6(25117, 24976), n =5(25077, 24942, 24785, 24331, 24269), n =3 (24860, 24820), and n =20(IFNB promoter) independent experiments. The average values in (C) were calculated from (Figure legend continues) The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. Proteins present in nuclear extracts of NHK bind speciﬁcally to the putative TFBS for AP-2, SMAD3/4, AP-1, and p63. (A) EMSAs were performed with nuclear extracts from NHK- and DY681-labeled oligonucleotides displaying putative TF BS as indicated above the gels. Unlabeled competitors were added in 100-fold excess. Speciﬁc shifts that were only competed by excess wt, and not by mt, oligonucleotide are indicated by arrows. (B) Complexes bound to the AP-2 oligonucleotide that can be supershifted by the addition of AP-2a (a) or AP-2g (g) Abs, but not by a control Ab (ctrl), are indicated by arrows. All experiments were performed with extracts from NHK derived from three different donors to ensure reproducibility and representative EMSAs are shown. by guest on September 25, 2021

The addition of 50 ng/ml TPA reduced IFNK expression 2-fold in In addition, the ISG IFIT1 was activated 3-fold by TGF-b NHK, whereas 1 mM ERKi increased its expression 4.7-fold and inhibited 1.9-fold by TGFBR1i (Fig. 3B). This suggested (Fig. 3A). These findings are consistent with the observation that that TGF-b–induced IFN-k is biologically active. This was further the inhibition of the EGFR tyrosine kinase activity or of MEK confirmed by an increase in pY701-STAT1 protein levels, which activity leads to an increase of IFN-k expression and ISG ex- indicates enhanced IFNAR1/2 signaling (Fig. 3C). Furthermore, pression in NHK (14, 15) because ERK1/2 are activated by EGFR transcriptional changes for IFIT1 and IFNK could be confirmed at via MEK (36, 37). These data also suggest that the putative AP-1 the protein level (Fig. 3C). The combination of TGF-b1 and ERKi BS is not activated by TPA, ERK1/2, or JNK. induced IFNK RNA 24.8-fold and displayed the highest levels of SMAD3 and 4 are crucial components of the TGF-b signaling IFN-k and IFIT1 proteins (Fig. 3C). network. Binding of TGF-b to the TGFBR1 and TGFBR2 induces TGFBR1 kinase activity, which then phosphorylates SMAD2 and The AP-1 and SMAD3/4 BS represent a b 3. Phospho-SMAD2 and 3 form complexes with SMAD4, which TGF- –responsive element then enter the nucleus and bind to SMAD3/4 BS to regulate The loss of activity of the mt_AP-1 reporter construct and the transcription (24). The addition of 20 ng/ml TGF-b1or10mMof treatment with T-5224 strongly suggested an involvement of JUN the TGFBR1 kinase inhibitor SB-431542 (labeled throughout the and/or FOS family members in IFNK gene activation. Published text as TGFBR1i) induced or repressed SERPINE1, an established evidence indicates that AP-1 BS can mediate TGF-b responsive- TGF-b target gene (38), 10- and 4.6-fold, respectively (Fig. 3B). ness of promoters (39–42). To test this, NHK were transfected This indicates that the TGF-b pathway is constitutively activated with the 25631, the deleted HS 2 (which lacks both the AP-1 in cultured NHK and can be further stimulated by adding exog- and SMAD3/4 BS), the mt_AP-1, or the mt_SMAD3/4 reporter enous TGF-b1. IFNK expression was induced 3.5-fold in NHK by constructs (Fig. 3D). The addition of TGF-b1 only stimulated the TGF-b1 and 3.6-fold repressed by TGFBR1i (Fig. 3B), strongly 25631 construct 6.6-fold but not mt_AP1, mt_SMAD3/4, or de- suggesting that IFN-k is a TGF-b target gene in keratinocytes. leted HS2. Consistent with this, the addition of TGFBR1i only

