Telomere dysfunction impairs epidermal stem cell specification and differentiation by disrupting BMP/pSmad/P63 signaling

Telomere shortening is associated with aging and age-associated diseases. Additionally, telomere dysfunction resulting from telomerase gene mutation can lead to premature aging, such as apparent skin atrophy and hair loss. However, the molecular signaling linking telomere dysfunction to skin atrophy remains elusive. Here we show that dysfunctional telomere disrupts BMP/pSmad/P63 signaling, impairing epidermal stem cell specification and differentiation of skin and hair follicles. We find that telomere shortening mediated by Terc loss up-regulates Follistatin (Fst), inhibiting pSmad signaling and down-regulating P63 and epidermal keratins in an ESC differentiation model as well as in adult development of telomere-shortened mice. Mechanistically, short telomeres disrupt PRC2/H3K27me3-mediated repression of Fst. Our findings reveal that skin atrophy due to telomere dysfunction is caused by a previously unappreciated link with Fst and BMP signaling that could be explored in the development of therapies.

Patients with mutations in the telomerase component (eg, Dyskerin, TERT, TERC) are frequently accompanied by symptoms of abnormal epidermis, such as hyperpigmentation, premature skin degradation, hair follicle shedding, skin atrophy, and dry skin. Mice with mutations in telomere-associated proteins or telomerase genes also show similar phenotypes, associated with telomere shortening. However, the underlying molecular signaling and mechanisms remain elusive. Here, we show that the differentiation of epidermis is disrupted resulting from short telomeres. Epidermal differentiation abnormalities can be a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Introduction
Telomeres consist of (TTAGGG)n DNA repeats and associated proteins that locate at chromosome ends, maintaining chromosomal stability and cell proliferation. The telomerase complex consists of a telomerase RNA component (TERC) and the reverse transcriptase catalytic subunit (TERT), and adds telomere repeats to chromosome ends to offset the loss of telomere sequences that occurs due to the end-replication problem, the inability of DNA polymerase to replicate fully the lagging DNA strand [1]. In the absence of telomerase, telomeres shorten progressively with cell division, ultimately leading to loss of telomere protection and a DNA damage response that induces senescence or cell death. Telomere shortening is closely tied to organism aging and premature aging and associated diseases [2][3][4][5]. Skin atrophy and hair loss are general phenomena associated with age [6]. Moreover, patients with the mutation of telomerase components (e.g. Dyskerin, TERT, TERC) exhibit telomere shortening and skin atrophy [7]. It has been shown that short telomeres impair differentiation and development of the epidermis, and cause skin atrophy and loss of hair follicles, in association with epidermal stem cell dysfunction with aging [8][9][10]. However, the molecular signaling underlying short telomeres-associated skin atrophy or degeneration and hair follicle loss remains elusive.
Embryonic stem (ES) cells are able to spontaneously differentiate into three embryonic germ layers ectoderm, mesoderm, and endoderm by standard test of embryoid body (EB) formation. This method has been extensively used to investigate signaling pathways that control ES cell differentiation towards various cell lineages [11][12][13], including epidermis [14][15][16]. Telomere length is critical for developmental pluripotency and differentiation capacity of ES cells or iPS cells [17][18][19][20]. We attempted to investigate how short telomere compromises epidermal lineage specification and differentiation initially by using ES cell lines with different telomere lengths, derived from Terc knockout (Terc -/-) mice [17]. We showed that telomere lengths affected differentiation of ES cells into epidermis. We further validated that short telomeres impeded epidermal differentiation in the adult telomerase-deficient, telomere shortened mice. Moreover, we investigated potential regulatory mechanisms of telomere length on epidermis differentiation.

