What is a gene variant and how do variants occur? Nthen MM, Magin TM. Reflecting a partial or complete loss of this crucial scaffolding role, genetic mutations altering the coding sequence of keratins account for a large number of epithelial fragility disorders. Experimental data were collected from biological replicates (three or more) and technical replicates (typically two). The gRNA was transcribed in vitro and purified prior to injection. Data in B and F represent meanSEM. These results suggest a link between receptor activation and immobilization. Keratins are a defining feature of epithelial cells and the predictable expression pattern in each lung epithelial cell type underlies their use for cell identification. A strict requirement for the heteropolymerization of types I and II keratin proteins during filament assembly underlies the pairwise transcriptional regulation of keratin genes. filament assembly. These data provide a new in vivo model for a fundamental mechanism integrating keratins, 14-3-3 and Hippo signaling (about which not that much is known in epidermis) during early stages of differentiation. Keratin expression is epithelial cell-type-specific, and influenced by developmental stage, cellular differentiation, and pathology. Both K14 WT and, albeit to a lesser extent, the 373A mutant interact with HA-tagged 14-3-3. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14s codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. (F)same as E, except that F-actin (via phalloidin) and Ser19-phosphorylated myosin light chain pMLC (Ser19) are stained. Ours is the first evidence that keratin proteins are directly involved in the regulation of keratinocyte differentiation in epidermis in situ. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The primary antibodies used are listed in Supplementary file 4 (Key Resources Table File). Keratinocytes transform as part of this dehydration process as the mass fraction of insoluble keratin protein composing the cell continues to rise as a function of this aging process. (A) Indirect immunofluorescence for Edu in tail skin section from WT and Krt14 C373A at 2 hr, 1 d, and 3 d after treatment with thymidine analog EdU. Second, the occurrence of K14-dependent disulfides may create an optimal binding interface for 14-3-3 and YAP in the cytoplasm residing proximal to the nucleus. Seattle (WA): University of 2005 Aug;125(2):239-43. Dermatol. Type I and II subtypes of keratin form polymers in an epithelial cell-type-specific manner, which is also influenced by developmental stage, cellular differentiation, and pathophysiology. 1) The reviewers felt that the analysis of mechanics (mostly data in Figure 6) were underdeveloped and gave a few suggestions for how this might be strengthened experimentally. More than 130 mutations in the KRT5 gene have been identified in people with epidermolysis bullosa simplex, a condition that causes the skin to be very fragile and to blister easily. Potential transgenic founders were screened using restriction digestion of PCR product extending beyond the repair template oligonucleotide and findings were confirmed by direct DNA sequencing (data not shown). Total RNA-seq data were loaded into the UCSC Genome Browser as bigwig files for visualization. The challenge, however, is finding the right formulation and condition to produce the corresponding structure that is of the right mechanical integrity and biochemical activity to carry out a specific task. The TM300 probe was warmed for 2 min prior to each measurement, and held on the area of interest for a minimum of 30 reads until the alpha level was below 0.2, per the manufacturers instructions. Together these observations link the anomalies observed in epidermal homeostasis and skin barrier to defects in terminal keratinocyte differentiation in Krt14 C373A mouse skin. Hard keratins (5% sulfur) found in hair, horns, feathers, nails, and tongue papillae [67,68] are classified as types Ia (acidic-hard) and IIa (basic-hard), whereas epidermal keratins such as the stratum corneum in skin, known as soft keratins (1% sulfur), are classified as Ib (acidic-soft) and IIb (basic-soft) [69]. HeLa cells were selected because they are epithelia-derived, and grow and transfect well. Dysfunctional and leaky blood vessels resulting from disruption of the endothelial cell (EC) barrier accompanies numerous diseases. They are produced within cells identified as keratinocytes that are sequestered to the stratum corneum layer, that outermost, regenerative layer of the skin. N=3 biological replicates per genotype. After addition of calcium (1 mM), which triggers differentiation and mimics a suprabasal state (Hennings et al., 1980), 73% of WT keratinocytes lose their nuclear YAP signal whereas 95% of Krt14 C373A keratinocytes exhibit predominantly nuclear YAP (Figure 5A,B). Amino acid composition of keratin found in hoof [13], hair [14], fingernail [15], and snake skin [14] The hoof is designated as high sulfur (high S) and low sulfur (low S). Consistent with the macroscopic appearance of skin tissue