n = 5 (del HS 1, del HS 2, del HS 3, mt_SMAD3/4), n = 4 (deleted 24661/24250, mt_AP-2, mt_AP-1), and n = 3 (mt_FOX, mt_HOX, mt_p63, mt_p63#2) independent experiments. Signiﬁcant deviations from the 25631 construct are denoted with asterisks. One-sample t test. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. del, deleted. 6 REGULATION OF IFN-k EXPRESSION IN KERATINOCYTES Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. ERK1/2 represses IFNK expression, whereas the AP-1 and SMAD BS constitute a TGF-b–responsive element, which confers production of biologically active IFN-k.(A and B) NHK were treated for 24 h with the indicated substances and then IFNK, IFIT1, and SERPINE1 transcripts were quantified by qPCR using PGK1 as a reference gene and are given relative to the vehicle control. Error bars indicate the SEM. The average values in (A) were calculated from n = 3 (TPA), n = 8 (ERKi), n = 4 (T-5224), and n = 3 (JNKi) and in (B) from n = 6 independent experiments. Significant deviations from the vehicle control are highlighted with asterisks. Paired t test: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. (C) Western blot analysis of NHK extracts treated for 24 h with the indicated substances using Abs against IFN-k, STAT1, phospho–Y701-STAT1, IFIT1, and tubulin as a loading control. The blot shown is representative of two independent experiments using extracts derived from different NHK donors. (D) NHK were transfected with the indicated reporter plasmids, TGF-b1 or TGFBRi were added 24 h later, and luciferase activities were determined 24 h after treatment. Bars represent the average of n = 4 independent experiments and error bars represent the SEM. Asterisks denote statistically significant differences from the vehicle control. Multiple t tests, corrected for multiple comparisons using the Holm–Sidak method. **p , 0.01, ****p , 0.0001. reduced the activity of 25631 construct 2.7-fold but not of the activation of the 25631 construct (Supplemental Fig. 2). Taken mutant reporter constructs (Fig. 3D). These data indicate that the together, this indicates that the ERKi does not target specific TF AP-1 BS is also required for TGF-b responsiveness. binding sites (TFBS) in the enhancer, but rather acts both on the To identify cis-elements required for activationbytheERKi,NHK enhancer and promoter of the IFNK gene. were transfected with the 25631, deleted HS 1, deleted 24661/24250, deleted HS 2, deleted HS 3, and 23540 constructs and incubated IFNK expression is diminished upon jun-B, SMAD4, and with ERKi or vehicle for 24 h. This revealed that the 25631 p63 knockdown construct was activated 3.1-fold, which is comparable to the en- To identify the TF responsible for IFNK expression, RNA inter- dogenous gene. Similar activation levels were observed for the ference was used. The levels of TFAP2A and -C could be effi- deletion constructs. The 23540 construct displayed only an acti- ciently reduced by siRNA, but IFNK expression was weakly vation of 1.7-fold, but this was not significantly different from the induced by siTFAP2A and not significantly altered by siTFAP2C, The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. IFNK transcription depends on junB, SMAD4, and p63. Transcript levels of IFNK (A) or the targets of the respective siRNAs (B) were determined 48 h post transfection by qPCR using PGK1 as a reference gene and are given relative to sicontrol. The average values were calculated from n = 3 (siTFAP2 family members, siAP-1 family members), n = 4 (siSMAD4, siTP63), and n = 5 (siTP53) independent experiments. Error bars indicate the SEM. Significant deviations from the control are highlighted with asterisks. (A and B) Paired t test: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. (C) NHK were treated with Nutlin-3a for 24 h and mRNA levels of IFNK were determined by qPCR using PGK1 as a reference gene and are given