Short telomere impairs epidermal stem cell specification and differentiation in vitro
To investigate the differentiation defects associated with short telomeres, we initially performed in vitro differentiation experiments by standard EB formation test using mouse ES cells with various telomere lengths due to telomerase (Terc -/-) deficiency (Fig 1A and 1B, S1A  Fig). Telomeres were longest in wild-type (WT) ES cells, shorter in heterozygous (Terc +/-) and early generation (G1) Terc -/-ES cells, and critically short or lost in late generation (G3 and G4) Terc -/-ES cells (Fig 1C and 1D), as we previously reported [17]. Late generation (G3 and G4) Terc -/cells also exhibited short telomeres by day15 of differentiation (Fig 1C and 1D). Upon differentiation, WT ES cells showed significantly reduced expression of pluripotency marker genes such as Oct4 and Nanog (S1B and S1C Fig). However, G3 and G4 Terc -/-ES cells maintained expression of Nanog and Oct4 at relatively high levels, and low methylation at Nanog promoter (S1D Fig), consistent with the finding using Tert -/-ES cells also with critically short telomeres [18]. Expression levels of genes related to endoderm, mesoderm and neuro-ectoderm did not differ between WT ES cells and ES cells with short telomeres, suggesting that shortening of telomeres does not significantly affect the differentiation of these germ layers (Fig 1E and 1G).
Notably, expression levels of genes important for epidermal ectoderm differentiation were consistently reduced in telomere shortened (G3/G4 Terc -/-) ES cells following differentiation as compared to WT ES cells (S1E and S1F Fig). During mouse embryo development, epidermal progenitors are specified at around embryonic day 8-12 (E8-12), later than that of neural induction. Expression of Keratin 14 (K14) was at very low level on day 8 of differentiation (Day 8) and was sharply increased on day 15 in WT cells. K14 was also low on day 8 in G3/G4 Terc -/cells, but dramatically reduced on day 15, as compared to WT cells. Consistently, expression levels of K5 (epidermal basal cell marker), K1 (epidermis marker of skin) and K4 (epidermis marker in stratified epithelia) [21], in the differentiated G3/G4 Terc -/cells were also significantly lower than that in WT cells (S1F Fig). p63 as one of the earliest genes for epidermal lineage is expressed as early as E7.5, identifies epidermal keratinocyte stem cells, and is required for epidermal differentiation [22][23][24]. p63 also is expressed earlier than does K14 during differentiation of human ES cells into keratinocytes [15]. Consistently, p63 expression was detectable in WT, Terc +/-, and G1 Terc -/cells by day 7-8 of differentiation, earlier than that of Keratins, but only minimal in G3/G4 Terc -/cells. p63 level was further increased by day 15 in WT, Terc +/-, and G1 Terc -/cells, but much lower in G3 and G4 Terc -/cells (S1E Fig). Consistent with the qPCR data, protein levels of both K14 and P63 at day 15 were also greatly reduced in G3/ G4 Terc -/cells as compared to WT cells ( Fig 1E). Immunofluorescence microscopy showed specific staining of P63 in the nuclei and K14 in cytoplasm and membrane in WT cells but much reduced staining in some G4 Terc -/cells ( Fig 1F). These data indicated that short telomeres lead to decreased expression of P63 and K14 and that telomere-shortened stem cells may fail to stratify in the differentiation into epidermal lineage.

Short telomere impairs epidermis in vivo
To examine the impacts of short telomeres on the differentiation capacity in vivo, we initially performed standard teratoma formation test [12,25]. Both WT and G4 Terc -/-ES cells were able to differentiate into three germ layers, including endoderm, mesoderm, and neural ectoderm revealed by histology (Fig 2A). However, epidermis lineage was reduced in teratomas differentiated from ES cells with short telomeres, in contrast to that of WT ES cells (Fig 2A  and 2B, S2A Fig). Structures in size or number identified by epidermis marker K14 or epidermal stem cell marker P63 were reduced in the sections of teratomas from G4 Terc -/-ES cells, compared with those from WT ES cells ( Fig 2B). Relative mRNA levels of p63 and K14 in teratomas derived from G4 Terc -/-ES cells also were lower than those from WT ES cells ( Fig 2C).
Similar phenotypes also can be observed in the adult G3 Terc -/mouse skin. Epidermis marked by co-immunostaining of P63 and K14 and by histology was thinner on average in skin of two-three month old G3 Terc -/mice, compared with age-matched WT mice ( Fig 2F  and 2G, S2B and S2C Fig). Additionally, hair follicles were readily seen in dermis of WT mice but fewer in G3 Terc -/mice (only 50% of WT mice) (Fig 2D and 2E, S3B Fig). Number of hair follicles was calculated based on at least 10 fields of view under microscopy. In WT mouse skin, the hair follicles are structurally intact with an average of 4 to 5 per field of view. However, hair follicles drop sharply in their numbers and loses the typical structure in the G3 Terc -/mouse skin (Fig 2D and 2E). Both in vitro and in vivo results validated that short telomeres reduce epidermal commitment.

