Keratinocytes produce lipids that are critical for the skin barrier, however, little is known about the impact of age on fatty acid (FA) biosynthesis in these cells.We have examined the relationship between keratinocyte FA composition, lipid biosynthetic gene expression, gene promoter methylation and age.Expression of elongase (ELOVL6 and 7) and desaturase (FADS1 and 2) genes was lower in adult versus neonatal keratinocytes, and was associated with lower concentrations of n-7, n-9 and n-10 polyunsaturated FA in adult cells. Consistent with these findings, transient FADS2 knockdown in neonatal keratinocytes mimicked the adult keratinocyte FA profile in neonatal cells.Interrogation of methylation levels across the FADS2 locus (53 genomic sites) revealed differential methylation of 15 sites in neonatal versus adult keratinocytes, of which three hypermethylated sites in adult keratinocytes overlapped with a SMARCA4 protein binding site in the FADS2 promoter. •Primary keratinocytes from adult donor skin synthesise less unsaturated fatty acids compared to keratinocytes from neonates.•FADS2 knockdown in neonatal keratinocytes switches cellular fatty acid composition to mimic adult keratinocytes.•In adult keratinocytes, the SMARCA4 binding site in the FADS2 promoter is hypermethylated.•Epigenetic regulation at the SMARCA4 binding site may affect fatty acid composition in skin cells. Fatty aThe cellular FA lipidome is determined by both dietary FA supply and metabolic enzyme activity, regulated within a complex feedback loop . Two majThere is a profound reduction in the total lipid content in skin from older adults . This isEpigenetic regulation of FA biosynthesis has been described. Consistent with the hypothesis that a FA profile change with age is due to altered biosynthesis, a recent study has shown that hypermethylation of CpG islands in the promoter region of the elongase gene ELOVL2 is associated with age in a number of cell types, particularly those with high proliferation rates .Here we have investigated the hypothesis that FA composition and concentration are different in keratinocytes from adult compared to neonatal donors due to specific differences in expression of FA biosynthesis gene expression. Our aim was to understand whether any molecular changes in FA profile in keratinocytes from different age donors are associated with epigenetic modification of specific FA biosynthesis gene promoter region.22.1Normal human epidermal keratinocytes (NHEK) from Lonza, Belgium were grown in serum-free keratinocyte KGM media supplemented with KGM Gold bullet kit  containing 60 μM calcium. Media was replaced every 48 h. To eliminate any passage-induced effects, all primary neonatal and adult NHEK were studied at passage 2. Senescence was detectable from passage 5.2.2Messenger RNA was extracted from NHEK using RNeasy mini plus kit (Qiagen) and RNA (500 ng) was converted to cDNA using a qScript cDNA synthesis kit (Quanta Bioscience). Quantitect primers were used for ELOVL1, 2, 3, 4, 5, 6, and 7, SCD-1, FADS1 and FADS2 expression, with YWHAZ as a housekeeping gene (Qiagen), using a Stratagene Mx3000P system. The relative gene expression was calculated against the input RNA.2.3Cell lysates  were prepared from proliferating NHEK cells (95% confluent) and spiked with 0.1 mg C13:0 and C17:0 before extraction of lipids as previously described by us [2.4Methylated lipids (1 μl) were injected on an Agilent 6890 GC in 0.9 ml/min helium (20:1 split) coupled to an Agilent 5973 MS and FAs were separated on an OmegaWax 250 column . Peaks were identified using standards from Supelco , Larodan  and by comparing mass spectral data to the NIST library. Peaks were quantitated as peak area and expressed relative to C16:0.2.5Accell Smartpool siRNA was purchased from Dharmacon  for targeting of ELOVL6, FADS1 and FADS2 genes, with positive (GAPDH) and vehicle control siRNA. Neonatal NHEK (n = 5 independent donors) were grown until 60% confluence, then treated with either ELOVL6, FADS1 or FADS2 siRNA (1 μM) as per manufacturer's protocol and grown for a further 96 h at which point media was changed and siRNA introduced for a further 72 h. Gene knockdown was confirmed using RT-PCR.2.6DNA was extracted using a DNAeasy blood and tissue kit (Qiagen). The genomic DNA was quantitated using Quant-iT™ PicoGreen® dsDNA Assay Kit (Life-Technologies), following the manufacturer's protocol using the high-range standard curve. Bi-sulphite conversion of the DNA and detection of methylated and non-methylated DNA was performed by Nxt-DX (Belgium). Genomic DNA was bisulfite-treated using the EZ DNA methylation kit  according to the manufacturer's specifications. The samples were then processed with the Infinium Methylation EPIC BeadChip kit according to the manufacturer's instructions. Quality checks were performed using density bean plots, probe detection p-values, and PCA analyses – no samples failed these checks. Beta-values are defined as beta = methylated/(methylated + unmethylated + offset); the offset of 100 is chosen to avoid dividing with small values. The beta values were normalized using SWAN normalization, and the M-values then calculated as M = logit(beta) = log(methylated/unmethylated). The M-values were used for statistical analyses in Genespring (version 14.8) and beta values used for subsequent filtering. In a targeted methylomics strategy, DNA methylation levels at 53 genomic sites located in and around the FADS2 gene were interrogated.2.7https://genome.ucsc.edu/cgi-bin/hgGateway) by blasting the source methylation probe sequences and visually inspecting for overlap of