relative to the vehicle control. Averages were calculated from n = 4 independent experiments. Error bars indicate the SEM. Significant deviations from the vehicle control are highlighted with asterisks. Paired t test: *p , 0.05, ***p , 0.001. (D) Western blot analysis of NHK extracts treated with 10 or 20 mM Nutlin-3a for 24 h using Abs against p53, p63, and GAPDH as a loading control. The blot shown is representative for two independent experiments using extracts derived from different NHK donors. (E) ChIP analyses were performed using chromatin isolated from NHK treated with TGF-b for 4 h. Binding of AP-2a, jun-B, SMAD4, or histone 3 (H3) to HS 1, HS 2, or SERPINE1 promoter DNA sequences was analyzed after immunoprecipitation with AP-2a, jun-B, SMAD4, or H3 Abs and then DNA sequences were quantified by qPCR. IgG served as a negative control. Bars represent the average of n = 3 independent experiments and are shown relative to chromatin input. Error bars indicate the SEM. respectively (Fig. 4A, 4B, Supplemental Fig. 3B). Because the that this knockdown diminishes both activators and inhibitors of EMSA experiments suggested that the AP-2 BS can interact with IFNK expression. both AP-2a and -2g, a combination of siTFAP2A and -C was Knockdown efficiencies for the AP-1 family members FOS, tested, but this also resulted only in a slight reduction to 79% of FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND were between IFNK mRNA levels, which was not significant (data not shown). 8 and 45% (Fig. 4B). Of those, only the knockdown of JUNB Knockdown of TFAP2E, another AP-2 family member, also had significantly reduced IFNK expression to 44%, whereas knock- no influence on IFNK mRNA levels (data not shown). EGFR down of FOSB induced IFNK expression 3.5-fold (Fig. 4A). A expression decreases upon TFAP2C knockdown in breast cancer knockdown of SMAD4 (the co-SMAD included in SMAD2/3/4 cells (43, 44). Indeed, knockdown of TFAP2A and -C separately trimers) reduced IFNK mRNA expression to 53% (Fig. 5). Be- or together significantly diminished EGFR mRNA expression cause p53 and p63 recognize similar consensus sequences, both (Supplemental Fig. 3A). Because the EGFR-MEK-ERK1/2 path- TP53 and TP63 were tested using siRNAs, which target all iso- way inhibits IFN-k expression, a possible explanation for the weak forms. This revealed a major effect for the TP63 knockdown, effects of the AP-2a or AP-2g knockdown on IFNK expression is whereas the knockdown of TP53 had only very minor effects, 8 REGULATION OF IFN-k EXPRESSION IN KERATINOCYTES

was determined by qPCR treated with TGFBR1i, TGF-b1, ERKi, or Nutlin-3a or transfected with siTFAP2A +-C or siTP63. TGF-b treatment did not significantly induce IFNA2, IFNB1,orIFNE (Fig. 5). IFNE, but not IFNA2 or IFNB1, expression was inhibited by TGFBR1i (Fig. 5). However, the reduction of IFNE by TGFBR1i was much weaker (1.8-fold) compared with IFNK (3.6-fold). In contrast to IFNK, ERKi reduced IFNA2 and IFNE expression ∼2-fold and had no effect on IFNB1 transcription. Consistent with this, the combination of ERKi and TGF-b, which greatly enhances IFN-k expression, did not induce IFNA2,-B1,or -E. Addition of Nutlin-3a weakly increased expression of IFNA2 and IFNE (1.9- and 1.4-fold, respectively), which is in sharp contrast to the pronounced repression of IFNK. The knockdown of TFAP2A +-C or TP63 had no effect on IFNE expression but induced both IFNA2 and -B1 to similar, yet not statistically significant, extents (Fig. 5). Taken together, these data strongly indicate that the regulation of IFNK expression by the TGF-b1, ERK1/2, p63, and p53 pathways in NHK is unique for this type I IFN. Downloaded from Activation of IFNK expression in HPV16-positive keratinocytes