Short telomere leads to excessive expression of Fst and represses BMP/ pSmad signaling
To understand the mechanisms underlying short telomeres-affecting ES cell differentiation towards epidermal lineage, we performed microarray analysis of G4 Terc -/-ES cells compared with WT ES cells. Interestingly, Follistatin (Fst), a negative regulator of Smad pathway which is critical in epidermis commitment, was expressed at higher level in G4 Terc -/than in WT ES cells at day 0 ( Fig 3A). qPCR analysis validated that expression levels of Fst were higher in G3 and G4 Terc -/than in WT ES cells (Fig 3B). Western blot also confirmed that Fst protein level was indeed higher in G4 Terc -/-ES cells than in WT ES cells during differentiation (Fig 3C, left  panel). Given that Fst is a secreted protein, we also examined Fst protein levels in the culture media for both cell lines. Fst protein was highly abundant in the culture media of G4 Terc -/-ES cells, but barely detectable in that of WT ES cells (Fig 3C, right panel). Furthermore, robust cytoplasmic and membrane staining of K14 and nuclear P63 were observed at day 15 in differentiated WT cells, but their expression levels were markedly reduced in differentiated G4 Terc -/cells, where higher Fst fluorescence signals with dotted staining still were readily visible in the cytoplasm of or around the differentiated cells, compared with lower Fst fluorescence in WT cells ( Fig 3D). Additionally, Fst protein level was higher in G4 Terc -/teratomas than in control teratomas (WT and Terc +/-) ( Fig 3E). Compared to WT teratomas, G4 Terc -/teratomas exhibited strong Fst immunofluorescence spotted inside or outside the cells, coincided with less and weak fluorescence staining of K14 and P63 (Fig 3F and 3G). These data provide further evidence that higher expression level of Fst is linked to short telomere.
Similar results were obtained from the skin of adult mice. Two-three month old G3 Terc -/mice displayed thinner epidermis and skin atrophy compared to the age-matched WT mice, consistent with previous studies [8,26]. K14 level also was reduced in the epidermis of G3 Terc -/adult mice, accompanied by increased expression of spotty Fst as compared to WT mouse epidermis (S3A Fig). Immunofluorescence staining of Fst in teratomas or tissues appears to be dotted in pattern, somewhat different from the immunostaining in cultured cells, probably because Fst can be locally confined with specific structure in tissues, whereas it diffuses in and around the cultured cells. In addition, G3 Terc -/mice displayed defective hair follicle development as evidenced by notably reduced number of hair follicles, with reduced expression of K14 and increased Fst, as well as impaired bulb and bulge at the basal follicles where progenitor cells reside, in contrast to the intact bulb (hair germ) and bulge in WT mice (S3B Fig). Taken together, short telomeres lead to excessive expression of Fst, which is incompatible with epidermal stem cell specification and stratification of skin and hair follicles.

Fst negatively regulates pSmad1/5/8 and p63
Collectively, these findings suggested that short telomere specifically prevents the transition from the common ectodermal progenitor state into the epidermis fate. Bone morphogenesis protein 4 (BMP4) signaling is known to be activated in the embryo at the time of ectodermal fate determination, inhibits premature neural differentiation while inducing epidermis development, and can act through phosphorylation and nuclear accumulation of Smad1/5/8 [27][28][29][30]. Differentiation of epidermal cells appears to be controlled, in part, by BMP4 [31]. Fst is an antagonist of BMP4. We next asked if the Fst-BMP-Smad1/5/8 signaling pathway plays a critical role in epidermal differentiation. In the wild-type ES cells, the up-regulation of BMP4, BMP7, Smad1, and the down-steam target (Gata1) during differentiation indicates that this pathway plays an important role in normal differentiation and development of epidermis (S4 Fig).
Compared with those of WT, Terc +/-, or G1 Terc -/-ES cells, levels of phosphorylated Smad1/5/8 were reduced in G3/G4 Terc -/-ES cells during differentiation (Fig 3H), suggesting that this pathway is suppressed by short telomeres. Gene expression upstream of this pathway seemed to be not affected by short telomeres. Downstream target genes of this signaling pathway such as Gata1 were expressed at lower levels in G4 Terc -/than in WT cells by day 8 and day 15 of differentiation (S4 Fig). These data suggested that short telomeres suppress BMP/ pSmad signaling following differentiation.
Above data imply that elevated expression of Fst resulting from short telomere might lead to reduction of pSmad1/5/8, P63 and K14, and thus defective epidermal stem cell specification and differentiation. To further validate this concept, we generated Fst overexpression (OE) ES cell line ( Fig 4A) and performed EB differentiation test using WT ES cell line as control. Western blot showed that Fst OE ES cells expressed p63 and K14 at reduced levels on day 8 and day 15 of differentiation, which was also confirmed by immunofluorescence microscopy ( Mechanism of short telomere-induced epidermal defects and 4C). Notably, in the differentiated Fst OE cell culture, areas with intensive Fst fluorescence indicative of high expression level exhibited minimal K14 staining, and yet areas with low Fst fluorescence displayed strong K14 or p63 staining ( Fig 4C). Hence, high Fst level is discordant with expression of P63 and K14. Consistently, pSmad1/5/8 was decreased in Fst OE cells ( Fig  4B). These data suggest a conserved but new role of Fst in negatively regulating pSmad-signaling pathway during epidermal ectoderm induction.
To test whether reducing Fst can de-repress down-stream genes/signaling for epidermis, we knocked down Fst by RNA interference in the differentiated G4 Terc -/cells. Effective knockdown of Fst by shRNA in differentiated G4 Terc -/cells up-regulated the levels of pSmad1/5/8 and P63 (Fig 4D and 4E). Fst downgregulation by RNA interference in the differentiated G4 Terc -/cells rescued P63 but not fully rescued K14 expression. This may be explained by three potential reasons. Changes in the expression level of K14 could be delayed following P63 expression during epidermal differentiation. Factors other than Fst alone also might be involved in regulation of K14 expression. Alternatively, the regulation of Fst-P63-K14 may slightly differ in differentiated ES cells compared with undifferentiated ES cells as model. Nevertheless, these data further support the notion that excessive expression levels of Fst negatively regulate pSmad1/5/8 signaling and p63, weakening epidermal stem cell specification and differentiation.

