Immunostaining of regenerated hair follicles.

<p>Indirect immunohistochemistry was performed on frozen sections of microdissected hair follicles using antibodies against (a) CD34, (b) SOX9, (c) LHX2, (d) CD200 and (e) K15, respectively. Alexa Fluor 488 (green) was used to localize selected molecular markers before counterstaining of nuclei with DAPI (blue). Scale bars = 50 µm.</p

Absence of pilosebaceous units in chimeric ESS.

<p>Nile red and ALP stainings were performed on epidermis and dermis of ESS controls and ESS with mDPC-GFP (a). In ESS controls, epithelium was thin enough to visualize the skin surface lipids on the serosal side of the epidermis (a). Contrary to host skin containing sebaceous glands (arrowheads), no sebaceous glands were detected in ESS controls and ESS with mDPC-GFP. ALP in the dermis corresponded to the area where the hair follicles were situated (a, arrows). Close examination confirmed that neofollicles were deficient of sebaceous glands (b). On the other hand, sebaceous glands (white arrowheads) were observed above the bulge regions of pelage hairs. Immunohistochemistry confirmed the human origin (HuNu) and demonstrated no co-localization between Mel-5 and GFP in the bulb (c). Skin barrier integrity was evaluated by TEWL (d). Significantly higher TEWL was observed in ESS with mDPC-GFP compared to host skin, but not different from human volunteers (NHS). Scale bars in (a) = 500 µm; (b) = 100 µm and (c) = 50 µm.</p

Characteristic features of hair induction <i>in vivo</i>.

<p>Compared to epithelium of ESS controls, which has fewer Ki67+ cells and no K6 staining (a), epithelium of ESS with mDPC-GFP contains numerous Ki67 with K6 expression (b). K10 stained positively in the suprabasal layer of ESS controls (c) and ESS with mDPC-GFP (d), but not the invaginating epidermis in ESS with mDPC-GFP (d). Similarly to embryonic development, LEF1 was restricted to the actively growing hair bulb (f, arrowhead) and placode (g, arrowheads). No nuclear LEF1 was observed in ESS controls (e). Corroborating these results, GFP+ dermal condensation was detected beneath the developing placode (h, arrowhead) and within the bulb (i, arrowhead). Dotted lines represent dermal-epidermal junctions. Scale bars = 100 µm.</p

Comparison of ESS models.

<p>Gene expression analyses of selected genes involved in Wnt/β-catenin pathway, fatty acid metabolism and skin cornification, including <i>LEF1, WNT10B, LOR, INV, SOX9, PRDM1, SCD</i> and <i>FABP3</i>, were compared between ESS controls and ESS with mDPC-GFP after normalization to NHS (a). Asterisks represent statistically significant differences between the ESS groups (<i>p</i><0.05). Morphological comparison of regenerated hair follicles in ESS with mDPC-GFP (b–d) and in newborn murine ESS was demonstrated (e–h). Scale bars = 50 µm, except in (b, e) = 100 µm and in (f) = 1 mm.</p

Analysis of chromatin accessibility in human epidermis identifies putative barrier dysfunction-sensing enhancers

<div><p>To identify putative gene regulatory regions that respond to epidermal injury, we mapped chromatin dynamics in a stratified human epidermis during barrier maturation and disruption. Engineered skin substitutes (ESS) cultured at the air-liquid interface were used as a model of developing human epidermis with incomplete barrier formation. The epidermal barrier stabilized following engraftment onto immunocompromised mice, and was compromised again upon injury. Modified formaldehyde-assisted isolation of regulatory elements (FAIRE) was used to identify accessible genomic regions characteristic of monolayer keratinocytes, ESS <i>in vitro</i>, grafted ESS, and tape-stripped ESS graft. We mapped differentiation- and maturation-associated changes in transcription factor binding sites enriched at each stage and observed overrepresentation of AP-1 gene family motifs in barrier-deficient samples. Transcription of <i>TSLP</i>, an important effector of immunological memory in response to allergen exposure, was dramatically elevated in our barrier-deficient samples. We identified dynamic DNA elements that correlated with <i>TSLP</i> induction and may contain enhancers that regulate <i>TSLP</i>. Two dynamic regions were located near the <i>TSLP</i> promoter and overlapped with allergy-associated SNPs rs17551370 and rs2289877, strongly implicating these loci in the regulation of <i>TSLP</i> expression in allergic disease. Additional dynamic chromatin regions ~250kb upstream of the <i>TSLP</i> promoter were found to be in high linkage disequilibrium with allergic disease SNPs. Taken together, these results define dynamic chromatin accessibility changes during epidermal development and dysfunction.</p></div

Engineered skin substitute (ESS) as a model to study human barrier maturation and injury.