FIGURE 5. The regulation by TGF-b1, ERK1/2, p53, and p63 pathways results in the induction of IFIT1 and reduces viral transcription is specific for IFNK. NHK were treated with the indicated substances for To address if IFN-k expression can also be activated under path- 24 h or transfected with the indicated siRNAs for 48 h. IFNA2, IFNB1, and ological conditions, three different human keratinocyte cell lines IFNE mRNA levels were determined by qPCR. Values given are relative to the vehicle control using PGK1 as a reference gene and represent averages with persistently, autonomously replicating HPV16 genomes (HK from n = 8 (TGF-b1, ERKi), n = 6 (TGFBRi, TGF-b1 + ERKi), n =4 HPV16) described previously (25, 26) were treated with TGF-b1, http://www.jimmunol.org/ (Nutlin-3a, siTP63), and n = 3 (siTFAP2A,-C) independent experiments. ERKi, or a combination thereof (Fig. 6). IFNK expression could Significant deviations from the control are highlighted with asterisks. be induced 6.2-fold by TGF-b, which is consistent with obser- Paired t test: *p , 0.05, **p , 0.01, ****p , 0.0001. vations by Woodby et al. (18), and enhanced compared with NHK (3.5-fold). In contrast, ERKi and ERKi + TGF-b induced IFNK expression 2.8-fold and 11.8-fold, respectively (Fig. 6), which is despite similar knockdown efficiencies (Fig. 4A, 4B, Supplemental weaker than in NHK (4.9- and 24.8-fold). Consistent with an Fig. 3B). To further elucidate if p53 is involved in IFNK expression, activation of the type I IFN signaling pathway, the ISG IFIT1 was Nutlin-3a, which increases p53 protein amounts by disrupting the significantly activated by all treatments and the extent of induction interaction between p53 and the E3 ubiquitin ligase MDM2, was correlated well with the levels of IFNK (Fig. 6). The analysis of by guest on September 25, 2021 used (45). Interestingly, p53 stabilization repressed IFNK expression, spliced viral transcripts E6*I and E1Ê4 revealed that TGF-b suggesting p53 is not involved in the activation of the gene (Fig. 4C). alone repressed both transcripts in a similar manner to 44 and In line with observations that p53 can inhibit p63 expression in 37%, respectively (Fig. 6). ERKi only significantly reduced E6*I, certain settings (46, 47), the levels of p63 protein were greatly di- but not E1Ê4, levels (Fig. 6), and these effects were weaker than minished upon Nutlin-3a treatment (Fig. 4D). These data suggest with TGF-b alone (66 versus 44%). The combination of TGF-b that IFNK is activated by p63 and repressed by p53 in keratinocytes. and ERKi repressed viral transcription that was slightly more AP-2a, jun-B, p63, and SMAD4 proteins bind to the efficient than TGF-b alone, but this was not significantly different IFNK enhancer in vivo (Fig. 6). In summary, these data show that IFNK can be more To establish that AP-2a, jun-B, SMAD4, and p63 regulate IFNK efficiently induced by the combination of TGF-b1 and ERKi in expression by binding to the enhancer, data mining was performed HPV16-positive cell lines and that this combination also represses for p63, and ChIP experiments were carried out for AP-2a, jun-B, viral transcription. and SMAD4. Analyzing two published NHK/p63 ChiP-sequencing datasets revealed that fragments Chr9:27,520,028-27,520,520 (48) Discussion and Chr9:27,519,197-27,520,919 (47), which encompass the p63 The skin is the first barrier an invading pathogen has to overcome. BS #1 and #2 (Chr9:27,520,318-27,520,411), are bound by p63 The major cell type in the epidermis are keratinocytes, which in vivo. ChIP experiments were carried out after stimulating NHK not only express a large variety of PRR to activate inducible IFN for 4 h with TGF-b1 using Abs to AP-2a, SMAD4, and jun-B. (50–52), but