The epidermis covering our skin and body maintains a vital and multidimensional barrier to water and the outside environment while renewing itself with rapid kinetics, even under normal physiological conditions (Kubo et al., 2012). The mechanisms through which new progenitor cells are produced at the base of this stratified epithelium, pace themselves through differentiation, and maintain tissue architecture and function in spite of a high rate of cell loss at the skin surface are only partially understood (Wells and Watt, 2018).

Keratin intermediate filaments are major protein constituents in epithelial cells and are encoded by a large family of 54 conserved genes that are individually regulated in a tissue- and differentiation-specific fashion (Schweizer et al., 2006). An outstanding question is the extent to which keratin, and other types of intermediate filaments (IFs), participate in basic processes such as cell differentiation and tissue homeostasis. The type I keratin 14 (K14) and type II K5 co-polymerize to form the prominent IF apparatus that occurs in the progenitor basal layer of epidermis and related complex epithelia (Nelson and Sun, 1983; Fuchs, 1995). Two main roles have so far been ascribed to K5-K14 IFs. First, to provide structural support and mechanical resilience to keratinocytes in the basal layer of epidermis and related epithelia (Coulombe et al., 1991a; Vassar et al., 1991; Fuchs and Coulombe, 1992). 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). Dominantly-acting missense alleles in either KRT5 or KRT14 underlie the vast majority of cases of epidermolysis bullosa simplex (EBS), a rare genetic skin disorder in which trivial trauma results in skin blistering secondary to the lysis of fragile basal keratinocytes (Bonifas et al., 1991; Coulombe et al., 1991b; Fuchs and Coulombe, 1992; Lane et al., 1992). 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.

Structural insight gained from solving the crystal structure of the interacting 2B regions of corresponding rod domain segments in human K5 and K14 highlighted the presence of a trans-dimer, homotypic disulfide bond involving cysteine (C) residue 367 (C367) in K14 (Coulombe and Lee, 2012). Conspicuously, residue C367 in K14 occurs within a four-residue interruption, or stutter, in the long-range heptad repeat of coil two in the central alpha-helical rod domain in virtually all IF proteins (Lee et al., 2012). We showed that K14 C367-dependent disulfides form in human and mouse skin keratinocytes (Lee et al., 2012), where they play a role in the assembly, organization and steady state dynamics of keratin IFs in live skin keratinocytes (Feng and Coulombe, 2015a; Feng and Coulombe, 2015b). We also showed that loss of the stutter cysteine alters K14’s ability to become part of the dense meshwork of keratin filaments that occurs in the perinuclear space of early differentiating keratinocytes (Lee et al., 2012; Feng and Coulombe, 2015a; Feng and Coulombe, 2015b). However, the physiological significance associated with the surprising properties conferred by a cysteine residue located in a mysterious conserved motif within the central rod domain of a keratin, namely K14, remained unclear.

Here, we report on studies involving a new mouse model that provides evidence that the stutter cysteine in K14 protein regulates entry into differentiation and thus the balance between proliferation and differentiation through regulated interactions with 14-3-3 adaptor proteins and YAP1, a terminal effector of Hippo signaling (Pocaterra et al., 2020). We also discuss evidence that this role likely applies to K10 and other type I keratins expressed in surface epithelia.

The distribution of cysteine residues in mouse K14 protein is schematized in Figure 1A. Codon C367 in KRT14 (human) occurs at position 373 in Krt14 (mouse), and is conserved in the orthologous keratin of several other species (Figure 1B). Moreover, this codon is also conserved in many other type I keratin genes expressed in skin (Strnad et al., 2011; Lee et al., 2012; Figure 1B). To address the physiological significance of the conserved stutter cysteine in K14, we generated Krt14 C373A mutant mice using CRISPR-Cas9 technology (Figure 1C) and verified its presence through allele specific DNA-sequencing (Figure 1D). Krt14 C373A mice are born in the expected mendelian ratio, are viable and fertile, and show a normal body weight when reaching adulthood (Figure 1E). Analysis of total skin proteins from several body sites showed that steady state levels of K14 protein are unaffected in Krt14 C373A relative to WT skin. By contrast, the pattern of K14-dependent, high molecular weight disulfide-bonded species is markedly altered, given fewer species that occur at lower levels (Figure 1F,G). This is so especially in ear and tail skin (Figure 1F,G), prompting us to focus on these two body sites in subsequent analyses. The residual K14-dependent disulfide bonding occurring in Krt14 C373A mutant skin (Figure 1F,G) likely reflects the participation of cysteines located in the N-terminal domain of K14 (see Figure 1A and Feng and Coulombe, 2015a). These findings indicate that mice homozygous for Krt14 C373A allele are viable and appear macroscopically normal, although biochemically they exhibit a strikingly altered pattern of K14-dependent disulfide bonding, particularly in ear and tail skin.

Figure 1

Download asset Open asset

The histology and barrier status of young adult Krt14 C373A mouse skin were analyzed next. By histology, the epidermis of Krt14 C373A mice is modestly but significantly thickened relative to WT in ear and tail skin (Figure 2A,B). Measurement of trans-epidermal water loss (TEWL) at the skin surface revealed an increase in Krt14 C373A mice relative to WT control. This is so both at baseline (7.02 ± 0.72 g/m2/h vs. 2.95 ± 0.49 g/m²/h) and after topical acetone application (19.20 ± 1.78 g/m2/h vs. 7.02 ± 0.72 g/m²/h) (Figure 2C), a standard challenge that puts the skin barrier under a mild and reversible stress (Denda et al., 1996). Skin barrier defects often trigger elevated expression of Danger-Associated Molecular Patterns (Lessard et al., 2013) (DAMPs, also known as alarmins). At baseline, DAMPs such as S100a8, S100a9, Mmp9, and Ptgs2 are upregulated by 4-fold or more at the mRNA level in Krt14 C373A skin compared to WT (Figure 2D). This aberrant state is markedly enhanced after a topical acetone challenge to the barrier (Figure 2E). Next, we analyzed cornified envelopes (CEs) isolated from epidermis, given that they are key contributors to skin barrier function (Eckhart et al., 2013). CEs harvested from WT mice appear relatively uniform in size and shape, are mostly oval-shaped, and feature clear and smooth outlines (Figure 2F,G). By contrast, CEs isolated from Krt14 C373A mice are smaller (85% of the area and 87% of the circumference of WT CEs), jagged, and less oval-shaped (aspect ratio of 1.3 compared to 1.1 in WT) (Figure 2F,G and Figure 2—figure supplement 1A,B). Thus, the morphological and molecular anomalies occurring in the epidermis are accompanied by significant defects in barrier function in Krt14 C373A skin.

Figure 2 with 1 supplement see all

Download asset Open asset

We next assessed keratinocyte proliferation, transit time, and apoptosis in order to identify possible causes for the increased thickness and barrier defect in Krt14 C373A epidermis. At 2 hr after a single pulse of the nucleotide analog Edu (Chehrehasa et al., 2009), a significantly greater fraction of keratinocytes are labeled in the basal layer of Krt14 C373A epidermis compared to WT (by ~1.6 fold; p=0.024) (Figure 3A,B), indicating that keratinocyte proliferation is enhanced at baseline in mutant mice. Following a 1 day chase after the Edu pulse, this difference is accentuated (>2 fold; p=0.01) and Edu-labeled nuclei now occur in the suprabasal layers of epidermis in both genotypes, reflecting keratinocyte exit from the basal layer (Figure 3A,B). Following a 3-day chase after the Edu pulse, nuclear labeling remains high and stable in the basal layer of epidermis in both genotypes, but a clear additional difference emerges as there are significantly more Edu-labeled nuclei in the suprabasal layers of mutant epidermis. At the 7-day mark, the fraction of labeled cells in the basal layer has subsided in both genotypes but, again, the suprabasal epidermis of Krt14 C373A skin shows far more labeled nuclei (Figure 3A,B). This pulse-chase experiment shows that keratinocytes in Krt14 C373A epidermis show enhanced proliferation confined to the basal layer at baseline, and that keratinocytes exhibit a faster pace of movement across the suprabasal layers as they progress through differentiation. A similar phenotype has been previously described for Krt10 null mice (Reichelt and Magin, 2002). We also observed a greater frequency (~3 fold) of TUNEL-positive nuclei in Krt14 C373A epidermis compared to WT, with apoptotic cell death confined to the suprabasal compartment (Figure 3C,D). We also examined expression of p63, given its role as master regulator of epidermal stratification and differentiation (Soares and Zhou, 2018). A distinct immunostaining pattern was observed in Krt14 C373A epidermis relative to WT (Figure 3E). Upon quantitation, significant differences prevailed in terms of frequency of keratinocytes labeled and their distance from the basal lamina (Figure 3F), with p63-positive staining showing a conspicuous elongated shape and extending higher up in the mutant epidermis. When combined, these findings suggest that the modest increase observed in epidermal thickness (Figure 1) masks a more pronounced defect in epidermal homeostasis under baseline conditions in Krt14 C373A mice.

