No animals were sacrificed as murine melanoma cell lines generated in 2019 were used (7). In brief, tissue samples from primary tumors (ear and tail) from two Tg(Grm1)Cyld^−/− and two Tg(Grm1)Cyld^+/+ male mice (age, 153 and 217 days) were collected and washed with Braunol^® (7.5%; B Braun Meisungen AG), followed by 1X PBS, 70% ethanol and 1X PBS. Tumor tissue was added to a mixture of DMEM (Sigma-Aldrich; Merck KGaA) and collagenase. Following incubation for 3 h at 37°C, the cell suspension was centrifuged (4 min at 600 g, room temperature) and the cells were seeded in T25 flasks, as previously described (7,13).

Murine melanoma cell lines (mCyld^+/+: EPv24 and EPv40 ear; mCyld^−/−: EC36 and EC111 ear) derived from tumor tissue of Tg(Grm1) model animals (13) were cultivated in DMEM (4,500 mg glucose/l, 110 mg sodium pyruvate/l and l-glutamine) with 10% fetal bovine serum, amphotericin B (2.5 μg/ml) and 5% penicillin/streptomycin (all Sigma-Aldrich; Merck KGaA) in a humidified atmosphere containing 8% CO₂ at 37°C. Cells were reseeded at a ratio 1:3 to 1:5 twice weekly.

Human primary melanoma cell line Mel Juso [provided by Dr Judith Johnson (Ludwig Maximilian University of Munich, Munich, Germany), stably transfected with control green fluorescent protein [human (h)CYLD⁻], CYLD (hCYLD⁺) or CYLD^C/S (catalytically inactive mutant of CYLD) vector (5,6) were cultivated (8% CO₂ at 37°C) in RPMI-1640 (Sigma-Aldrich; Merck KGaA) with NaHCO₃ and the aforementioned supplements. For cell counting the light microscope AE2000 from Motic Incorporation Ltd. was used (×4 or ×20 magnification).

For inhibitor studies, murine and human melanoma cells were treated with Chaetocin (inhibitor of histone methyltransferase SUV39H1; 10 nM; Absource Diagnostics GmbH), the Jumonji histone demethylase inhibitor JIB04 (500 nM; Sigma-Aldrich; Merck KGaA) and CM272 [dual EHMT2/DNA methyltransferase (DNMTs) inhibitor; 200 nM; kindly provided by Matías A Ávila (14)] for 24 h at 37°C.

For simplification, the following nomenclature has been used in the rest of the text: murine Cyld-expressing (mCyld^+/+) and Cyld-deficient (mCyld^−/−) cells; human CYLD-expressing (hCYLD⁺) and CYLD-deficient (hCYLD⁻) and CYLD mutant (hCYLD^C/S) cells.

Total RNA and cDNA of the murine and human melanoma cells were generated as previously described (7). RT-qPCR was performed with LightCycler 480 system (Roche Diagnostics GmbH) to analyze mRNA expression, as previously described (15). For each reaction, 1.0 cDNA template, 0.5 forward and reversed primers each (20 μM each) and 10.0 μl SYBR Green I (Roche Diagnostics GmbH) were used in a total volume of 20 μl. The primers were as follows: β-actin forward, 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′ and reverse, 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3′; cadherin (CDH)1 forward, 5′-ACG TAT CAG GGT CAA GTG CC-3′ and reverse, 5′-CCT GAC CCA CAC CAA AGT CT-3′; suppressor of cytokine signaling (SOCS)1 forward, 5′-TAA CCC GGT ACT CCG TGA CT-3′ and reverse, 5′-CTC ACC CTC CAC AAC CAC TC-3′; methylthioadenosine phosphorylase (MTAP) forward, 5′-ATC GTG ACC ACA GCT TGC GGG-3′ and reverse, 5′-TCT GCC CGG GAG CTG AA-3′ and euchromatic histone lysine methyltransferase 2 (EHMT2) forward, 5′-TAC CCA TCC CCT GTG TCA AT-3′ and reverse, 5′-TCC TTG TCA TAC CAG CAT CG-3′. All samples were analyzed in duplicate and normalized to β-actin.