Repair of Terc rejuvenates telomeres and rescues Fst/P63/K14 signaling
Then, we tested whether rejuvenating telomeres in ES cells with short telomeres can repress Fst. Using CRISPR/Cas9 technology, we successfully knocked in Terc in G4 Terc -/-ES cells and obtained several Terc-repaired ES cell lines (two lines are shown in Fig 5A). These Terc repaired (TR) ES cell lines exhibited much longer telomeres than did their parental G4 Terc -/-ES cell line after culture for 10 passages. Yet, their telomeres were still shorter than those of WT cells as revealed by qPCR and QFISH ( Fig 5B and 5C), presumably because of inadequate passages, even though the telomerase activity was recovered ( Fig 5D). Frequency of telomere loss was significantly reduced in Terc repaired ES cell lines, in contrast to that of G4 Terc -/-ES cells (Fig 5C). We repeated the in vitro differentiation assay with WT, G4 Terc -/-, and Terc repaired ES cell lines. On day 15 of differentiation, telomere length was also rescued in Terc repaired cells compared with Terc -/cells ( Fig 5B). Terc repaired cells showed reduced level of Fst and noticeably increased protein levels of P63 and K14 as compared to those of G4 Terc -/cells (Fig 5E), which were confirmed by immunofluorescence microscopy (Fig 5F and 5G). These results suggested that epidermal differentiation could be rescued by repairing Terc and restoration of telomere length.

Fst is regulated by PRC2-mediated repression
The critical question was how short telomeres result in excessive Fst expression. Fst gene is located at the subtelomere region of the long arm of chromosome 13, whose expression might be regulated by telomere position effect (TPE) [32]. To reveal the telomere state of chromosome 13, we performed immunofluorescence microscopy to detect the chromosome 13 using the Overexpression (OE) of Fst in ES cells decreases expression of P63 and K14 by immunofluorescence microscopy. While WT ES cells transfected with construct Plch37 served as control express Fst at only a low level, ES cells stably overexpressing Fst express Fst at a much higher level by immunofluorescence microscopy. Following differentiation of ES cells overexpressing Fst, immunofluorescence staining of cytoplasmic K14 and nuclear P63 is dramatically reduced, compared with that of Plch37 plasmid controls. Scale bar = 20 μm. (D&E) Knockdown (KD) of Fst in G4 Terc -/cells at day 15 of differentiation leads to increased mRNA levels of P63 by qPCR (D) and also elevated protein levels of pSmad1/5/8 and P63 by Western blot (E). Right panel, quantification of protein levels using ImageJ software, normalized to β-actin. � , p<0.05; �� , p<0.01; ��� p<0.001, compared to WT. T1 and T3 are two independent interference RNA sequences targeting Fst.
Loss of telomeric repeats leads to a change in the heterochromatic architecture with decreased H3K9me3 abundance at telomeres/subtelomeres [33]. We tested whether epigenetic modifications are implicated in regulation of Fst. Both DNA methyltransferases Dnmt3a and 3b were expressed at lower levels in G4 Terc -/-ES cells than in WT cells, but Dnmt3b expressed at higher levels following differentiation (S5A Furthermore, we performed ChIP-qPCR analysis to examine the abundance of H3K9me3/2, H3K9ac and H3K27me3 at Fst promoter loci using β-actin as a control. Enrichment of H3K9me3, H3K9Ac, and H3K9me2 at Fst promoter was low and showed no significant difference between WT and G4 Terc -/-ES cells (Fig 6B). However, H3K27me3 was highly enriched at Fst promoter. Importantly, H3K27me3 level was markedly reduced at all five loci of Fst promoter in G4 Terc -/-, compared with that of WT ES cells (Fig 6B). By luciferase reporter assay, the Fst promoter activity was higher in G4 Terc -/than in WT ES cells (S7A Fig). We further examined expression levels of Eed, Suz12, Ezh1 and Ezh2 which are catalytic components of Polycomb repressive complex PRC2 and potentially tri-methylate H3K27 to repress gene expression and that are shown to play important roles in skin stem cell function and differentiation [34,35]. Expression levels of Ezh1 and Ezh2 are reduced in G4 Terc -/-ES cells as compared to WT ES cells (S7B Fig). Telomere-repaired ES cells partially restored Ezh1/2 expression, together with increased H3K27me3 enrichment at Fst promoter (S7C and S7D Fig). Pluripotent marker genes Nanog and Oct4 were also down-regulated during differentiation of Terc repaired G4 ES cells, like those of WT ES cells (S7E and S7F Fig). It is interesting to note that Ezh2 expression level in one Tercrepaired ES cell line (A49 TR3) was not recovered well, and coincidently this clone had relatively shorter telomere than that of WT ES cells (Fig 5C). These results further suggest that short telomeres reduce H3K27me3 enrichment at Fst promoter, likely together with reduced Ezh1 and Ezh2 levels, de-repress Fst, and these together may contribute to excessive expression of Fst. Excessive Fst further down-regulates p63/K14 through disrupting BMP4/pSmad signaling (Fig 6C).  . K14), in the specification and differentiation of epidermis and hair follicles. In the event of telomere shortening or loss, abundance of H3K27me3 at Fst foci is reduced, and this causes increased expression of Fst, which impairs BMP4-pSmad signaling, and consequently reduces P63 and K14 expression, epidermal stem cell specification and differentiation. https://doi.org/10.1371/journal.pgen.1008368.g006