Figure 3 with 2 supplements see all

Download asset Open asset

We next assessed markers relevant to keratinocyte proliferation to identify possible causes for the defective barrier of Krt14 C373A skin relative to WT. We examined the distribution of K14 (basal cell layer), K10 (early differentiation), filaggrin and loricrin (late differentiation) in tail skin sections from young adult mice. The staining for filaggrin and loricrin were markedly decreased (~62% and 48% reductions, respectively) while the staining for K10 was modestly decreased (~37% reduction) in Krt14 C373A epidermis relative to WT (Figure 3G,H). We also examined markers of adherens junction (E-cadherin), desmosomes (desmoplakin), tight junctions (claudin 3) since epidermal differentiation entails a tightly coordinated rearrangement of intercellular junctions. Claudin 3 staining was decreased by ~33% in Krt14 C373A epidermis, consistent with the barrier defect. The signals for E-cadherin and desmoplakin appeared slightly increased (Figure 3I), an occurrence that may reflect the modest epidermal thicknening (Figure 3J). In contrast to tail and ear epidermis, several markers including K14, K10, loricrin and filaggrin appear normal in the thin epidermis of back skin (Figure 3—figure supplement 1A–C), consistent with the markedly lower yield of K14-dependent disulfide bonding in this body site (Figure 1). Together these observations link the anomalies observed in epidermal homeostasis and skin barrier to defects in terminal keratinocyte differentiation in Krt14 C373A mouse skin.

We previously showed that replacing Cys with Ala at position 367 in human K14 does not abrogate 10 nm filament formation but leads to a reduction in the perinuclear clustering of keratin filaments in cultured keratinocytes (Feng and Coulombe, 2015b). Transmission electron microscopy of epoxy-embedded skin tissue sections was next used to assess whether similar changes occur in vivo. In basal keratinocytes of WT epidermis, keratin IFs typically occur as bundles near the nucleus. In Krt14 C373A basal keratinocytes, however, keratin IFs are absent from the perinuclear region and appear redistributed towards the cell periphery (Figure 3—figure supplement 2A,B). Consistent with the macroscopic appearance of skin tissue there is no ultrastructural evidence of cell fragility in Krt14 C373A epidermis (Figure 3—figure supplement 2A,B and data not shown). We find that nuclei feature a more ellipsoid shape along with a greater frequency of cytoplasmic invaginations (by ~1.4 fold in basal keratinocytes and by ~1.7 fold in suprabasal keratinocytes, respectively) compared to WT controls; Figure 3—figure supplement 2C). The occurrence of ultrastructural anomalies in the perinuclear keratin IF network in Krt14 C373A basal keratinocytes extend previous live imaging observations (Feng and Coulombe, 2015b) and point to the possibility that the mechanical properties of the nuclear envelope or nucleus are altered in these cells.

To identify potential pathways regulated by K14-dependent disulfides, we performed K14 co-immunoprecipitation (co-IP) assays followed by mass spectrometry (MS) analysis in protein extracts prepared from newborn WT keratinocytes in primary culture in the presence of 1 mM Ca²⁺, a condition that induces keratinocyte differentiation (Hennings et al., 1980). This screen identified 14-3-3σ and other 14-4-3 isoforms as major interacting partners for K14 in WT cell cultures (Figure 4A and Supplementary file 1). There is a strong precedent for interactions between 14-3-3 proteins and keratins, including K18 (Liao and Omary, 1996; Ku et al., 1998), K17 (Kim et al., 2006), which occur in a phosphorylation-dependent fashion. Co-immunoprecipitation assays confirmed that transfected, HA tagged-14-3-3σ physically interact with endogenous WT K14 and Krt14373A mutant protein in mouse keratinocytes in primary culture (Figure 4B and data not shown). 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-K14’s established role in skin pigmentation (Gu and Coulombe, 2007).

Figure 4 with 1 supplement see all

Download asset Open asset

14-3-3σ was deemed of interest because it regulates the proliferation and differentiation of keratinocytes in epidermis (Herron et al., 2005; Li et al., 2005). The latter is achieved in part by modulating the cellular localization of YAP (Li et al., 2005; Sun et al., 2015), a terminal effector of Hippo signaling (Schlegelmilch et al., 2011; Silvis et al., 2011; Sambandam et al., 2015). Hippo is an evolutionary conserved pathway with a primary role in regulating growth and homeostasis in organs and tissues (Pocaterra et al., 2020). We next assessed the distribution of 14-3-3σ and YAP using indirect immunofluorescence of tissue sections prepared from WT and Krt14 C373A tail skin. 14-3-3σ occurs mostly as aggregates in suprabasal keratinocytes of Krt14 C373A epidermis, which is in striking contrast to the diffuse distribution observed in WT controls (Figure 4C). Consistent with previous reports (Schlegelmilch et al., 2011; Sambandam et al., 2015), a strong signal for YAP occurs in both the nucleus and cytoplasm in basal keratinocytes, and otherwise YAP occurs as a weaker and diffuse signal in the cytoplasm (but is not seen in the nucleus) of suprabasal keratinocytes in WT epidermis (Figure 4D). In Krt14 C373A epidermis, strikingly, YAP localizes preferentially to nuclei in both basal and suprabasal keratinocytes, in a consistent fashion (Figure 4D). The latter finding suggests that YAP-dependent gene expression may be altered in mutant mouse skin. Follow-up RT-qPCR assays show that the steady state levels for several known YAP target gene mRNAs, including Cyr61, Zeb1, Ctgf and Snail2, are markedly elevated in Krt14 C373A relative to WT skin (Figure 4E). By western immunoblotting, the levels of endogenous YAP1 and Ser^127-phosphorylated YAP1 are similar in WT and Krt14 C373A skin (Figure 4F,G). Together these findings point to a misregulation of 14-3-3σ and YAP as likely contributors to the epidermal phenotype exhibited in the ear and tail skin of Krt14 C373A mice.

Next we asked whether the misregulation of YAP subcellular partitioning also occurs in primary culture. Keratinocytes were isolated from WT and Krt14 C373A newborn pups, cultured in the absence or presence of calcium (Hennings et al., 1980), and analyzed using microscopy-based readouts. 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 4—figure supplement 1A,B). In the absence of calcium, the staining for YAP is concentrated in the nucleus in both WT and Krt14 C373A keratinocytes (Figure 5A,B). 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). Western immunoblotting confirmed that, as expected, both K10 and filaggrin proteins occur at lower levels in calcium-treated primary cultures of Krt14 C373A relative to WT keratinocytes (Figure 4—figure supplement 1C,D), suggesting a delay or a defect in terminal differentiation. Moreover, PLA assays yielded evidence for decreased interactions between 14-3-3σ and YAP in Krt14 C373A keratinocytes in primary culture, relative to WT controls (Figure 4—figure supplement 1E,F), thereby extending the immunoprecipitation findings shown in Figure 4B. We conclude that the abnormal retention of YAP to the nucleus and abnormal differentiation of Krt14 C373A keratinocytes are both preserved outside of the skin tissue setting, further suggesting that these properties are linked and inherent to keratinocytes.