Murine and human melanoma cells were lysed in RIPA buffer (Roche Diagnostics GmbH) for 15 min at 4°C and cell debris was separated via centrifugation at 600 x g and 4°C for 10 min. Protein concentration was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 35 μg total lysate per lane was separated on 10.00-12.75% SDS-PAGE gels and subsequently transferred onto a PVDF membrane. Following blocking for 1 h at room temperature with 5% BSA/0.1% TBS-T (Sigma-Aldrich; Merck KGaA) or 5% non-fat dry milk/TBS-T (0.1% Tween-20), the membrane was incubated overnight at 4°C with the following antibodies: β-actin (1:3,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA), histone 3 (H3) K9me2 (1:1,000; cat. no. ab3251; Abcam), H3K9me3 (1:1,000; cat. no. 07-442; Merck KGaA), H3 (1:1,000; cat. no. 4499; Cell Signaling, Technology, Inc.), EHMT2 (1:1,000; cat. no. 3306; Cell Signaling Technology, Inc.) and CYLD [1:1,000; cat. nos. 12797 (mCYLD) and 4495 (hCYLD), Cell Signaling Technology, Inc.].

After washing three times with TBS-T (0.1% Tween-20), membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary anti-rabbit (cat. no. 7074) or anti-mouse (7076) antibody (both 1:3,000; both Cell Signaling Technology, Inc.). Finally, the membrane was washed three times in TBS-T and the immunoreaction was visualized using Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Inc.) and Chemostar chemiluminescence imager (Intas Pharmaceuticals, Ltd.). Signal intensity was quantified using LabImage software Version 4.2.1 (Kapelan Bio-Imaging GmbH).

For isolation of histone extracts, Histone Extraction kit was used (cat. no. ab113476; Abcam). A total of 1x10⁷ melanoma cells (murine or human) were seeded in T75 flasks and harvested after 24 h incubation (8% CO₂ at 37°C) with a cell scraper. Following isolation according to the manufacturer's instructions, protein concentration was quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). For western blot analysis, 10 μg purified histone was used. Aliquots were stored at -80°C.

Murine melanoma cells were seeded in a T75 flask (750,000 cells/flask) 24 h (8% CO₂ at 37°C) before treatment. Cells were treated with 5 μM AZA (Sigma-Aldrich; Merck KGaA) for 72 h (8% CO₂ at 37°C) and harvested for qPCR and western blot analysis.

To investigate proliferation, colony formation ability was assessed. A total of 300 cells (murine and human melanoma cell lines) was seeded in a 6-well plate and cultivated for 10 days (8% CO₂ at 37°C). After washing twice with PBS, cells were fixed and stained with 6.0 glutaraldehyde and 0.5% crystal violet (Sigma-Aldrich; Merck KGaA), respectively, for 1 h at room temperature). The colony (>50 cells) size was analyzed using cellSens dimension-software (V1.12) and an IX83 fluorescence microscope (both Olympus Corporation).

The EpiQuik™ Chromatin Accessibility Assay kit was used according to the manufacturer's instructions (BioCat GmbH). Briefly, 1×10⁶ cells (murine or human melanoma cells) were collected 24 h after seeding. Following cell lysis and chromatin isolation, one half of the lysed cell was digested with a nuclease mix (Nse) and the other was left untreated (No-Nse). Subsequently, DNA was purified and analyzed via qPCR as aforementioned. Fold enrichment (FE) was calculated as follows: FE=2^{(Nse Ct-No-Nse Ct)}/-2^{(Nse Ct-No-Nse Ct)} ×100%. FE>400% indicated closed chromatin and FE<1,600% indicated open chromatin (values between 400 and 1,600% represent partially open chromatin). FE was normalized to Cyld-deficient cells and the control.

Histone H3 Modification Multiplex Assay kit (cat. no. ab185910, Abcam) was used to quantify 21 H3 modifications according to the manufacturer's instructions. A total of 50 ng/well histone extract from Cyld-expressing (Cyld^+/+) and -deficient (Cyld^−/−) murine cell lines, 50 ng/well histone extract was used. H3 lysine mono-di- and trimethylation was measured at sites K4, K9, K27, K36 and K79.