Discussion
Based on the data obtained from both ES cell differentiation in vitro and in vivo, we propose that functional telomere is important for suppressing Fst to prevent its overexpression and to maintain normal expression of P63 and K14 during epidermal stem cell specification and differentiation. Short telomere disrupts PRC2-H3K27me3-mediated repression of Fst, which leads to excessive Fst expression. Consequently, excessive Fst suppresses BMP/pSmad signaling, reducing P63 and keratins and resulting in epidermal differentiation defects and skin atrophy. This model links dysfunctional telomeres to skin atrophy and hair follicle loss by disrupting Fst/BMP/pSmad/P63/K14 signaling.
This study also provides additional evidence in supporting that ES cell differentiation model is a powerful alternative tool to discover novel signaling and mechanisms that are involved in in vivo cell lineage specification at very early developmental stages that might not be readily revealed in live mouse model and particularly in humans [11]. The differentiation assay used in our study shows that the dynamics of P63 and K14 in mouse ES cell is similar to that of human ES cells and mouse embryonic skin development [14]. Our results also confirmed that P63 is a master regulator for K14, K5 and other epidermal genes [36,37], and that BMP4/pSmad signaling pathway can activate P63 [38]. BMP4 negatively regulates neural induction and promotes epidermogenesis during differentiation of mouse ES cells [31], whereas blocking BMP signaling facilitates differentiation of human ES cells into neural lineages [28].
Mounting evidence supports the notion that telomere dysfunction is accompanied by symptoms of abnormal epidermis [10,[39][40][41][42]. Mice with critically short telomeres exhibit symptoms, including epidermal abnormalities such as poor wound healing, ulcerative skin lesions, early hair loss and early hair graying [2,8,10,43]. We show that short telomeres lead to reduced expression of P63 and declined epidermal stratification and formation, linking to skin atrophy. Study of P63-null mice demonstrates important roles of P63 in orchestrating first epidermal stratification [44,45]. p63-null mice exhibit striking defects in embryonic epidermal morphogenesis [46], and also suffer from diminished stem cell renewal capacity [47]. Moreover, TAp63 serves to maintain adult skin stem cells and prevents premature tissue aging [45,48,49]. Hence, P63 is required to maintain epidermal stem cell renewal while allowing K14 expression and epidermal differentiation [24]. Short telomeres cause stem cell failure [50], and also impair the ability of epidermal stem cells to mobilize out of the hair follicle niche, and thus skin and hair growth [26]. On the other hand, hyper-long telomeres are advantageous for skin regeneration compared with normal length telomeres [51]. Our data provide novel molecular mechanisms of linking short telomeres to reduced pSmad signaling and P63 and thus declined epidermal differentiation.
Moreover, excessive Fst expression resulting from short telomere negatively regulates BMP/ pSmad/P63 pathways in the epidermal stem cell specification and differentiation. It has been reported that Fst is an antagonist of BMPs by blocking binding of BMP with its receptor [52]. Excessive Fst may compete with BMPs and inhibit BMP-pSmad signaling. Telomere re-elongation successfully achieved by CRISPR/Cas9-mediated knock-in of Terc represses Fst and recovers expression of P63 and K14. Consistently, telomerase reintroduction into mice with critically short telomeres is sufficient to elongate telomeres in skin keratinocytes and to correct epidermal hair follicle stem cell defects, and rescues skin and hair growth defects [26]. These data also may explain the early findings that Fst-knockout mice die within hours of birth but show thicker epidermis [53]. Likewise, deletion of Fst results in enhanced keratinocyte proliferation in the tail epidermis of these animals and an earlier onset of keratinocyte hyperproliferation at the wound edge after skin injury, suggesting that Fst regulates epidermal homeostasis and also wound repair [54].
In agreement, Fst-overexpression transgenic mice are characterized by a thinner dermis and epidermis, reduced density of the dermis and smaller hair follicles, indicative of skin atrophy, and a severe delay in wound healing observed after injury [55]. Moreover, mice that overexpress Fst are smaller compared with their control littermates, and their body weight is significantly reduced. This phenotype is similar to that of late generation Terc -/mice [2,56] (also shown in this study), and these mice also exhibit severely impaired wound healing [2]. Coincidently, p63 −/− mice have an impaired wound-healing response as well [48]. Together, these data support the idea that abnormal Fst/p63 signaling is implicated in short telomeresassociated skin atrophy and wound healing. Excitingly, mouse ES cells with hyper-long telomeres generate healthier chimera mice that also have longer telomeres and exhibit delayed aging and high capacity for skin wound healing [51].