also constitutively express IFN-k (7). Its constitutive This revealed that AP-2a binding to the HS 1 region is 16-fold– expression enables expression of different ISG, such as PRR and enriched compared with the isotype control (IgG; Fig. 4E). In HS components of IFN signaling pathways (8, 9, 16). Thus, IFN-k 2, jun-B as well as SMAD4 proteins were found enriched 18-fold expression most likely supports immediate reactions to infections. and 4-fold relative to IgG, respectively. Similar enrichment of In this study, we show that IFN-k expression is controlled by an jun-B and SMAD4 binding was found at a published BS in the enhancer element located 5 kb upstream of the IFNK gene, which SERPINE1 promoter [Fig. 4E, (49)]. These results suggest that consists of functional BS for AP-2a/g, jun-B, SMAD3/4, and p63. AP-2a, jun-B, SMAD4, and p63 bind to the IFNK enhancer Although we cannot exclude that additional regulatory elements in vivo and activate IFNK expression. for IFNK expression exist, the identified TF provide a framework for both the cell type–specific expression of IFN-k and its mod- b The regulation by TGF- , ERK1/2, p53, and p63 pathways is ulation by extracellular ligands and signal transduction path- specific for IFNK ways in keratinocytes. Functional elements in the IFNK enhancer To address if the identified pathways also regulate other type I IFNs differ significantly from those in the IFNB enhancer. The size of such as IFNA2, IFNB1, and IFNE in NHK, their RNA expression the IFNB enhancer is only 55 bp, and it is comprised of tightly The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 6. TGF-b1 and ERKi induce IFNK expression in HPV16-positive keratinocytes. RNA levels of IFNK, IFIT1, HPV16 E6*I, and HPV16 E1^E4 transcripts were determined by qPCR analyses after treatment of three different HPV16-positive cell lines with the indicated substances for 24 h (n = 3 for each cell line). Values given are relative to the vehicle control, using PGK1 as a reference. Error bars indicate the SEM. Signiﬁcant deviations from the vehicle control are highlighted with asterisks. Paired t test: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

clustered TFBS that form an enhanceosome (53), whereas the which DNp63a is the most abundant isoform in NHK (59). In line by guest on September 25, 2021 IFNK enhancer consists of multiple elements spread over 900 bp. with this, our immunoblotting analysis using an Ab that recog- The only common motif among both enhancers are functional nizes all p63 isoforms detected only one band with a molecular BS for the AP-1 TF family. However, the IFNK AP-1 BS mass of ∼70 kDa, which decreased upon siRNA treatment and (TGAATCA) belongs to the TPA response element (TRE) family thus most likely represents DNp63a. In summary, these findings and is recognized by jun-B, whereas the IFNB AP-1 BS (TGA- strongly support the concept that the combination of AP-2a/g, CATAG) belongs to the cAMP response element family and is p63, jun-B, and SMAD3/4 BS confers keratinocyte specificity and bound by Jun/ATF-2 (32, 54). Our data indicate that the AP-1 BS TGF-b inducibility of the IFNK enhancer (Fig. 7). and SMAD3/4 BS both mediate TGF-b responsiveness of IFNK Interestingly, IFNK expression cannot only be activated but also expression, consistent with findings that AP-1 TPA response ele- be inhibited. Our findings that TPA represses and an ERKi acti- ment sequences can be found in the proximity of SMAD2/3 re- vates IFNK expression are consistent with observations that in- sponse elements (39–42, 49). Furthermore, SERPINE1 expression hibition of EGFR or MEK increases IFNK expression (14, 15), requires the binding of both jun-B and SMAD2/3 to be strongly strongly suggesting that