DNA-seq was performed using input samples of corresponding chromatin immunoprecipitation (ChIP) experiments. Chromatin from 1×10⁷ cells (mCyld^−/− and mCyld^+/+) of each sample was crosslinked in 1:10 volume of fixation buffer (50 mM HEPES/KOH, pH 7.9, 11% formaldehyde) for 10 min at room temperature and quenched by 0.125 M glycine. Following two washes with PBS and PMSF, cells were scraped and centrifuged at 4°C and 3,500 x g for 10 min. The supernatant was discarded and the pellet was resuspended in 15 ml lysis buffer 1 (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1X Roche Complete, EDTA-free protease inhibitor) and incubated for 10 min on ice. Following centrifugation of 5 min at 4°C and 3,500 x g, the supernatant was discarded and the pellet resuspended in 15 ml lysis buffer 2 (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1X Roche Complete, EDTA-free protease inhibitor). The suspension was incubated on ice for an additional 10 min and examined under the light microscope (magnification: x200) for quality assessment of isolated nuclei from cytoplasmic fractions. The nuclei were pelleted at 4°C and 3,500 x g for 5 min. The supernatant was discarded and the pellet was resuspended in sonication buffer (1x10⁷ cells/450 μl sonication buffer: 16.7 mM Tris-HCl, pH 8.0, 1.2 mM EDTA, 16,7 mM NaCl, 0.01% SDS, 1.10% Triton X-100, 1X Roche Complete, EDTA-free protease inhibitor). Cross-linked chromatin was sheared to an average DNA fragment size of 200-700 bp using the Covaris ME220 Focused-ultrasonicator (Covaris, Inc.). Then, 20 μl supernatant was used for DNA purification using the QIAquick PCR purification kit (Qiagen GmbH). Library preparation was performed with purified DNA using the TruSeq^® ChIP Sample Preparation kit according to the manufacturer's instructions (Illumina, Inc.). Quality and quantity of the final libraries were checked for size (200-500 bp) by TapeStation 4200 using the High-Sensitivity DNA kit (Agilent Technologies, Inc.) and concentration was determined by Qubit 4 Fluorometer (Thermo Fisher Scientific, Inc.). Each library was diluted to 4 nM and pooled to a final concentration of 5 nM. DNA libraries were sequenced for 50 cycles on an Illumina HiSeq4000 with single-end module (HiSeq 3000/4000 SBS kit, 50 cycles; cat. no. FC-410-1001; llumina, Inc.). Sequence tags of all experiments were mapped to the current mouse reference sequence (GRCm38/mm10) using Bowtie 2 (v 2.2.7) (16,17). Only uniquely mapped tags were used for CNV determination via Control-FREEC (v11.6) (18).

Total RNA samples were isolated from mCyld^−/− and mCyld^+/+ cell lines using Total RNA kit I (Omega Bio-Tek, Inc.) according to manufacturer's instructions. All RNA samples were examined for integrity and purity by TapeStation 4200 (Agilent Technologies, Inc.).

Library preparation was performed with two biological replicates using the TruSeq^® Stranded Total RNA Library Prep Human/Mouse/Rat kit (cat. no. 20020596, Illumina, Inc.) according to the manufacturer's instructions. The resulting libraries were checked for size (200-500 bp) by TapeStation 4200 using the High-Sensitivity DNA kit (Agilent Technologies, Inc.) and concentration was determined by Qubit 4 Fluorometer (Thermo Fisher Scientific, Inc.). Each library was diluted to 4 nM and pooled to a final concentration of 5 nM. Paired-end seq was performed using a HiSeq4000 with paired-end module (Illumina, Inc.) according to the manufacturer's instructions. The samples were sequenced from each side of a fragment ~75 bp long with an average number 20 million reads per sample. Following quality check using FastQC (Babraham Bioinformatics; bioinformatics.babraham. ac.uk/projects/fastqc/) paired-end reads were aligned to the mouse reference genome (GRCm38, mm10) using the STAR alignment software (v 2.5.2a) (17,19). Following mapping, only reads that mapped to a single unique location were selected for further analysis. The mapped reads were used for variant calling by Genome Analysis Toolkit (GATK; v4.1; gatk. broadinstitute.org) (20). Single nucleotide variants (SNVs) were identified with a cut-off of 1% minor allele frequency and a minimum of 10 variant reads. Variants were annotated using SnpEff 5.0e (21). Intronic variants were discarded and all exon variants were analyzed to determine their effect on the resulting coding sequence (non-synonymous or synonymous). Plots of contingency tables were produced using variant calling output and R script Genotype-Variants (v1.1; github. com/cfarkas/Genotype-variants) of the genotype-variant pipeline as described by Farkas et al (22).