Short telomeres can change expression of many genes and signaling pathways particularly with cell differentiation, as shown by the transcription profile data. We identified unique alterations of down-stream genes under regulation by the major TGFβ superfamily during differentiation of ES cells into epidermal lineage. Fst happens to be an evident negative regulator upstream in this pathway, and is up-regulated when telomere is short. We searched for mechanisms underlying short telomere-induced activation of Fst, and tested the hypothesis that repressive histone modification or DNA methylation may underlie telomere suppression of Fst. By ChIP-qPCR assay with selective related antibodies, we show that PRC2-mediated repression involving Ezh1/2 and H3K27me3 makes a major contribution to suppressing Fst. In fact, the regulatory region of Fst gene is characteristic of bivalent genes whose promoters are enriched for both activating mark by H3K4me3 and repressing mark by H3K27me3 and Ezh2, primed for differential expression upon differentiation [57][58][59]. In mice, PRC2 has been found to be enriched in the progenitor cells of developing epidermis, regulates epidermal specification in mouse embryos and maintains hair follicle homeostasis [60,61]. H3K27me3 marks are enriched in a subset of epidermal differentiation gene promoters in undifferentiated cells and disappear on a subset of epidermal gene promoters upon differentiation [62]. Moreover, Ezh1 and Ezh2 repress premature differentiation and H3K27me3 is involved in early lineage specification of embryonic epidermis differentiation [60]. Interestingly, Ezh1/2 null skin progenitors show reduced H3K27me3 abundance and significant up-regulation of Fst [61]. Identification of hair follicle stem cell signature genes showed that Fst also is one of genes involving transit-amplifying (TA) progeny repressed by H3K27me3, whereas BMP4 signaling is activated during this process likely induced by epigenetic shift to control by H3K4me3 and H3K79me2 [63]. Consistently, short telomere reduces H3K27me3 enrichment at Fst promoter, which leads to elevated Fst expression and defective epidermal specification and differentiation. Tercrepaired G4 Terc -/-ES cells rejuvenate telomere length to various degrees and partly restore H3K27me3-mediated suppression of Fst. Nanog and Oct4 are down-regulated following differentiation of Terc-repaired G4 Terc -/-ES cells like WT cells. Coincidentally, Tert -/-ES cells also have critically short telomeres and disrupted PRC2 function and low H3K27me3 enrichment at Nanog promoter, leading to defective suppression of Nanog during differentiation [18]. Fst-BMP4 signal pathway is known as a critical regulator for epidermal differentiation initiation and induced expression of p63, which may coordinate with BMP4 to accelerate epidermal specification by regulating accumulation of H3K27me3 [64]. Deletion of p63 resulted in a significant decrease in signal of H3K27me3 mark [64]. We show that short telomeres can up-regulate Fst via reducing H3K27me3 at Fst promoter and decrease pSmad, resulting in declined expression of p63. These findings suggest a complex feedback mechanism between H3K27me3 and Fst-BMP4-P63. Fst, BMP4, P63, and H3K27me3 are key players in the orchestra that regulates epidermal differentiation.
Another interesting phenomenon for Fst promoter is its hypomethylated state. Additionally, methylation levels at subtelomeres of chromosome 13 where Fst gene is located are also drastically reduced in G4 Terc -/-ES cells, compared with those of WT ES cells. Coincidently, Fst and other subtelomeric genes such as Tcstv1/3 in chromosome 13 are expressed at higher levels in G4 Terc -/-ES cells, but down-regulated in Terc-repaired G4 Terc -/-ES cells like WT cells (S8 Fig). DNA hypomethylation could lead to decreased levels of H3K27me3 in ordinarily unmethylated regions [18,65]. Our data suggests that H3K9me3-mediated gene silencing does not play a direct role in repressing Fst. We indeed find a global reduction of H3K9me3 in G4 Terc -/-ES cells in which H3K9me3 also shows reduced co-localization with telomeres. These data suggest that telomere shortening-induced reduction of H3K9me3 at telomeres/subtelomeres may have a general impact on gene de-repression instead of a direct impact on Fst gene. Taken together, short or loss of telomere disrupts PRC2 function involving H3K27me3 and de-represses Fst. Elevated Fst inhibits pSmad/P63 signaling, leading to defective epidermal stem cell specification, stratification and differentiation. Rejuvenating telomere length can rescue these defects. We do not exclude the possibility that additional signaling pathways may also be involved in and/or cooperate with aberrant Fst/pSmad/P63 signaling in defective epidermal differentiation resulting from telomere dysfunction. Targeting Fst/pSmad/P63 pathway may have implications in ameliorating skin and hair degeneration associated with aging and telomere shortening.