Figure 5 with 1 supplement see all

Download asset Open asset

The five cysteine residues present in human K14 are conserved in the mouse ortholog (Lee et al., 2012), and cysteines at positions 4, 40, and 367 in human K14 participate in disulfide bonding (Feng and Coulombe, 2015a). We next asked whether the aberrant YAP localization in Krt14 C373A mutant epidermis is Cys373-specific. To this end we devised a rescue assay using Krt14 null mouse keratinocytes in primary culture, transfection of GFP-K14 or mutants thereof, and analysis of YAP subcellular partitioning. Consistent with previous findings (Sambandam et al., 2015), only 19.5% of GFP-K14WT-expressing keratinocytes exhibit nuclear YAP in the presence of calcium (Figure 5—figure supplement 1A,B). By contrast, cells expressing either a GFP-K14 cysteine free (CF) mutant, or a GFP-C367A single mutant, feature an abnormally high nuclear retention of YAP (83.8% and 81.9%, respectively) (Figure 5—figure supplement 1A,B). Restoring Cys367 in the K14-CF backbone (GFP-K14CF-C367 construct) rescued the abnormal nuclear retention of YAP, given that only 27.3% of transfected cells show YAP in the nucleus (Figure 5—figure supplement 1A,B). These findings directly implicate the stutter cysteine (C367 in human K14) as a calcium-dependent regulator of the subcellular partitioning of YAP in skin keratinocytes.

In an effort to directly relate K14 to the activity of YAP1 as a transcriptional regulator, we next conducted luciferase reporter assays in a heterologous cell culture setting. HeLa cells were selected because they are epithelia-derived, and grow and transfect well. HeLa cells normally show low levels of K14 expression (Moll et al., 1982) but respond well to K5-K14 co-transfection (Figure 5—figure supplement 1C). Transfection of a Ccn1 (Cyr61) gene promoter-driven Firefly luciferase plasmid, previously shown to effectively report on YAP transcriptional activity (Ma et al., 2017), led to a strong (~8 fold) induction of luciferase activity normalized to cells transfected with a reference Renilla luciferase plasmid (Figure 5C). Consistent with K14’s ability to retain YAP in the cytoplasm, Ccn1 promoter activity was significantly attenuated by co-expression of WT K14 along with assembly partner WT K5 (Figure 5C). In striking contrast, activity of the Ccn1 promoter construct was much less affected when cysteine-free K14 (K14 CF) was co-expressed with WT K5 (Figure 5—figure supplement 1C). Of note, the organization of filaments in HeLa cells co-expressing either K5 and K14WT or K5 and K14CF is comparable (Figure 5—figure supplement 1C). The findings from this heterologous assay substantiate the notion that K14-containing filaments impact YAP activity as predicted, and that this property depends on the presence of Cys residues in K14.

YAP is a key effector of mechanosensing and mechanotransduction (Dupont et al., 2011; Benham-Pyle et al., 2015; Panciera et al., 2017). Cells experiencing tension often respond by enhancing determinants such as F-actin stress fibers, acto-myosin contraction, the recruitment of α-catenin and vinculin to adherens junctions (Leckband and de Rooij, 2014; Yap et al., 2018), expression of lamin A/C in the nucleus (Swift et al., 2013), and frequency of binucleation (Cao et al., 2017). Monitoring the status of such elements provides a test for altered mechanosensing or mechanotransduction. In tail skin tissue sections, the immunostaining for α-catenin and for lamin A/C but not that for desmoglein 1 are markedly increased in Krt14 C373A mice relative to WT controls (Figure 6A,B). Use of the a-18 antibody that recognizes a mechanosensitive epitope on α-catenin (Shimoyama et al., 1992) confirms that epidermal keratinocytes are under altered tensile stress in Krt14 C373A epidermis (Figure 6C). In the setting of primary culture, we observed a greater incidence of multi-nucleated keratinocytes in Ca²⁺-treated Krt14 C373A newborn skin keratinocytes compared to WT control (14.3% versus 2.8%; Figure 6D). We also find that the immunofluorescence signal for α-catenin, lamin A/C, F-actin stress fibers and phosphorylated myosin light chain II (pMLC Ser19) are each increased in Krt14 C373A relative to WT (Figure 6E,F). Collectively these findings strongly suggest that, consistent with YAP misregulation (see Figure 5), Krt14 C373A mutant keratinocytes show alterations in mechanosensing and/or mechanotransduction relative to WT.

Figure 6

Download asset Open asset

Our study establishes that residue cysteine 373 in mouse K14 partakes in regulating the balance between keratinocyte proliferation and differentiation in epidermis in vivo, and ultimately barrier function, in skin. 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. Our findings support the conclusion that K14-dependent disulfide bonding involving the conserved ‘stutter cysteine’ residue impacts cell architecture, mechanosensing and Hippo signaling at an early stage of epithelial differentiation in the epidermis.

A model that conveys the significance our findings is given in Figure 7A. Consistent with the literature, the model posits that Hippo signaling is inactive in most keratinocytes in the basal layer of epidermis (Schlegelmilch et al., 2011; Beverdam et al., 2013; Sambandam et al., 2015; Totaro et al., 2017), with YAP localizing to the nucleus given a specific level of cellular crowding and/or integrin-mediated adhesion to the extracellular matrix (Panciera et al., 2017; Elbediwy and Thompson, 2018). In basal keratinocytes, K5-K14 filaments are organized in loose bundles that run alongside the nucleus (Coulombe et al., 1989; Lee et al., 2012) while 14-3-3σ occurs at low levels (Dellambra et al., 2000; Reichelt and Magin, 2002; Kim et al., 2006). The first prediction of our model is that reception of differentiation-promoting cues triggers K14-dependent disulfide bonding and creates binding sites for 14-3-3 on K5-K14 filaments. The latter likely occurs via site-specific phosphorylation, on K14 (e.g., Inaba et al., 2018). These events foster the known reorganization of keratin filaments into a prominent perinuclear network (Lee et al., 2012), along with the binding and sequestration of YAP1 to the cytoplasm, thus activating Hippo signaling as keratinocytes initiate terminal differentiation (Figure 7A). Several reports converged in establishing a role for mitochondria-derived reactive oxygen species as a key trigger towards the initiation of terminal differentiation in keratinocytes of epidermis (Tamiji et al., 2005; Bause et al., 2013; Hamanaka et al., 2013; Sun et al., 2015). Such species could non-enzymatically mediate K14-dependent disulfide bonding in the perinuclear cytoplasm of keratinocytes (Suzuki et al., 2019).

Figure 7 with 1 supplement see all

Download asset Open asset

Many type I keratins expressed in epidermis (e.g., K10, K16, K17) feature a cysteine residue at the location corresponding to codon 373 in mouse K14 and codon 367 in human K14 (Lee et al., 2012). Four lines of evidence support the contention that K10, in particular, is a strong candidate for K14-like regulation of YAP1 subcellular partitioning and Hippo signaling in the epidermis. First, KRT10 (human) and Krt10 (mouse) expression is turned on at the earliest stage of terminal differentiation (Woodcock-Mitchell et al., 1982; Schweizer et al., 1984), and the K10 protein features a structurally and biochemically equivalent Cys residue at position 401 and is capable of interacting with 14-3-3 (Wilker et al., 2007; Huang et al., 2010). 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. Third, we previously showed that K10 partakes in the formation of disulfide-dependent, dimer-sized species in skin keratinocytes (Lee et al., 2012). Fourth the Krt10 null mouse, described >15 years ago (Reichelt and Magin, 2002; Reichelt et al., 2004), exhibits a strong phenotype consisting of hyperproliferation, faster keratinocyte transit time, and impaired differentiation in the epidermis which, molecularly, correlates with a marked upregulation of 14-3-3sigma and c-Myc. MYC has since then been shown to be a bona fide YAP1 target gene (Schütte et al., 2014; Kim et al., 2017; Cai et al., 2018). 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.