there is no ultrastructural evidence of cell fragility in Krt14 C373A epidermis (Figure 3figure supplement 2A,B and data not shown). Next, we analyzed cornified envelopes (CEs) isolated from epidermis, given that they are key contributors to skin barrier function (Eckhart et al., 2013). But these connections are not made in the paper. The YAP1 and TEAD genes (human) are themselves poised to be positively regulated by YAP in progenitor keratinocytes (Figure 7figure supplement 1; Supplementary file 2). The loss of cysteine 373 results in profound alterations in the i) pattern of K14-dependent disulfide bonding in epidermis; ii) regulation of 14-3-3sigma and YAP in early-stage differentiating keratinocytes; and iii) several mechanosensitive readouts in the young adult epidermis in situ and newborn skin keratinocytes in primary culture. See our, URL of this page: https://medlineplus.gov/genetics/gene/krt5/. D-F: Studies involving newborn skin keratinocytes in primary culture. (C) Schematic diagram of the strategy used to generate Krt14 C373A mice using the Crispr/Cas9 system. The reviewers have opted to remain anonymous. (G) Indirect immunofluorescence for K14 (green), K10 (red), filaggrin, and loricrin from tail skin sections of WT and Krt14 C373A mice. The predicted mouse K14 protein sequence (UniProtKB Q61781) was analyzed using publicly accessible algorithms written to predict 14-3-3 binding sites and phosphorylation events, including 14-3-3-Pred (Madeira et al., 2015) and Scansite 4.0 (Obenauer et al., 2003). All mouse studies were reviewed and approved by the Institutional Animal Use and Care Committee (IACUC) at both Johns Hopkins University and the University of Michigan. Does this change in 14-3-3 cooperate with changes in mechanics to promote nuclear YAP, or alternatively (or in addition) is YAP driving the change in mechanics? Fengrong Wang, Pierre A. Coulombe, in Methods in Enzymology, 2016. This mutation replaces the amino acid proline with the amino acid leucine at protein position 25 (written as Pro25Leu or P25L). (D) Sanger sequencing showing the TGC to GCA transversion at codon 373 (cysteine to alanine) in the Krt14 gene. Briefly, adult mouse ear skin or adult mouse tail skin (1 cm length) were boiled at 95 C (in place of hot water bath) for 20 min in 2 ml CE isolation buffer containing 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM dithiothreitol (DTT), and 2% sodium dodecyl sulfate (SDS). EdU staining was performed using the Click-iT Plus EdU Alexa Fluor 488 Imaging Kit (catalog no. Such mutant alleles may also affect skin pigmentation (Gu and Coulombe, 2007), establishing the relevance of both roles of K5-K14 in both healthy and diseased skin. K14-dependent disulfide bonding is low in the absence of calcium and rises of the course of days after adding calcium to primary cultures of WT mouse keratinocytes (Figure 4figure supplement 1A,B). For immunoprecipitation, aliquots of cell lysate were incubated with a K14 antibody, and immune complexes were captured using the Protein G Sepharose (17-0618-01, GE Healthcare). Readings were obtained using a TEWAMETER TM300 (Courage and Khazaka, Kln, Germany) from adult WT and Krt14 C373A mice at baseline and after the last topical treatment with acetone. Accordingly, our model (Figure 7A) predicts that the newly-defined role for K14 in regulating the subcellular partitioning of YAP and onset of keratinocyte differentiation in epidermis is picked up and maintained by K10 as differentiation proceeds. It is hard to call them eukaryocytes, but they clearly start out as such. N=3 biological replicates. Dashed lines depict the dermo-epidermal interface. Modulation of keratin expression also depends upon both the type and stage of differentiation in epithelia. As highlighted by the color code used in frames (a) and (b), individual type I and type II keratin genes belonging to the same subgroup, based on the primary structure of their protein products, tend to be clustered in the genome. This is so especially in ear and tail skin (Figure 1F,G), prompting us to focus on these two body sites in subsequent analyses. Epub 2010 Mar 5. Second, to regulate the distribution of melanin with an impact on skin pigmentation and tone (Uttam et al., 1996; Betz et al., 2006; Gu and Coulombe, 2007). A major function fulfilled by keratin filaments is to act as a resilient yet pliable scaffold, which endows epithelial cells with the ability to sustain mechanical and nonmechanical stresses. Firefly relative light unit (RLU) was normalized to internal Renilla RLU per well. 14-3-3 was deemed of interest because it regulates the proliferation and differentiation of keratinocytes in epidermis (Herron et al., 2005; Li et al., 2005). From an environmental point of view, the success of a top-down approach of recycling keratinous biowastes from both human and animal sources, through the creation of high value keratin based products, is a highly green and sustainable strategy that will be welcome by society and consumers. Since keratin fragments are released from tumors and in other