Ethics statement
All animal experiments were approved by the Institutional Animal Care and Use Committee at Nankai University (License number 20140006). All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Nankai University. All efforts were made to minimize the number of animals used by the experimental design.

Mice
Two-three month old Terc deficient (Terc -/-) mice and wild-type mice in C57Bl/6 background, and immunodeficient mice were used in this study. Mice were housed and cared for in a pathogen-free facility at Nankai University.

Terc repair in Terc -/-ES cell line by CRISPR/Cas9
pSpCas9(BB)-2A-Puro (PX459) was a gift from Feng Zhang (plasmid # 48139, Addgene). Guide RNAs were designed using the online design tool available at http://crispr.genomeengineering.org/. PX459 was digested with BbsI and then gel purified. Two pairs of oligos including targeting sequences were annealed and cloned into the BbsI-digested PX459 vector. The Terc donor sequences were obtained based on mouse genomic sequence and the information provided in the original paper [43]. The Terc donor vector contained Terc flanked by 5'(Left) and 3'(Right) homology arms. The DNA fragments are individually amplified by proper primers and then cloned into the vector with proper enzymes. G4 Terc -/-ES cell line A49 was transfected with two PX459 and Terc donor plasmids using lipofectamine 2000 transfection reagent (Invitrogen). Twenty-four hours later, 2 μg/ml puromycin was added into the culture medium for 7 days, clones were picked and the genomic DNA was extracted. PCR was performed with several pairs of primers to detect and obtain the genomic knock-in Terc repaired cell lines.

In vitro differentiation of ES cells
ES cells are allowed to aggregate and form three-dimensional colonies known as embryoid bodies (EBs) [66]. Differentiation of ES cells was accomplished in a two-step process: (1) Embryoid body (EB) formation was obtained by using cell suspension and hanging drop method. Undifferentiated ES cells were trypsinized to obtain a single cell suspension, and EBs were formed in ES cell culture medium without LIF, in a definite number of cells in "hanging drops" for 4 days. (2) Then, EB were transferred to 24-well microwell plates with one EB per well. Daily microscopic observations were conducted to detect beating EBs. 10~15 EBs were transferred to 6-well microwell plates per well for protein, RNA, and DNA sample collection.

Teratoma formation assay and histological analysis
Approximately 2×10 6 ES cells with different telomere length were injected subcutaneously into dorsal flanks of immunodeficient mice. Four weeks after the injection, the mice were humanely sacrificed and the teratomas were surgically dissected from the mice. Samples were weighed, fixed in PBS containing 3.7% formaldehyde, and embedded in paraffin. Sections were stained with hematoxylin and eosin for histological examination.

Telomere measurement by qPCR
Cells were washed in PBS and stored at -20˚C until subsequent DNA extraction. Genome DNA was prepared using DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA). Average telomere length was measured from total genomic DNA using a real-time PCR assay, as previously described [68], but modified for measurement of mouse telomere [69]. PCR reactions were performed on the iCycler iQ5 2.0 Standard Edition Optical System (Bio-Rad, Hercules, CA), using telomeric primers, primers for the reference control gene (mouse 36B4 single copy gene) and PCR settings as previously described [70]. For each PCR reaction, a standard curve was made by serial dilutions of known amounts of DNA. The telomere signal was normalized to the signal from the single copy gene to generate a T/S ratio indicative of relative telomere length. Equal amounts of DNA (20 ng) were used for each reaction. The primers for telomere measurement by qPCR are listed in S2 Table. Telomere quantitative fluorescence in situ hybridization (QFISH) Telomere length and function (telomere integrity and chromosome stability) were estimated by telomere quantitative FISH [17,43]. Briefly, cells were incubated with 0.5 μg/ml nocodazole for 1.5 h to enrich cells at metaphases. Chromosome spreads were made by standard method. Metaphase-enriched cells were exposed to hypotonic treatment with 75 mM KCl solution, fixed with methanol: glacial acetic acid (3:1) and spread onto clean slides. Telomeres were denatured at 80˚C for 3 min and hybridized with FITC-labeled telomere (CCCTAA) peptide nucleic acid (PNA) probe (0.5 μg/ml) (Panagene, Korea). Chromosomes were stained with 0.5 μg/ml DAPI. Fluorescence from chromosomes and telomeres was digitally imaged on a Zeiss microscope with fluorescein isothiocyanate (FITC)/DAPI filters, using AxioCam and AxioVision software 4.6. Telomere length shown as telomere fluorescence intensity was integrated using the TFL-TELO program (a gift kindly provided by Peter Lansdorp).

Telomerase activity by TRAP assay
Telomerase activity was measured by the Stretch PCR method according to the manufacturer's instruction using TeloChaser Telomerase assay kit (T0001, MD Biotechnology). Briefly, about 2.5 × 10 4 cells from each sample were lysed. Lysis buffer served as negative controls. PCR products of cell lysates were separated on non-denaturing TBE-based 12% polyacrylamide gel electrophoresis and visualized by ethidium bromide staining.

Telomere Restriction Fragment (TRF) measurement
TRF analysis was performed using a commercial kit (TeloTAGGG Telomere Length Assay, catalog no. 12209136001, Roche Life Science). Cells were pretreated with RNaseA and Proteinase K (PCR Grade, 03115879001, Roche Life Science), followed by extraction using phenol: chloroform: isoamyl alcohol, digested with MboI (R0147, NEB) at 37˚C overnight and electrophoresed through 1% agarose gels in 0.5 × TBE at 14˚C using a CHEF Mapper pulsed field electrophoresis system (Bio-rad). Auto algorithm was used to separate DNA samples with a size range from 5 to 150 kb. The gel was blotted and probed using reagents in the kit.