Our findings extend previous reports linking 14-3-3σ to the regulation of YAP during terminal differentiation in epidermis (Sambandam et al., 2015; Sun et al., 2015). They suggest that the network of keratin filaments proximal to the nucleus acts as a key docking site for 14-3-3/YAP complexes during keratinocyte delamination and differentiation, taking on a role that has been held to this point by integrin-based adhesion sites (Elbediwy et al., 2016) and adherens junctions (via alpha-catenin; (Schlegelmilch et al., 2011; Silvis et al., 2011). In addition, a disulfide bonding-rich perinuclear network of keratin filaments could also afford protection to the nucleus and the genome during delamination (Lee et al., 2012), thus extending the known role of K5-K14 IFs in providing mechanical support in basal keratinocytes (Coulombe et al., 1991a; Ramms et al., 2013). Besides, keratinocyte delamination and differentiation in epidermis together entail a profound reorganization of cell-cell and cell-matrix adhesion, and of F-actin and microtubule organization (Sumigray and Lechler, 2015; Muroyama and Lechler, 2017; Miroshnikova et al., 2018; Rübsam et al., 2018; Wickström and Niessen, 2018). How all of these effectors and their inputs are integrated to result in a redistribution of intracellular tension and/or compressive forces (Miroshnikova et al., 2018; Rübsam et al., 2018), along with proper regulation of YAP function and Hippo signaling, awaits further investigation.

YAP’s role as a transcription factor requires its binding to TEAD protein, which is itself stably bound to the promoter of its target genes in a sequence-specific fashion in the nucleus (Vassilev et al., 2001). Data available through ENCODE for human foreskin keratinocytes in primary culture in the absence or presence of calcium (see Materials and methods) provides insight into the rationale for keratin protein involvement towards the regulation of YAP-driven transcription and Hippo signaling. 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, for example 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 7—figure supplement 1; Supplementary file 2). ENCODE data convey that the chromatin surrounding these genes becomes non-accessible in the presence of calcium (Figure 7—figure supplement 1 and 2), which promotes differentiation (Hennings et al., 1980). Keratin genes expressed in differentiating keratinocytes of epidermis, including the type I Krt10 and type Krt1 and Krt2, do not feature proximal TEAD binding sites (Figure 7—figure supplement 1; Supplementary file 2) and thus are not likely to be transcribed in a TEAD/YAP-dependent fashion in epidermis. The YAP1 and TEAD genes (human) are themselves poised to be positively regulated by YAP in progenitor keratinocytes (Figure 7—figure supplement 1; Supplementary file 2). SFN, ITGB1, CCN1 and TP63 (human) are among the additional genes of interest that we included in this analysis (Supplementary file 2). Accordingly, the YAP/TEAD axis may be set up to promote unabated cell proliferation in a fast-renewing epithelium such as epidermis until two post-translational events converging on K14 protein, phosphorylation and disulfide bonding, would lead to YAP protein sequestration in the cytoplasm and inactivation of the YAP/TEAD axis as part of a regulated negative feedback loop, enacted at the time of entry into differentiation in epidermis (Figure 7B).

Several mechanisms could account for the functional interplay between K14-dependent disulfide bonding, 14-3-3σ, and the regulation of YAP’s subcellular partitioning in keratinocytes. First, the perinuclear enrichment of keratin IFs, which is promoted by K14-dependent disulfide bonding, is poised to increase the local concentration of binding sites for 14-3-3σ and YAP near the nucleus (simple mass action law). Second, the occurrence of K14-dependent disulfides may create an optimal binding interface for 14-3-3σ and YAP in the cytoplasm residing proximal to the nucleus. Third, the nucleus is known to function as a mechanosensor (Cao et al., 2017), and local forces impacted by the perinuclear network of keratin filaments could alter the mechanical gating of YAP across nuclear pores (Elosegui-Artola et al., 2017). These three mechanisms, and possibly others, could act in combination. How K14 (and possibly K10), 14-3-3σ, YAP, and other crucial effectors bind each other as part of this newly defined signaling axis, its regulation, and its significance have now emerged as open issues of high significance for future studies.

All of the data generated or analyzed during this study are included in the manuscript and supporting files.

1. 1. Afgan E
   2. Baker D
   3. van den Beek M
   4. Blankenberg D
   5. Bouvier D
   6. Čech M
   7. Chilton J
   8. Clements D
   9. Coraor N
   10. Eberhard C
   11. Grüning B
   12. Guerler A
   13. Hillman-Jackson J
   14. Von Kuster G
   15. Rasche E
   16. Soranzo N
   17. Turaga N
   18. Taylor J
   19. Nekrutenko A
   20. Goecks J

   (2016) The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update

   Nucleic Acids Research 44:W3–W10.

   https://doi.org/10.1093/nar/gkw343

   • PubMed
   • Google Scholar
2. 1. Bause AS
   2. Matsui MS
   3. Haigis MC

   (2013) The protein deacetylase SIRT3 prevents oxidative stress-induced keratinocyte differentiation

   Journal of Biological Chemistry 288:36484–36491.

   https://doi.org/10.1074/jbc.M113.472324

   • PubMed
   • Google Scholar
3. 1. Benham-Pyle BW
   2. Pruitt BL
   3. Nelson WJ

   (2015) Cell adhesion mechanical strain induces E-cadherin-dependent Yap1 and β-catenin activation to drive cell cycle entry

   Science 348:1024–1027.

   https://doi.org/10.1126/science.aaa4559

   • PubMed
   • Google Scholar
4. 1. Betz RC
   2. Planko L
   3. Eigelshoven S
   4. Hanneken S
   5. Pasternack SM
   6. Bussow H
   7. Van Den Bogaert K
   8. Wenzel J
   9. Braun-Falco M
   10. Rutten A
   11. Rogers MA
   12. Ruzicka T
   13. Nöthen MM
   14. Magin TM
   15. Kruse R

   (2006) Loss-of-function mutations in the keratin 5 gene lead to Dowling-Degos disease

   The American Journal of Human Genetics 78:510–519.

   https://doi.org/10.1086/500850

   • PubMed
   • Google Scholar
5. 1. Beverdam A
   2. Claxton C
   3. Zhang X
   4. James G
   5. Harvey KF
   6. Key B

   (2013) Yap controls stem/progenitor cell proliferation in the mouse postnatal epidermis

   Journal of Investigative Dermatology 133:1497–1505.

   https://doi.org/10.1038/jid.2012.430

   • PubMed
   • Google Scholar
6. 1. Bonifas JM
   2. Rothman AL
   3. Epstein EH

   (1991) Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities

   Science 254:1202–1205.

   https://doi.org/10.1126/science.1720261

   • PubMed
   • Google Scholar
7. 1. Bunick CG
   2. Milstone LM

   (2017) The X-Ray crystal structure of the keratin 1-Keratin 10 Helix 2B Heterodimer Reveals Molecular Surface Properties and Biochemical Insights into Human Skin Disease

   Journal of Investigative Dermatology 137:142–150.

   https://doi.org/10.1016/j.jid.2016.08.018

   • PubMed
   • Google Scholar
8. 1. Cai J
   2. Song X
   3. Wang W
   4. Watnick T
   5. Pei Y
   6. Qian F
   7. Pan D

   (2018) A RhoA-YAP-c-Myc signaling Axis promotes the development of polycystic kidney disease

   Genes & Development 32:781–793.

   https://doi.org/10.1101/gad.315127.118

   • PubMed
   • Google Scholar
9. 1. Cao J
   2. Wang J
   3. Jackman CP
   4. Cox AH
   5. Trembley MA
   6. Balowski JJ
   7. Cox BD
   8. De Simone A
   9. Dickson AL
   10. Di Talia S
   11. Small EM
   12. Kiehart DP
   13. Bursac N
   14. Poss KD

   (2017) Tension creates an endoreplication wavefront that leads regeneration of epicardial tissue