Schematic illustrations were abstracted and modified from 'Les Laboratoires Servier-smart' Medical Art (smart.servier.com/). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.

Data are presented as the mean ± SEM of ≥3 independent experiments, unless otherwise stated. Statistical significance was determined using Student's unpaired t-test or one-way ANOVA followed by Bonferroni's post hoc test. Data were analyzed using GraphPad Prism Version 9 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Melanoma is one of the tumors with the highest mutation burden among solid tumors (23). To investigate whether CYLD affects melanoma-associated mutations, sequencing analyses were performed. No common CNVs were detected in mCyld^−/− cell and mCyld^+/+ cells (Fig. 1). Therefore, SNVs were investigated to identify potential mutations underlying CYLD-dependent changes in tumor development of Tg(Grm1)Cyld mice. Variant calling was performed using Mutect2 of GATK (v4.2) to discover somatic variants in the RNA-seq dataset mCyld^−/− vs. mCyld^+/+ cells. Few common somatic mutations were determined and all but one variants (pre-mRNA processing factor 39 on chromosome 12) in mCyld^−/− were located on chromosome 8 near the CYLD genomic location (Fig. 2). In summary, no Cyld-dependent significant copy number gains or losses and just a few somatic mutations were detected.

Besides genomic mutations, epigenetic dysregulation has also been linked to tumor development and progression in a large number of studies (9-12). To investigate the role of CYLD in controlling methylation of CpG islands, expression of known hypermethylated genes (MTAP, CDH1 and SOCS1) in human melanoma was investigated (24,25). No significant differences in MTAP, CDH1 and SOCS1 mRNA expression were observed between mCyld^+/+ and mCyld^−/− cell lines (Fig. 3A), indicating that CYLD was not involved in DNA methylation. DNA demethylation was induced by treatment with AZA to investigate CYLD-dependent alterations in gene expression. AZA treatment was associated with a notable (but not significant) increase in CYLD protein expression (Fig. S1). mRNA expression of hypermethylated genes was not significantly altered in mCyld^+/+ and mCyld^−/− cells following AZA treatment (Fig. 3B), confirming the aforementioned results.

In summary, these results indicated that CYLD had no effect on DNA methylation.

Epigenetic events affect the accessibility of chromatin via formation of heterochromatin and euchromatin (26). To determine whether CYLD affected chromatin structure, chromatin accessibility was assessed using the EpiQuik chromatin accessibility kit. This assay showed that mCyld^+/+ cells exhibited a more open chromatin structure compared with mCyld^−/− cells (Fig. 4A).

Heterochromatin is associated with certain H3 modifications (27); to the best of our knowledge, however, there are no studies regarding H3 modification and CYLD. Therefore, H3 modification assay was performed. Although modifications were notably higher in mCyld^−/− compared with mCyld^+/+ cells, this was only significant for H3K9me1- and H3K27me3- modifications (Fig. 4B). Taken together, these data suggested that CYLD was involved in chromatin structure and histone methylation.

The aforementioned results suggested an association between CYLD, chromatin structure and histone methylation. Because aberrant histone modification disrupts epigenetic balance and contributes to melanoma progression (28), the effect of histone demethylase/methyltransferase inhibitors on CYLD-dependent proliferation were analyzed.

Treatment with Chaetocin, a lysine-specific histone methyltransferase, leads to inhibition of the histone methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), which is involved in formation of hetero-chromatin by di-and trimethylating H3K9 (29,30). Here, treatment resulted in significantly decreased H3K9 dimethylation only in mCyld^−/− cells; trimethylation of H3K9 was not significantly affected (Fig. 5A). A previous study has demonstrated that Chaetocin exerts anti-proliferative effects in vivo in melanoma cells (31). However, clonogenic assay revealed no significant change in colony size between mCyld^−/− and mCyld^+/+ cells (Fig. 5B).

Using the established human CYLD melanoma system, no significant changes in H3K9 di- and trimethylation levels were observed via western blot analysis after treatment with Chaetocin (Fig. 5C). Consistent with the results in the murine cell lines, proliferation was not significantly altered by Chaetocin in human cell lines (Fig. 5D). Treatment with Chaetocin had no significant effect on CYLD protein expression in murine and human melanoma cells (Fig. S2A and B).