<p>(A) ESS derived from primary human keratinocytes and fibroblasts were grafted onto 14 female NIH-III mice after two weeks in air-liquid interface (ALI) culture. Image was taken two weeks after surgery when bandages were removed. (B) Grafts were allowed to heal for an additional four weeks. (C) Histology of grafted ESS at the junction of the grafted mouse epidermis. Arrowhead indicates grafted human ESS, arrow points to native mouse skin. (D) Tape-stripping was performed on 7 of the mice, with the other 7 as grafted controls. Transepidermal water loss (TEWL) was used as an indicator of barrier integrity. (E) qRT-PCR using long-form <i>TSLP</i> -specific primers of monolayer keratinocytes, ESS at ALI, grafted ESS, and in grafted ESS 3 hours after tape-stripping. Data are normalized to GAPDH and shown as fold change over keratinocytes; error bars = standard deviation. (F) Sonicated ESS DNA run on a 1% agarose gel. Sonication was performed in buffer containing 0, 0.1%, 0.5%, 1%, and 5% SDS. (G) FAIRE workflow for human epidermis. Modifications from the published protocol are shown with red text.</p

Dynamic FAIRE peaks in barrier maturation and function.

<p>(A) Venn diagram demonstrating overlap of FAIRE peaks in each sample and 5 relevant peak classes based on tissue pattern: Epidermal (n = 2094), Three-dimensional (3D; n = 2827), Stable (n = 5268), Intact Barrier (n = 19437), and Barrier-deficient (n = 15599). Peaks present in any non-epidermal cell type were filtered out prior to analysis. (B) Venn diagram showing overlap of annotated genes for each sample type. The nearest gene was assigned to each peak using Homer annotatePeaks. (C) FAIRE signals with representative peaks from four categories: Epidermal, dotted line; Three-dimensional, dashed line; Intact barrier, pink line; and Barrier-deficient peaks, blue line.</p

Allergy-associated SNPS in LD with dynamic peaks in the <i>TSLP</i> TAD.

<p>Locations of dynamic peaks containing SNPs that are in LD with allergic disease associated SNPs. R² indicates LD based on 1000 genomes phase 1 CEU data. Abbreviations: AR- allergic rhinitis; AD- atopic dermatitis; EoE- eosinophilic esophagitis; CR- chronic rhinitis; eQTL- expression quantitative trait locus.</p

Dynamic FAIRE peaks in the <i>TSLP</i> transcriptionally associated domain (TAD) associate with allergic disease-associated SNPs.

<p>(A) The most likely <i>TSLP</i> regulatory region was identified using published Hi-C data from several cell types to infer the TAD around <i>TSLP</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.ref039" target="_blank">39</a>]. (B) GWAS SNPs located within the <i>TSLP</i> TAD (n = 16); all are associated with allergic disease (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.t001" target="_blank">Table 1</a> and Methods). (C) FAIRE peaks within the <i>TSLP</i> TAD designated by category: Housekeeping peaks which were present in our samples as well as in at least 3 ENCODE FAIRE cell types (grey); epidermal peaks, present in our samples but not in non-epidermal ENCODE cell types (light blue); Three-dimensional (3D) peaks present only in stratified epidermis (black); and Barrier-deficient peaks that were present in the context of insufficient epidermal barrier (ESS and Tape-strip) but absent in an intact barrier (Graft) or culture monolayer (keratinocytes, NHEK; dark blue). Representative ESS signal is shown below, along with ENCODE H3K4me1. Color designations for the most prominent cell types in the H3K4me1 track are indicated in the legend. (D) FAIRE signals from ENCODE NHEK, cultured Keratinocytes, ESS, Grafted ESS, and Tape-stripped ESS are shown for the <i>TSLP</i> locus (right panel) as well as a distal region ~200-250kb upstream of <i>TSLP</i> (left panel) containing allergy-associated SNPs and dynamic peaks. Two Barrier-deficient peaks (dashed boxed regions, right panel) overlap rs17551370 and rs2278277, respectively. Rs17551370 is associated with allergic rhinitis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.ref042" target="_blank">42</a>] and rs2289277 is a known expression quantitative trait locus (eQTL) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.ref043" target="_blank">43</a>] for <i>TSLP</i> and associated with changes in IgE levels [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.ref044" target="_blank">44</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.ref045" target="_blank">45</a>]. Four distal (left panel) Epidermal, 3D, and Barrier-deficient FAIRE peaks are in high (R² = 0.8–1) linkage disequilibrium (LD) with allergy-associated SNPs. Peaks are boxed in the same color as the SNP they are in LD with. Full LD plots and analysis are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.s004" target="_blank">S4 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.t001" target="_blank">Table 1</a>.</p

Transcription factor (TF) motif enrichment in chromatin associated with different stages in epidermal development or function.

<p>(A) Negative log of the p-value of over 3000 TF binding motifs from the Cis-BP catalog in Keratinocytes (x-axis) and ESS (y-axis). P-value indicates motif enrichment over a scrambled dinucleotide background (see text for details). Gene families with high significance are color-coded as shown in the legend. (B) Comparison between ESS and Grafted ESS and (C) Grafted ESS and Tape-stripped graft. (D) Representative motifs identified in a direct comparison of enrichment between data sets: ESS and Keratinocytes (EvK), ESS and Graft (EvG), Tape-strip and Graft (TSvG), Keratinocytes and ESS (KvE), Graft and ESS (GvE), and Graft and Tape-strip (GvTS). Full lists of motifs and p-values can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184500#pone.0184500.s008" target="_blank">S1 Appendix</a>.</p