   Developmental Cell 42:600–615.

   https://doi.org/10.1016/j.devcel.2017.08.024

   • Google Scholar
10. 1. Chehrehasa F
    2. Meedeniya AC
    3. Dwyer P
    4. Abrahamsen G
    5. Mackay-Sim A

    (2009) EdU, a new thymidine analogue for labelling proliferating cells in the nervous system

    Journal of Neuroscience Methods 177:122–130.

    https://doi.org/10.1016/j.jneumeth.2008.10.006

    • PubMed
    • Google Scholar
11. 1. Chung BM
    2. Murray CI
    3. Van Eyk JE
    4. Coulombe PA

    (2012) Identification of novel interaction between annexin A2 and keratin 17: evidence for reciprocal regulation

    The Journal of Biological Chemistry 287:7573–7581.

    https://doi.org/10.1074/jbc.M111.301549

    • PubMed
    • Google Scholar
12. 1. Coulombe PA
    2. Kopan R
    3. Fuchs E

    (1989) Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation

    The Journal of Cell Biology 109:2295–2312.

    https://doi.org/10.1083/jcb.109.5.2295

    • PubMed
    • Google Scholar
13. 1. Coulombe PA
    2. Hutton ME
    3. Letal A
    4. Hebert A
    5. Paller AS
    6. Fuchs E

    (1991a) Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses

    Cell 66:1301–1311.

    https://doi.org/10.1016/0092-8674(91)90051-Y

    • Google Scholar
14. 1. Coulombe PA
    2. Hutton ME
    3. Vassar R
    4. Fuchs E

    (1991b) A function for keratins and a common thread among different types of epidermolysis bullosa simplex diseases

    The Journal of Cell Biology 115:1661–1674.

    https://doi.org/10.1083/jcb.115.6.1661

    • PubMed
    • Google Scholar
15. 1. Coulombe PA
    2. Lee CH

    (2012) Defining keratin protein function in skin epithelia: epidermolysis bullosa simplex and its aftermath

    Journal of Investigative Dermatology 132:763–775.

    https://doi.org/10.1038/jid.2011.450

    • PubMed
    • Google Scholar
16. 1. Dellambra E
    2. Golisano O
    3. Bondanza S
    4. Siviero E
    5. Lacal P
    6. Molinari M
    7. D'Atri S
    8. De Luca M

    (2000) Downregulation of 14-3-3sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes

    Journal of Cell Biology 149:1117–1130.

    https://doi.org/10.1083/jcb.149.5.1117

    • PubMed
    • Google Scholar
17. 1. Denda M
    2. Wood LC
    3. Emami S
    4. Calhoun C
    5. Brown BE
    6. Elias PM
    7. Feingold KR

    (1996) The epidermal hyperplasia associated with repeated barrier disruption by acetone treatment or tape stripping cannot be attributed to increased water loss

    Archives of Dermatological Research 288:230–238.

    https://doi.org/10.1007/BF02530090

    • PubMed
    • Google Scholar
18. 1. Dupont S
    2. Morsut L
    3. Aragona M
    4. Enzo E
    5. Giulitti S
    6. Cordenonsi M
    7. Zanconato F
    8. Le Digabel J
    9. Forcato M
    10. Bicciato S
    11. Elvassore N
    12. Piccolo S

    (2011) Role of YAP/TAZ in mechanotransduction

    Nature 474:179–183.

    https://doi.org/10.1038/nature10137

    • PubMed
    • Google Scholar
19. 1. Eckhart L
    2. Lippens S
    3. Tschachler E
    4. Declercq W

    (2013) Cell death by cornification

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1833:3471–3480.

    https://doi.org/10.1016/j.bbamcr.2013.06.010

    • Google Scholar
20. 1. Elbediwy A
    2. Vincent-Mistiaen ZI
    3. Spencer-Dene B
    4. Stone RK
    5. Boeing S
    6. Wculek SK
    7. Cordero J
    8. Tan EH
    9. Ridgway R
    10. Brunton VG
    11. Sahai E
    12. Gerhardt H
    13. Behrens A
    14. Malanchi I
    15. Sansom OJ
    16. Thompson BJ

    (2016) Integrin signalling regulates YAP and TAZ to control skin homeostasis

    Development 143:1674–1687.

    https://doi.org/10.1242/dev.133728

    • Google Scholar
21. 1. Elbediwy A
    2. Thompson BJ

    (2018) Evolution of mechanotransduction via YAP/TAZ in animal epithelia

    Current Opinion in Cell Biology 51:117–123.

    https://doi.org/10.1016/j.ceb.2018.02.003

    • PubMed
    • Google Scholar
22. 1. Elosegui-Artola A
    2. Andreu I
    3. Beedle AEM
    4. Lezamiz A
    5. Uroz M
    6. Kosmalska AJ
    7. Oria R
    8. Kechagia JZ
    9. Rico-Lastres P
    10. Le Roux A-L
    11. Shanahan CM
    12. Trepat X
    13. Navajas D
    14. Garcia-Manyes S
    15. Roca-Cusachs P

    (2017) Force triggers YAP nuclear entry by regulating transport across nuclear pores

    Cell 171:1397–1410.

    https://doi.org/10.1016/j.cell.2017.10.008

    • Google Scholar
23. 1. Feng J
    2. Liu T
    3. Qin B
    4. Zhang Y
    5. Liu XS

    (2012) Identifying ChIP-seq enrichment using MACS

    Nature Protocols 7:1728–1740.

    https://doi.org/10.1038/nprot.2012.101

    • PubMed
    • Google Scholar
24. 1. Feng X
    2. Coulombe PA

    (2015a) Complementary roles of specific cysteines in keratin 14 toward the assembly, organization, and dynamics of intermediate filaments in skin keratinocytes

    Journal of Biological Chemistry 290:22507–22519.

    https://doi.org/10.1074/jbc.M115.654749

    • PubMed
    • Google Scholar
25. 1. Feng X
    2. Coulombe PA

    (2015b) A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes

    Journal of Cell Biology 209:59–72.

    https://doi.org/10.1083/jcb.201408079

    • PubMed
    • Google Scholar
26. 1. Fuchs E

    (1995) Keratins and the skin

    Annual Review of Cell and Developmental Biology 11:123–154.

    https://doi.org/10.1146/annurev.cb.11.110195.001011

    • PubMed
    • Google Scholar
27. 1. Fuchs E
    2. Coulombe PA

    (1992) Of mice and men: genetic skin diseases of keratin

    Cell 69:899–902.

    https://doi.org/10.1016/0092-8674(92)90607-E

    • PubMed
    • Google Scholar
28. 1. Garrod KR
    2. D. Cahalan M

    (2008) Murine skin transplantation

    Journal of Visualized Experiments 2:55105.

    https://doi.org/10.3791/634

    • Google Scholar
29. 1. Gu LH
    2. Coulombe PA

    (2007) Keratin function in skin epithelia: a broadening palette with surprising shades

    Curr Opin Cell Biol.  19:13–23.

    https://doi.org/10.1016/j.ceb.2006.12.007

    • Google Scholar
30. 1. Hamanaka RB
    2. Glasauer A
    3. Hoover P
    4. Yang S
    5. Blatt H
    6. Mullen AR
    7. Getsios S
    8. Gottardi CJ
    9. DeBerardinis RJ
    10. Lavker RM
    11. Chandel NS

    (2013) Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development

    Science Signaling 6:ra8.

    https://doi.org/10.1126/scisignal.2003638

    • PubMed
    • Google Scholar
31. 1. Heinz S
    2. Benner C
    3. Spann N
    4. Bertolino E
    5. Lin YC
    6. Laslo P
    7. Cheng JX
    8. Murre C
    9. Singh H
    10. Glass CK

    (2010) Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities

    Molecular Cell 38:576–589.

    https://doi.org/10.1016/j.molcel.2010.05.004

    • PubMed
    • Google Scholar
32. 1. Hennings H
    2. Michael D
    3. Cheng C
    4. Steinert P
    5. Holbrook K
    6. Yuspa SH