the EGFR-MEK-ERK1/2 pathway nega- activated by TGF-b (49). Taken together, this strongly suggests tively regulates IFN-k expression. The mechanistic basis for this that IFNK is a bona fide TGF-b target in normal keratinocytes. In addition, AP-2a/g and p63 are important for constitutive IFNK expression. Interestingly, genome-wide analyses of keratinocytes revealed that AP-2 BS are significantly enriched near SMAD2/3 BS and that knockdown of TFAP2A changes the global transcription profile induced by TGF-b (41). Our data show for the first time, to our knowledge, that p63 directly activates IFNK but not other type I IFNs in keratinocytes. Interestingly, p63 regulates keratinocyte differentiation and activation of the keratinocyte- specific C40 enhancer in conjunction with AP-2a/g (55, 56). In line with these findings, expression of ΔNp63a, the predominant p63 isoform in basal keratinocytes (57–59), and AP-2a together with GRHL2 and c-myc is sufficient to reprogram mouse mes- enchymal cells into keratinocyte-like cells (60). A large body of additional evidence also indicates that AP-2a/g and p63 are crucial players that regulate many aspects of keratinocyte biology FIGURE 7. Model for the regulation of IFNK expression in keratinocytes. (57, 58, 61–65). The TP63 gene encodes multiple p63 proteins, of Numbers indicate the distance in nt from the start codon of the IFNK gene. 10 REGULATION OF IFN-k EXPRESSION IN KERATINOCYTES effect remains unclear. Using reporter constructs, we were unable 4. Iversen, M. B., and S. R. Paludan. 2010. Mechanisms of type III interferon expression. J. Interferon Cytokine Res. 30: 573–578. to identify an ERK1/2-responsive element in the IFNK enhancer, 5. Hermant, P., C. Francius, F. Clotman, and T. Michiels. 2013. IFN-ε is consti- indicating that ERK1/2 does not inhibit IFNK expression via jun- tutively expressed by cells of the reproductive tract and is inefficiently secreted B, SMAD3/4, AP-2a/g, or p63. Further evidence that ERKi does by fibroblasts and cell lines. PLoS One 8: e71320. 6. Fung, K. Y., N. E. Mangan, H. Cumming, J. C. Horvat, J. R. Mayall, S. A. Stifter, not primarily activate the TGF-b pathway in NHK comes from the N. De Weerd, L. C. Roisman, J. Rossjohn, S. A. Robertson, et al. 2013. Inter- observation that the TGF-b target gene SERPINE1 is inhibited by feron-ε protects the female reproductive tract from viral and bacterial infection. ERKi (Fig. 3B). A previous study reported that IRF1 is induced Science 339: 1088–1092. 7. LaFleur, D. W., B. Nardelli, T. Tsareva, D. Mather, P. Feng, M. Semenuk, by PD168393, an EGFR inhibitor, and that knockdown of IRF1 K. Taylor, M. Buergin, D. Chinchilla, V. Roshke, et al. 2001. Interferon-k,a attenuated IFN-k expression (15). Because the IFNK enhancer is novel type I interferon expressed in human keratinocytes. J. Biol. Chem. 276: 39765–39771. not ERK1/2 responsive, we believe it is unlikely that IRF1 directly 8. Reiser, J., J. Hurst, M. Voges, P. Krauss, P. Mu¨nch, T. Iftner, and F. Stubenrauch. binds to the enhancer. However, we cannot exclude that functional 2011. High-risk human papillomaviruses repress constitutive kappa interferon IRF1 BS exist outside the IFNK enhancer. transcription via E6 to prevent pathogen recognition receptor and antiviral-gene expression. J. Virol. 85: 11372–11380. TGF-b is involved in wound healing (66). Therefore, activation 9. Rincon-Orozco, B., G. Halec, S. Rosenberger, D. Muschik, I. Nindl, of IFN-k expression by the TGF-b pathway might be relevant A. Bachmann, T. M. Ritter, B. Dondog, R. Ly, F. X. Bosch, et al. 2009. Epi- in vivo when wounding occurs because this increases the likeli- genetic silencing of interferon-k in human papillomavirus type 16-positive cells. Cancer Res. 69: 8718–8725. hood that basal keratinocytes may encounter pathogens, such as 10. Hardy, M. P., C. M. Owczarek, L. S. Jermiin, M. Ejdeba¨ck, and P. J. Hertzog. HPV (67). 