the resulting genomic location with OReg Anno transcription factor binding site (TFBS) data [Statistical analysis was performed using GraphPad Prism  Qlucore  or GeneSpring  where stated. For the methylation data, replicate sample data was averaged, imported into Genespring and a moderated T-test performed on the data for probes annotated to FADS2 gene (53 probes). A false discovery rate (FDR) of p < 0.05 was considered significant. Binding sites for transcription factor binding proteins were assessed in the UCSC browser (BS) data .2.8The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.33.1A heat map of lipid levels M confirm3.2ELOVL6 and 7 were expressed at lower levels in adult NHEK compared to neonatal NHEK  transcriptional start site (TSS). All other hypermethylated sites demonstrated a similar difference (i.e. delta beta) between the neonatal and adult female samples as they did between the neonatal and adult male sample. In addition, 4 of the sites were located in the FADS2 promoter (<200 bp upstream of the TSS) and 9 probes  overlapped with SMARCA4 binding sites ; Fig. 4J4Our work has shown that NHEK from healthy adults have lower unsaturated FA levels and reduced expression of FADS1 and 2, ELOVL6 and 7 compared to neonatal NHEK. Only FADS2 knockdown in neonatal NHEK was able to recapitulate the adult NHEK FA profile, indicating that lower expression of FADS2 is a major contributor to the differences in FA composition. To understand why FADS2 expression was lower in adult NHEK, we explored epigenetic modification of promotor and identified that binding sites for the helicase, SMARCA4, were hypermethylated in the promoter of FADS2 in adult NHEK.To our knowledge, this is the first report of differential FA level and gene expression with age in skin cells. An earlier study described FADS2 gene expression in young, old, ultraviolet (UV)-protected and UV-exposed skin with FADS2 being consistently down-regulated in intrinsic skin ageing and photo-ageing; however, functional effects on lipid composition were not explored .Lower FADS2 expression in skin is described in inflammatory skin conditions . Dermatitis also develops after FADS2-targeted disruption in the skin  further The epigenome becomes increasingly divergent with age due increasing exposure to stochastic events. In general, a progressive genome demethylation is observed with age , howeverPreviously, common age-associated hypermethylation sites have been described for ELOVL2, SFMBT1, KLF14, PENK and FHL2 despite differences in sample tissue types or age distributions ,20. HoweDetailed analysis of the FADS2 promoter confirmed that SMARCA4 binding sites overlapped with nine FADS2 probes, and three methylation sites overlapped with a SMARCA4 binding site. All nine FADS2 probes showed increased methylation levels in adults, which normally contributes to lower gene expression. This is in keeping with the lower FADS2 mRNA that we observed in adult NHEK.SMARCA4 is a master regulator of epidermal differentiation, mediating the effects of p63 in skin . As a heIn skin, the ELOVLs are responsible for extending FA chain length to create a functionally diverse unsaturated FA pool upstream and downstream from FADS2. While we identified lower ELOVL7 in adult NHEK, which normally catalyses the extension of saturated FA chains, this was not paralleled by any differences between adult and neonatal NHEK FA levels. ELOVL6 is important for C18:1n7 production. Despite a lack of difference between neonatal and adult NHEK C18:1n7 levels, we investigated the effect of ELOVL6 knockdown. We found that its regulation was tightly coupled to FADS1 and 2, indicating that FADS gene expression differences were likely to be the major determinates of FA profiles.The observed lower levels of PUFA in adult NHEK compared to neonatal NHEK are attributed to lower FADS expression . In supp15 times lower than other FA genes) for keratin-7 were negligible, and therefore we suggest that NHEK possess a novel n-10 FA profile.C16:1n-10 is a secretion product from sebaceous cells following C16:0 desaturation by FADS2 or from linoleic acid metabolism . C16:1n-ELOVL6 is also essential for C18:1n-9 synthesis and C18:1n-9 can be further metabolised by FADS2 in keratinocytes to form other n-9 FAs . CoordinThis study is limited by the availability of commercial cells i.e. neonatal and adult NHEK and potential for gender bias. Therefore, we undertook principal component analysis which confirmed that age rather than gender was the major influence on FA profile and methylation.In conclusion, novel PUFA profile differences between adult and neonatal NHEK are associated with lower activity in the n-7, n-9 and n-10 FA biosynthetic pathways and desaturation via FADS2. Lower FADS2 gene expression was associated with hypermethylation of the FADS2 promotor at the binding site of the helicase SMARCA4. Further work is needed to understand the contribution of epigenetic regulation at the SMARCA4 binding site for barrier function in health and disease.HRG and GJ conceived the project. CP, DH, DM acquired the data, assisted by LJW, KEB, DT, MIF and FLL for analysis; CP and HRG wrote the core manuscript text and all authors contributed to revising it critically for important intellectual content and final approval of the version to be submitted.None.2, Barrett K.E.2, Hyliands D.2, Talbot D.2, Fowler M.I.2, Kawatra T.2, Gunn D.A.2, Lim FL.2, Wainwright L.J.2, Jenkins G.2, are employed by Unilever R&D.Messenger D.J.1,3 received part funding from 10.13039/100007190Unilever R&D and 10.13039/501100000268BBSRC to undertake this study.Griffiths H.R.