    (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture

    Cell 19:245–254.

    https://doi.org/10.1016/0092-8674(80)90406-7

    • PubMed
    • Google Scholar
33. 1. Herron BJ
    2. Liddell RA
    3. Parker A
    4. Grant S
    5. Kinne J
    6. Fisher JK
    7. Siracusa LD

    (2005) A mutation in stratifin is responsible for the repeated epilation (Er) phenotype in mice

    Nature Genetics 37:1210–1212.

    https://doi.org/10.1038/ng1652

    • PubMed
    • Google Scholar
34. 1. Hobbs RP
    2. DePianto DJ
    3. Jacob JT
    4. Han MC
    5. Chung BM
    6. Batazzi AS
    7. Poll BG
    8. Guo Y
    9. Han J
    10. Ong S
    11. Zheng W
    12. Taube JM
    13. Čiháková D
    14. Wan F
    15. Coulombe PA

    (2015) Keratin-dependent regulation of aire and gene expression in skin tumor keratinocytes

    Nature Genetics 47:933–938.

    https://doi.org/10.1038/ng.3355

    • PubMed
    • Google Scholar
35. 1. Huang WG
    2. Cheng AL
    3. Chen ZC
    4. Peng F
    5. Zhang PF
    6. Li MY
    7. Li F
    8. Li JL
    9. Li C
    10. Yi H
    11. Li XH
    12. Yi B
    13. Xiao ZQ

    (2010) Targeted proteomic analysis of 14-3-3sigma in nasopharyngeal carcinoma

    The International Journal of Biochemistry & Cell Biology 42:137–147.

    https://doi.org/10.1016/j.biocel.2009.10.001

    • PubMed
    • Google Scholar
36. 1. Inaba H
    2. Yamakawa D
    3. Tomono Y
    4. Enomoto A
    5. Mii S
    6. Kasahara K
    7. Goto H
    8. Inagaki M

    (2018) Regulation of keratin 5/14 intermediate filaments by CDK1, Aurora-B, and Rho-kinase

    Biochemical and Biophysical Research Communications 498:544–550.

    https://doi.org/10.1016/j.bbrc.2018.03.016

    • PubMed
    • Google Scholar
37. Preprint

    1. Jaskula-Ranga V
    2. Zack DJ

    (2016) grID: a CRISPR-Cas9 guide RNA database and resource for genome-editing

    bioRxiv.

    https://doi.org/10.1101/097352

    • Google Scholar
38. 1. Kerns ML
    2. Hakim JM
    3. Lu RG
    4. Guo Y
    5. Berroth A
    6. Kaspar RL
    7. Coulombe PA

    (2016) Oxidative stress and dysfunctional NRF2 underlie pachyonychia congenita phenotypes

    Journal of Clinical Investigation 126:2356–2366.

    https://doi.org/10.1172/JCI84870

    • PubMed
    • Google Scholar
39. 1. Kim S
    2. Wong P
    3. Coulombe PA

    (2006) A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

    Nature 441:362–365.

    https://doi.org/10.1038/nature04659

    • Google Scholar
40. 1. Kim J
    2. Kim YH
    3. Kim J
    4. Park DY
    5. Bae H
    6. Lee DH
    7. Kim KH
    8. Hong SP
    9. Jang SP
    10. Kubota Y
    11. Kwon YG
    12. Lim DS
    13. Koh GY

    (2017) YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation

    Journal of Clinical Investigation 127:3441–3461.

    https://doi.org/10.1172/JCI93825

    • PubMed
    • Google Scholar
41. 1. Ku NO
    2. Liao J
    3. Omary MB

    (1998) Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins

    The EMBO Journal 17:1892–1906.

    https://doi.org/10.1093/emboj/17.7.1892

    • PubMed
    • Google Scholar
42. 1. Kubo A
    2. Nagao K
    3. Amagai M

    (2012) Epidermal barrier dysfunction and cutaneous sensitization in atopic diseases

    Journal of Clinical Investigation 122:440–447.

    https://doi.org/10.1172/JCI57416

    • PubMed
    • Google Scholar
43. 1. Kumar V
    2. Bouameur JE
    3. Bär J
    4. Rice RH
    5. Hornig-Do HT
    6. Roop DR
    7. Schwarz N
    8. Brodesser S
    9. Thiering S
    10. Leube RE
    11. Wiesner RJ
    12. Vijayaraj P
    13. Brazel CB
    14. Heller S
    15. Binder H
    16. Löffler-Wirth H
    17. Seibel P
    18. Magin TM

    (2015) A keratin scaffold regulates epidermal barrier formation, mitochondrial lipid composition, and activity

    Journal of Cell Biology 211:1057–1075.

    https://doi.org/10.1083/jcb.201404147

    • PubMed
    • Google Scholar
44. 1. Lane EB
    2. Rugg EL
    3. Navsaria H
    4. Leigh IM
    5. Heagerty AH
    6. Ishida-Yamamoto A
    7. Eady RA

    (1992) A mutation in the conserved Helix termination peptide of keratin 5 in hereditary skin blistering

    Nature 356:244–246.

    https://doi.org/10.1038/356244a0

    • PubMed
    • Google Scholar
45. 1. Leckband DE
    2. de Rooij J

    (2014) Cadherin adhesion and mechanotransduction

    Annual Review of Cell and Developmental Biology 30:291–315.

    https://doi.org/10.1146/annurev-cellbio-100913-013212

    • PubMed
    • Google Scholar
46. 1. Lee CH
    2. Kim MS
    3. Chung BM
    4. Leahy DJ
    5. Coulombe PA

    (2012) Structural basis for heteromeric assembly and perinuclear organization of keratin filaments

    Nature Structural & Molecular Biology 19:707–715.

    https://doi.org/10.1038/nsmb.2330

    • PubMed
    • Google Scholar
47. 1. Lessard JC
    2. Piña-Paz S
    3. Rotty JD
    4. Hickerson RP
    5. Kaspar RL
    6. Balmain A
    7. Coulombe PA

    (2013) Keratin 16 regulates innate immunity in response to epidermal barrier breach

    PNAS 110:19537–19542.

    https://doi.org/10.1073/pnas.1309576110

    • PubMed
    • Google Scholar
48. 1. Li Q
    2. Lu Q
    3. Estepa G
    4. Verma IM

    (2005) Identification of 14-3-3sigma mutation causing cutaneous abnormality in repeated-epilation mutant mouse

    PNAS 102:15977–15982.

    https://doi.org/10.1073/pnas.0508310102

    • PubMed
    • Google Scholar
49. 1. Liao J
    2. Omary MB

    (1996) 14-3-3 proteins associate with phosphorylated simple epithelial keratins during cell cycle progression and act as a solubility cofactor

    The Journal of Cell Biology 133:345–357.

    https://doi.org/10.1083/jcb.133.2.345

    • PubMed
    • Google Scholar
50. 1. Ma X
    2. Zhang H
    3. Xue X
    4. Shah YM

    (2017) Hypoxia-inducible factor 2α (HIF-2α) promotes Colon cancer growth by potentiating Yes-associated protein 1 (YAP1) activity

    Journal of Biological Chemistry 292:17046–17056.

    https://doi.org/10.1074/jbc.M117.805655

    • PubMed
    • Google Scholar
51. 1. Madeira F
    2. Tinti M
    3. Murugesan G
    4. Berrett E
    5. Stafford M
    6. Toth R
    7. Cole C
    8. MacKintosh C
    9. Barton GJ

    (2015) 14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides

    Bioinformatics 31:2276–2283.

    https://doi.org/10.1093/bioinformatics/btv133

    • PubMed
    • Google Scholar
52. 1. Miroshnikova YA
    2. Le HQ
    3. Schneider D
    4. Thalheim T
    5. Rübsam M
    6. Bremicker N
    7. Polleux J
    8. Kamprad N
    9. Tarantola M
    10. Wang I
    11. Balland M
    12. Niessen CM
    13. Galle J
    14. Wickström SA