2004. Characterization of the type I interferon locus and identification of novel genes. Genomics 84: 331–345. A recent study suggested that TGF-b1 activates IFNK expres- 11. Nardelli, B., L. Zaritskaya, M. Semenuk, Y. H. Cho, D. W. LaFleur, D. Shah, sion only in HPV16-positive keratinocytes, but not in uninfected S. Ullrich, G. Girolomoni, C. Albanesi, and P. A. Moore. 2002. Regulatory effect Downloaded from keratinocytes, by inducing the demethylation of a CpG island in of IFN-k, a novel type I IFN, on cytokine production by cells of the innate immune system. J. Immunol. 169: 4822–4830. the IFNK promoter (18). In contrast, our data provide evidence 12. DeCarlo, C. A., A. Severini, L. Edler, N. G. Escott, P. F. Lambert, M. Ulanova, that the TGF-b signaling pathway regulates IFN-k expression in and I. Zehbe. 2010. IFN-k, a novel type I IFN, is undetectable in HPV-positive uninfected keratinocytes and that this is independent from pro- human cervical keratinocytes. Lab. Invest. 90: 1482–1491. 13. Albertini, S., I. Lo Cigno, F. Calati, M. De Andrea, C. Borgogna, V. Dell’Oste, moter methylation but requires SMAD4 and jun-B BS in the en- S. Landolfo, and M. Gariglio. 2018. HPV18 persistence impairs basal and DNA hancer. As pointed out above, the EGFR-MEK-ERK1/2 pathway ligand-mediated IFN-b and IFN-l1 production through transcriptional repression http://www.jimmunol.org/ of multiple downstream effectors of pattern recognition receptor signaling. inhibits IFN-k expression. Because keratinocytes are cultivated in J. Immunol. 200: 2076–2089. the presence of EGF, it is possible that differences in cell culture 14. Lulli, D., M. L. Carbone, and S. Pastore. 2017. The MEK inhibitors trametinib conditions account for the lack of induction of IFNK by TGF-b and cobimetinib induce a type I interferon response in human keratinocytes. Int. J. Mol. Sci. 18: E2227. reported by Woodby and colleagues (18). Our data further indicate 15. Lulli, D., M. L. Carbone, and S. Pastore. 2016. Epidermal growth factor receptor that the combination of TGF-b and ERKi induces higher levels of inhibitors trigger a type I interferon response in human skin. Oncotarget 7: IFN-k than TGF-b alone also in HPV16-positive cells. Although 47777–47793. 16. Sarkar, M. K., G. A. Hile, L. C. Tsoi, X. Xing, J. Liu, Y. Liang, C. C. Berthier, we do not see a huge difference in the extent of repression of viral W. R. Swindell, M. T. Patrick, S. Shao, et al. 2018. Photosensitivity and type I transcription, there is not only a significant increase in IFNK,but IFN responses in cutaneous lupus are driven by epidermal-derived interferon

kappa. Ann. Rheum. Dis. 77: 1653–1664. by guest on September 25, 2021 also in ISG transcription. Because IFN-k can also act on immune 17. Scarponi, C., B. Nardelli, D. W. Lafleur, P. A. Moore, S. Madonna, O. De Pita`, cells, such as monocytes and dendritic cells, and induce several G. Girolomoni, and C. Albanesi. 2006. Analysis of IFN-k expression in path- cytokines without a costimulatory signal (11), an increase in the ologic skin conditions: downregulation in psoriasis and atopic dermatitis. J. Interferon Cytokine Res. 26: 133–140. local IFN-k concentration might be beneficial to induce and/or 18. Woodby, B. L., W. K. Songock, M. L. Scott, G. Raikhy, and J. M. Bodily. 2018. enhance immune responses to HPV, which would be consistent Induction of interferon kappa in human papillomavirus 16 infection by with immune-modulatory actions of other type I IFN (1, 2). In- transforming growth factor beta-induced promoter demethylation. J. Virol. 