The counterpart of histone lysine methyltransferases are histone lysine demethylases (KDMs), including family members KDM3 [Jumonji Domain-Containing Protein 1A (JMJD1)], KDM4 (JMJD2) and KDM2 (JmjC domain-containing histone demethylation protein 1) (32). To the best of our knowledge, there are no inhibitors targeting specific KDMs. Nevertheless, there are broad-spectrum KDM inhibitors, such as the small molecule JIB04, which increases H3K9me2 levels (33). Since the aforementioned results excluded an effect of CYLD on H3K9 trimethylation on proliferation, cells were treated with JIB04; this treatment had no significant effect on levels of H3K9me2 in mCyld^−/− or mCyld^+/+ cells (Fig. 6A). Clonogenic assay revealed no significant changes in colony size following JIB04 treatment in mCyld^−/− compared with mCyld^+/+ cells (Fig. 6B). There was no significant change in H3K9me2 levels (Fig. 6C) or colony size (Fig. 6D) in hCYLD⁻ compared with hCYLD⁺ cells. CYLD protein expression was not significantly affected by treatment with JIB04 (Fig. S2C and D).

Selective inhibitor, CM272 suppresses activity of EHMT2 (also known as G9a) (14). This inhibitor contributes to decreased dimethylation of H3K9 and may hinder melanoma growth (14). To the best of our knowledge, however, no data concerning the role of CYLD in this context are available. Western blot analysis confirmed a significant decrease H3K9me2 levels following CM272 treatment only in mCyld^−/− cell lines (Fig. 7A). Clonogenic assay revealed a significant decrease in colony size in mCyld^−/− cells, which was consistent with H3K9me2 western blot results (Fig. 7B and C). mCyld^+/+ cells exhibited a significantly increased colony size (Fig. 7C), as well as significantly decreased CYLD expression following CM272 treatment (Fig. 7D). In conclusion, CM272 treatment of CYLD-expressing cells led to decreased expression and tumor suppressive function of CYLD. These results were confirmed in human cells: hCYLD⁻ cells exhibited significantly decreased H3K9me2 levels (Fig. 7E) and proliferation following CM272 treatment (Fig. 7F and G). hCYLD⁺ cells showed decreased CYLD expression following CM272 treatment (Fig. 7H) but this was not significant.

It was hypothesized that treatment of Cyld-deficient cells with CM272 would lead to looser chromatin structure. Chromatin accessibility analysis of mCyld^−/− cells showed more open chromatin structure following CM272 treatment compared with control cells (Fig. 7I) but this was not significant.

Catalytically inactive CYLD mutant cells (hCYLD^C/S) exhibited similar trends to hCYLD⁻ cells. There was a significant decrease in H3K9me2 levels (Fig. S3A) and colony size (Fig. S3B), whereas CYLD expression was not significantly altered following CM272 treatment (Fig. S3C). Moreover, hCYLD^C/S cells exhibited more compact chromatin structure, similar to hCYLD⁻ cells, whereas hCYLD⁺ cells exhibited a more open chromatin structure (Fig. S3D).

Together, these data showed that the inhibitors Chaetocin and JIB04 exerted no CYLD-dependent effect on cell proliferation. The inhibitor CM272 had anti-proliferative effects on Cyld-deficient cells but pro-proliferative effects on Cyld-expressing cells. The pro-proliferative effects were dependent on enzymatic function of CYLD.

Histone lysine methyltransferase EHMT2 specifically mono- and dimethylates H3K9 and catalyzes trimethylation of H3K27 (34). Thus, the present study investigated expression levels of this methyltransferase and its primary methylation site H3K9me2. Western blot analysis demonstrated notably enhanced EHMT2 expression in mCyld^−/− cells (Fig. 8A). mCyld^−/− cells showed a significant increase in H3K9me2 compared with Cyld^+/+ cells (Fig. 8B). Same investigations in our human cell system verified the negative association between CYLD and EHMT2 expression as well as a positive association between H3K9me2 and EHMT2 (Fig. 8C and D). hCYLD^C/S cells showed similar H3K9me2 and EHMT2 expression to hCYLD⁻ cells (Fig. S3E and F).