    (2018) Adhesion forces and cortical tension couple cell proliferation and differentiation to drive epidermal stratification

    Nature Cell Biology 20:69–80.

    https://doi.org/10.1038/s41556-017-0005-z

    • PubMed
    • Google Scholar
53. 1. Moll R
    2. Franke WW
    3. Schiller DL
    4. Geiger B
    5. Krepler R

    (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells

    Cell 31:11–24.

    https://doi.org/10.1016/0092-8674(82)90400-7

    • PubMed
    • Google Scholar
54. 1. Muroyama A
    2. Lechler T

    (2017) A transgenic toolkit for visualizing and perturbing microtubules reveals unexpected functions in the epidermis

    eLife 6:e29834.

    https://doi.org/10.7554/eLife.29834

    • PubMed
    • Google Scholar
55. 1. Nekrasova O
    2. Harmon RM
    3. Broussard JA
    4. Koetsier JL
    5. Godsel LM
    6. Fitz GN
    7. Gardel ML
    8. Green KJ

    (2018) Desmosomal cadherin association with Tctex-1 and cortactin-Arp2/3 drives perijunctional actin polymerization to promote keratinocyte delamination

    Nature Communications 9:1053.

    https://doi.org/10.1038/s41467-018-03414-6

    • PubMed
    • Google Scholar
56. 1. Nelson WG
    2. Sun TT

    (1983) The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies

    The Journal of Cell Biology 97:244–251.

    https://doi.org/10.1083/jcb.97.1.244

    • PubMed
    • Google Scholar
57. 1. Obenauer JC
    2. Cantley LC
    3. Yaffe MB

    (2003) Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs

    Nucleic Acids Research 31:3635–3641.

    https://doi.org/10.1093/nar/gkg584

    • PubMed
    • Google Scholar
58. 1. Ohbayashi N
    2. Fukuda M

    (2012) Role of rab family GTPases and their effectors in melanosomal logistics

    Journal of Biochemistry 151:343–351.

    https://doi.org/10.1093/jb/mvs009

    • PubMed
    • Google Scholar
59. 1. Panciera T
    2. Azzolin L
    3. Cordenonsi M
    4. Piccolo S

    (2017) Mechanobiology of YAP and TAZ in physiology and disease

    Nature Reviews Molecular Cell Biology 18:758–770.

    https://doi.org/10.1038/nrm.2017.87

    • PubMed
    • Google Scholar
60. 1. Pocaterra A
    2. Romani P
    3. Dupont S

    (2020) YAP/TAZ functions and their regulation at a glance

    Journal of Cell Science 133:jcs230425.

    https://doi.org/10.1242/jcs.230425

    • PubMed
    • Google Scholar
61. 1. Ramírez F
    2. Ryan DP
    3. Grüning B
    4. Bhardwaj V
    5. Kilpert F
    6. Richter AS
    7. Heyne S
    8. Dündar F
    9. Manke T

    (2016) deepTools2: a next generation web server for deep-sequencing data analysis

    Nucleic Acids Research 44:W160–W165.

    https://doi.org/10.1093/nar/gkw257

    • PubMed
    • Google Scholar
62. 1. Ramms L
    2. Fabris G
    3. Windoffer R
    4. Schwarz N
    5. Springer R
    6. Zhou C
    7. Lazar J
    8. Stiefel S
    9. Hersch N
    10. Schnakenberg U
    11. Magin TM
    12. Leube RE
    13. Merkel R
    14. Hoffmann B

    (2013) Keratins as the main component for the mechanical integrity of keratinocytes

    PNAS 110:18513–18518.

    https://doi.org/10.1073/pnas.1313491110

    • PubMed
    • Google Scholar
63. 1. Reichelt J
    2. Furstenberger G
    3. Magin TM

    (2004) Loss of keratin 10 leads to mitogen-activated protein kinase (MAPK) activation, increased keratinocyte turnover, and decreased tumor formation in mice

    Journal of Investigative Dermatology 123:973–981.

    https://doi.org/10.1111/j.0022-202X.2004.23426.x

    • PubMed
    • Google Scholar
64. 1. Reichelt J
    2. Magin TM

    (2002)

    Hyperproliferation, induction of c-Myc and 14-3-3sigma, but no cell fragility in keratin-10-null mice

    Journal of Cell Science 115:2639–2650.

    • PubMed
    • Google Scholar
65. 1. Rübsam M
    2. Broussard JA
    3. Wickström SA
    4. Nekrasova O
    5. Green KJ
    6. Niessen CM

    (2018) Adherens junctions and desmosomes coordinate mechanics and signaling to orchestrate tissue morphogenesis and function: an evolutionary perspective

    Cold Spring Harbor Perspectives in Biology 10:a029207.

    https://doi.org/10.1101/cshperspect.a029207

    • PubMed
    • Google Scholar
66. 1. Sambandam SAT
    2. Kasetti RB
    3. Xue L
    4. Dean DC
    5. Lu Q
    6. Li Q

    (2015) 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    Journal of Investigative Dermatology 135:1621–1628.

    https://doi.org/10.1038/jid.2015.42

    • PubMed
    • Google Scholar
67. 1. Schlegelmilch K
    2. Mohseni M
    3. Kirak O
    4. Pruszak J
    5. Rodriguez JR
    6. Zhou D
    7. Kreger BT
    8. Vasioukhin V
    9. Avruch J
    10. Brummelkamp TR
    11. Camargo FD

    (2011) Yap1 acts downstream of α-catenin to control epidermal proliferation

    Cell 144:782–795.

    https://doi.org/10.1016/j.cell.2011.02.031

    • PubMed
    • Google Scholar
68. 1. Schütte U
    2. Bisht S
    3. Heukamp LC
    4. Kebschull M
    5. Florin A
    6. Haarmann J
    7. Hoffmann P
    8. Bendas G
    9. Buettner R
    10. Brossart P
    11. Feldmann G

    (2014) Hippo signaling mediates proliferation, invasiveness, and metastatic potential of clear cell renal cell carcinoma

    Translational Oncology 7:309–321.

    https://doi.org/10.1016/j.tranon.2014.02.005

    • PubMed
    • Google Scholar
69. 1. Schweizer J
    2. Kinjo M
    3. Fürstenberger G
    4. Winter H

    (1984) Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: basal cells with properties of terminally differentiating cells

    Cell 37:159–170.

    https://doi.org/10.1016/0092-8674(84)90311-8

    • PubMed
    • Google Scholar
70. 1. Schweizer J
    2. Bowden PE
    3. Coulombe PA
    4. Langbein L
    5. Lane EB
    6. Magin TM
    7. Maltais L
    8. Omary MB
    9. Parry DA
    10. Rogers MA
    11. Wright MW

    (2006) New consensus nomenclature for mammalian keratins

    The Journal of Cell Biology 174:169–174.

    https://doi.org/10.1083/jcb.200603161

    • PubMed
    • Google Scholar
71. 1. Shimoyama Y
    2. Nagafuchi A
    3. Fujita S
    4. Gotoh M
    5. Takeichi M
    6. Tsukita S
    7. Hirohashi S

    (1992)

    Cadherin dysfunction in a human Cancer cell line: possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesiveness

    Cancer Research 52:5770–5774.