92: e01714-17. terestingly, genome-wide analyses of HR-HPV–induced cancers 19. Evans, M. R., C. D. James, M. L. Bristol, T. J. Nulton, X. Wang, N. Kaur, have revealed that both the EGFR-MEK-ERK1/2 and the TGF- E. A. White, B. Windle, and I. M. Morgan. 2019. Human papillomavirus 16 E2 regulates keratinocyte gene expression relevant to cancer and the viral life cycle. b/SMAD4 pathways are significantly mt (68). It is tempting to [Published erratum appears in 2019 J. Virol. 93: e01067-19.] J. Virol. 93: speculate that one consequence of this is to limit IFN-k expres- pii e01941-18. sion. In addition, several skin disorders are characterized by an 20. Habiger, C., G. Ja¨ger, M. Walter, T. Iftner, and F. Stubenrauch. 2015. Interferon kappa inhibits human papillomavirus 31 transcription by inducing Sp100 pro- aberrant type I IFN signature (69), and this often correlates with teins. J. Virol. 90: 694–704. an altered IFN-k expression. IFN-k expression is upregulated in 21. Lo Cigno, I., M. De Andrea, C. Borgogna, S. Albertini, M. M. Landini, A. Peretti, K. E. Johnson, B. Chandran, S. Landolfo, and M. Gariglio. 2015. The skin from allergic contact dermatitis and lichen planus–affected nuclear DNA sensor IFI16 acts as a restriction factor for human papillomavirus skin but is downregulated or absent from psoriatic or atopic der- replication through epigenetic modifications of the viral promoters. J. Virol. 89: matitis lesions (17). Very recently, increased IFN-k expression has 7506–7520. 22. Stepp, W. H., J. M. Meyers, and A. A. McBride. 2013. Sp100 provides intrinsic been linked to the pathology of cutaneous lupus erythematosus immunity against human papillomavirus infection. MBio 4: e00845-13. (16). Hence, our findings may be used to manipulate IFN-k ex- 23. Stepp, W. H., J. D. Stamos, S. Khurana, A. Warburton, and A. A. McBride. 2017. pression in pathological settings. Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle. PLoS Pathog. 13: e1006660. 24. Derynck, R., and E. H. Budi. 2019. Specificity, versatility, and control of TGF-b family signaling. Sci. Signal. 12: eaav5183. Disclosures 25. Straub, E., M. Dreer, J. Fertey, T. Iftner, and F. Stubenrauch. 2014. The viral The authors have no financial conflicts of interest. E8Ê2C repressor limits productive replication of human papillomavirus 16. J. Virol. 88: 937–947. 26. Straub, E., J. Fertey, M. Dreer, T. Iftner, and F. Stubenrauch. 2015. Character- ization of the human papillomavirus 16 E8 promoter. J. Virol. 89: 7304–7313. References 27. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real- 1. Fensterl, V., S. Chattopadhyay, and G. C. Sen. 2015. No love lost between time RT-PCR. Nucleic Acids Res. 29: e45. viruses and interferons. Annu. Rev. Virol. 2: 549–572. 28. Creyghton, M. P., A. W. Cheng, G. G. Welstead, T. Kooistra, B. W. Carey, 2. McNab, F., K. Mayer-Barber, A. Sher, A. Wack, and A. O’Garra. 2015. Type I E. J. Steine, J. Hanna, M. A. Lodato, G. M. Frampton, P. A. Sharp, et al. 2010. interferons in infectious disease. Nat. Rev. Immunol. 15: 87–103. Histone H3K27ac separates active from poised enhancers and predicts devel- 3. Honda, K., H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, opmental state. Proc. Natl. Acad. Sci. USA 107: 21931–21936. Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi. 2005. IRF-7 is the master 29. De Santa, F., I. Barozzi, F. Mietton, S. Ghisletti, S. Polletti, B. K. Tusi, H. Muller, regulator of type-I interferon-dependent immune responses. Nature 434: J. Ragoussis, C.-L. Wei, and G. Natoli. 2010. A large fraction of extragenic RNA 772–777. pol II transcription sites overlap enhancers. PLoS Biol. 8: e1000384. The Journal of Immunology 11

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