pathological conditions affecting epithelial cells, they may also serve as disease markers. Further inspection of the top 100 MS-identified proteins in this targeted proteomics screen (Supplementary file 1) reveals, as expected, the presence of desmosomal proteins, known keratin-interacting proteins (e.g., annexins; (Chung et al., 2012), and several proteins with known roles in organelle transport and organization (e.g., rab family members; Ohbayashi and Fukuda, 2012), which is consistent with K5-K14s established role in skin pigmentation (Gu and Coulombe, 2007). These cells were tested routinely using a commercial luminescence assay (MycoAlert, Lonza) and found to be mycoplasma-free. At this point we do not have direct mechanical measurements comparing WT and Krt14 C373A skin keratinocytes. Thank you for submitting your article "Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3 and YAP1" for consideration by eLife. (A-C) Studies involving tail skin sections from young adult WT and Krt14 C373A mice. The combination of DNAse one hypersensitivity mapping, ATAC-seq mapping, and presence of TEAD binding sites (in either orientation) on DNA (5- CATTCC-3; Heinz et al., 2010) suggests that keratin genes expressed in dividing basal cells of epidermis, forexample the type I Krt14 and Krt15 and type II Krt5, are likely YAP target genes given the presence of consensus-matching TEAD binding sites occurring in open chromatin regions in the absence of calcium (Figure 7figure supplement 1; Supplementary file 2). A revised nomenclature was produced in 2006 to accommodate a large number of novel keratins (uncovered mainly through whole genome sequencing efforts) while complying with the human and mouse genome nomenclature committees. The ImageJ software (NIH) was used for western blot signal quantitation. and de novo mutations in the KRT5 and KRT14 genes, phenotype/genotype These changes are coupled to a redistribution of tension-related forces and cell-cell adhesion complexes as basal keratinocytes delaminate and move from the basal to the suprabasal compartment of epidermis (Miroshnikova et al., 2018; Nekrasova et al., 2018; Wickstrm and Niessen, 2018). KRT5 in a unique Chinese family with generalized Dowling-Degos disease. After understanding how keratin subtypes interact, deciphering the conditions needed to organize and assemble them in an orchestrated way will be key. To identify putative TEAD family binding sites, we loaded a file of genome-wide locations of predicted motifs from HOMER (Heinz et al., 2010) into the public Galaxy server and selected motifs corresponding to TEAD family members. Skin barrier defects often trigger elevated expression of Danger-Associated Molecular Patterns (Lessard et al., 2013) (DAMPs, also known as alarmins). Second, the crystal structure of the interacting 2B segments of K1-K10 (Bunick and Milstone, 2017) shows an overall fold identical to our original K5(2B)-K14(2B) structure (Lee et al., 2012), including the presence of a trans-dimer, homotypic disulfide bond mediated by the stutter cysteine (C401) in K10. putative role in vesicle transport. Alignment files of ATAC-seq data were downloaded with the following identifiers: ENCFF111ULL, ENCFF654ZNI, ENCFF205KDV, ENCFF479UTZ, ENCFF374VWZ, ENCFF588PIS. DNase-seq data from human newborn foreskin keratinocytes were produced by the Stamatoyannopoulos laboratory at University of Washington (Project: Roadmap, Award 01ES017156) and downloaded from the ENCODE portal (www.encodeproject.org) as a coverage file with the identifier ENCFF380PKB. RNA and ATAC sequencing data from male newborn human foreskin keratinocytes at 0, 3 and 6 days of calcium-induced differentiation were produced by the Greenleaf and Snyder laboratories at Stanford University (Project: GRR, Award: U01HG007919) and downloaded from the ENCODE portal. The multifaceted roles of SEKs are increasingly appreciated due to findings obtained from transgenic mouse models and human studies that identified SEK variants in several digestive diseases. -Keratins have an average molecular mass in the range of 4060kDa, are low in sulfur, partly crystalline, and form an -helical secondary structure [64]. S.H. Thin sections were cut (5070 nm thick), counter-stained with uranyl acetate and lead citrate, and examined using a Hitachi HU-12A transmission electron microscope. Under normal circumstances, keratin is not found in the extracellular space of viable tissues in humans. Keratins are the cytoskeletal intermediate filament proteins of epithelial cells. HeLa cells were transfected such that the cell density in each well was 3040% the following morning. The signals for E-cadherin and desmoplakin appeared slightly increased (Figure 3I), an occurrence that may reflect the modest epidermal thicknening (Figure 3J). This new data is consistent with the reviewers prediction and with the observation that there is considerably less evidence for K14-dependent disulfide bonding in adult back skin tissue as reported in Figure 1C. The latter yielded compelling results that have been added to Figure 6.