    • PubMed
    • Google Scholar
72. 1. Silvis MR
    2. Kreger BT
    3. Lien WH
    4. Klezovitch O
    5. Rudakova GM
    6. Camargo FD
    7. Lantz DM
    8. Seykora JT
    9. Vasioukhin V

    (2011) α-catenin is a tumor suppressor that controls cell accumulation by regulating the localization and activity of the transcriptional coactivator Yap1

    Science Signaling 4:ra33.

    https://doi.org/10.1126/scisignal.2001823

    • PubMed
    • Google Scholar
73. 1. Soares E
    2. Zhou H

    (2018) Master regulatory role of p63 in epidermal development and disease

    Cellular and Molecular Life Sciences 75:1179–1190.

    https://doi.org/10.1007/s00018-017-2701-z

    • PubMed
    • Google Scholar
74. 1. Strnad P
    2. Usachov V
    3. Debes C
    4. Gräter F
    5. Parry DA
    6. Omary MB

    (2011) Unique amino acid signatures that are evolutionarily conserved distinguish simple-type, epidermal and hair keratins

    Journal of Cell Science 124:4221–4232.

    https://doi.org/10.1242/jcs.089516

    • PubMed
    • Google Scholar
75. 1. Sumigray KD
    2. Lechler T

    (2015) Cell adhesion in epidermal development and barrier formation

    Current Topics in Developmental Biology 112:383–414.

    https://doi.org/10.1016/bs.ctdb.2014.11.027

    • PubMed
    • Google Scholar
76. 1. Sun BK
    2. Boxer LD
    3. Ransohoff JD
    4. Siprashvili Z
    5. Qu K
    6. Lopez-Pajares V
    7. Hollmig ST
    8. Khavari PA

    (2015) CALML5 is a ZNF750- and TINCR-induced protein that binds stratifin to regulate epidermal differentiation

    Genes & Development 29:2225–2230.

    https://doi.org/10.1101/gad.267708.115

    • PubMed
    • Google Scholar
77. 1. Suzuki T
    2. Muramatsu A
    3. Saito R
    4. Iso T
    5. Shibata T
    6. Kuwata K
    7. Kawaguchi SI
    8. Iwawaki T
    9. Adachi S
    10. Suda H
    11. Morita M
    12. Uchida K
    13. Baird L
    14. Yamamoto M

    (2019) Molecular mechanism of cellular oxidative stress sensing by Keap1

    Cell Reports 28:746–758.

    https://doi.org/10.1016/j.celrep.2019.06.047

    • PubMed
    • Google Scholar
78. 1. Swift J
    2. Ivanovska IL
    3. Buxboim A
    4. Harada T
    5. Dingal PC
    6. Pinter J
    7. Pajerowski JD
    8. Spinler KR
    9. Shin JW
    10. Tewari M
    11. Rehfeldt F
    12. Speicher DW
    13. Discher DE

    (2013) Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation

    Science 341:1240104.

    https://doi.org/10.1126/science.1240104

    • PubMed
    • Google Scholar
79. 1. Tamiji S
    2. Beauvillain JC
    3. Mortier L
    4. Jouy N
    5. Tual M
    6. Delaporte E
    7. Formstecher P
    8. Marchetti P
    9. Polakowska R

    (2005) Induction of apoptosis-like mitochondrial impairment triggers antioxidant and Bcl-2-dependent keratinocyte differentiation

    Journal of Investigative Dermatology 125:647–658.

    https://doi.org/10.1111/j.0022-202X.2005.23885.x

    • PubMed
    • Google Scholar
80. 1. Totaro A
    2. Castellan M
    3. Battilana G
    4. Zanconato F
    5. Azzolin L
    6. Giulitti S
    7. Cordenonsi M
    8. Piccolo S

    (2017) YAP/TAZ link cell mechanics to Notch signalling to control epidermal stem cell fate

    Nature Communications 8:15206.

    https://doi.org/10.1038/ncomms15206

    • Google Scholar
81. 1. Uttam J
    2. Hutton E
    3. Coulombe PA
    4. Anton-Lamprecht I
    5. Yu QC
    6. Gedde-Dahl T
    7. Fine JD
    8. Fuchs E

    (1996) The genetic basis of epidermolysis bullosa simplex with mottled pigmentation

    PNAS 93:9079–9084.

    https://doi.org/10.1073/pnas.93.17.9079

    • PubMed
    • Google Scholar
82. 1. Vassar R
    2. Coulombe PA
    3. Degenstein L
    4. Albers K
    5. Fuchs E

    (1991) Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease

    Cell 64:365–380.

    https://doi.org/10.1016/0092-8674(91)90645-F

    • PubMed
    • Google Scholar
83. 1. Vassilev A
    2. Kaneko KJ
    3. Shu H
    4. Zhao Y
    5. DePamphilis ML

    (2001) TEAD/TEF transcription factors utilize the activation domain of YAP65, a src/Yes-associated protein localized in the cytoplasm

    Genes & Development 15:1229–1241.

    https://doi.org/10.1101/gad.888601

    • PubMed
    • Google Scholar
84. 1. Wang H
    2. Yang H
    3. Shivalila CS
    4. Dawlaty MM
    5. Cheng AW
    6. Zhang F
    7. Jaenisch R

    (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering

    Cell 153:910–918.

    https://doi.org/10.1016/j.cell.2013.04.025

    • PubMed
    • Google Scholar
85. 1. Wang F
    2. Zieman A
    3. Coulombe PA

    (2016) Skin keratins

    Methods in Enzymology 568:303–350.

    https://doi.org/10.1016/bs.mie.2015.09.032

    • PubMed
    • Google Scholar
86. 1. Wells JM
    2. Watt FM

    (2018) Diverse mechanisms for endogenous regeneration and repair in mammalian organs

    Nature 557:322–328.

    https://doi.org/10.1038/s41586-018-0073-7

    • PubMed
    • Google Scholar
87. 1. Wickström SA
    2. Niessen CM

    (2018) Cell adhesion and mechanics as drivers of tissue organization and differentiation: local cues for large scale organization

    Current Opinion in Cell Biology 54:89–97.

    https://doi.org/10.1016/j.ceb.2018.05.003

    • PubMed
    • Google Scholar
88. 1. Wilker EW
    2. van Vugt MA
    3. Artim SA
    4. Huang PH
    5. Petersen CP
    6. Reinhardt HC
    7. Feng Y
    8. Sharp PA
    9. Sonenberg N
    10. White FM
    11. Yaffe MB

    (2007) 14-3-3sigma controls mitotic translation to facilitate cytokinesis

    Nature 446:329–332.

    https://doi.org/10.1038/nature05584

    • PubMed
    • Google Scholar
89. 1. Woodcock-Mitchell J
    2. Eichner R
    3. Nelson WG
    4. Sun TT

    (1982) Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies

    The Journal of Cell Biology 95:580–588.

    https://doi.org/10.1083/jcb.95.2.580

    • PubMed
    • Google Scholar
90. 1. Yap AS
    2. Duszyc K
    3. Viasnoff V

    (2018) Mechanosensing and mechanotransduction at Cell-Cell junctions

    Cold Spring Harbor Perspectives in Biology 10:a028761.

    https://doi.org/10.1101/cshperspect.a028761

    • PubMed
    • Google Scholar
91. 1. Zhang Y
    2. Liu T
    3. Meyer CA
    4. Eeckhoute J
    5. Johnson DS
    6. Bernstein BE
    7. Nusbaum C
    8. Myers RM
    9. Brown M
    10. Li W
    11. Liu XS

    (2008) Model-based analysis of ChIP-Seq (MACS)

    Genome Biology 9:R137.

    https://doi.org/10.1186/gb-2008-9-9-r137

    • PubMed
    • Google Scholar

Author details

1. Yajuan Guo

   1. Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States
   2. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

2. Catherine J Redmond

   Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, United States

   Contribution

   Conceptualization, Data curation, Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9474-2803

3. Krystynne A Leacock

   Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Margarita V Brovkina

   Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5580-5872

5. Suyun Ji

   Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, United States

   Contribution

   Formal analysis

   Competing interests

   No competing interests declared

6. Vinod Jaskula-Ranga

   Department of Ophthalmology, Johns Hopkins School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Data curation, Methodology

   Competing interests

   No competing interests declared

7. Pierre A Coulombe

   1. Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, United States
   2. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, United States
   3. Department of Dermatology, University of Michigan Medical School, Ann Arbor, United States
   4. Rogel Cancer Center, Michigan Medicine, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology

   For correspondence

   coulombe@umich.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0680-2373

• 5,302

  views

• 586

  downloads

• 45

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.