Immunofluorescence microscopy
Tail or back skin tissues obtained from wild-type (WT) or G3 Terc deficient mice, or teratomas were fixed overnight in 3.7% paraformaldehyde at 4˚C, dehydrated through graded alcohols and xylene, and embedded in paraffin. After deparaffinizing, rehydrating and washing in PBS, sections were incubated with 3% H 2 O 2 for 10 min at room temperature to block endogenous peroxidase, subjected to high pressure antigen recovery sequentially in 0.01% citrate buffer for 3 min, blocked with 5% goat serum in PBS for 2 h at room temperature, and then incubated with the primary antibodies against K14 (ab7800, Abcam), Fst (ab64490, Abcam) or P63 (ab124762, Abcam) overnight at 4˚C, washed and incubated for 2 h with appropriate fluorescence-conjugated secondary antibodies (Goat anti Mouse IgG (H+L), FITC, 115-095-003, Jackson; Goat anti Rabbit IgG (H+L), Alexa Fluor 594, 111-585-003, Jackson). For immunostaining of ES cells and in vitro differentiated cells, they were washed twice in PBS, then fixed in freshly prepared 3.7% paraformaldehyde in PBS (pH 7.4), permeabilized in 0.1% Triton X-100 (Sigma-Aldrich, Saint Louis, MO) in blocking solution (3% goat serum plus 0.5% BSA in PBS) for 30 min, washed and left in blocking solution for 1 h. Cells were then incubated overnight at 4˚C with primary antibodies and then secondary antibodies as described above. Blocking solution without the primary antibody served as negative control. Nuclei were counterstained with 0.5 μg/ml Hoechst 33342 in Vectashield mounting medium. Fluorescence was imaged using a Zeiss fluorescence microscope (Axio Imager Z1) and using the same exposure time for each group. ImageJ software (https://imagej.net/) was used for relative quantity measurement of fluorescence intensity. Region-of-interest (ROI) tool was used to select the cell or background, and the fluorescence intensity of ROIs achieved. Background with the same threshold was subtracted for each image.

Immunofluorescence-telomere FISH (IF-FISH)
IF-FISH was performed based on an established protocol [71]. Briefly, immunostaining of the cells was performed as described above. After washing the excess of secondary antibody with PBS, cells were fixed in 4% formaldehyde for 2 min, dehydrated with ethanol, and incubated with FITC-telomeric PNA probe as described earlier for telomere QFISH. Fluorescence was imaged using the Zeiss fluorescence microscope.

Bisulfite genomic sequencing
DNA methylation by bisulfite sequencing Genomic DNA was extracted from cells using DNeasy & Blood Tissue Kit (Qiagen) according to the manufacturer's instructions. Bisulfite treatment of DNA was performed with the EpiTect Bisulfite Kit (Qiagen). Bisulfite converted DNA was amplified by seminested PCR, using HS EX Taq DNA Polymerase (Takara). Primer sequences are detailed in S3 Table. PCR products were recovered from stained gels (EasyPure Quick Gel Extraction Kit,Transgen), cloned into a pEASY-T1 Simple Cloning vector (Transgen) and then sequenced.

Overexpression of Fst or p63
The plasmid pEASY-T1-Fst-overexpression (OE) and pEASY-T1-p63-OE were constructed by amplification of Fst or p63 cDNA by PCR and cloning it into pEASY-T1 simple cloning vector (TransGen). Following digestion with XhoI and NotI, Fst or p63 sequences were inserted into Plch37 plasmid. Then the recombinant plasmids were transfected into J1 ES cells or MEF. At 48 h after transfection with 2 μg plasmid using lipofectamine 2000 (Invitrogen), cells were collected for protein and RNA extraction. For obtaining stably transfected cell lines with Fst overexpression, cells were transfected with 2 μg plasmid using lipofectamine 2000 (Invitrogen) and selected by 1.5 μg/ml puromycin for 7-10 days, and clones were picked.

Fst RNAi
shRNA sequences were synthesized (S4 Table), and cloned into pSIREN-RetroQ, according to manufacturer's instructions. The shRNAs without sequence homology to mouse genes served as a negative control. The RNAi retrovirus was packaged using Plat-E cells and then infected cells during differentiation.

Genome-wide gene expression by microarray analysis
Microarray was performed by CapitalBio Corporation (Beijing, China) using Affymetrix 430 2.0 oligonucleotide mouse arrays designed from GenBank, dbEST, and RefSeq sequences based on the UniGene database. The analysis was carried out based on the software and method provided by CapitalBio (http://www.capitalbio.com). Only probe sets showing at least 1.8-fold change were retained in the final list. The detection call indicates whether a transcript was reliably detected (P, Present) or not (A, Absent). We performed hierarchical clustering with the differentially expressed genes using cluster software (version 3) and by applied mean centering and normalization of genes and arrays prior to average linkage clustering.

Luciferase reporter assay
The Fst promoter (~2000bp) was cloned into pGL3-basic vector, following digestion with XhoI and HindIII. 2×10 5 ES cells per 12 well were transfected with 1 μg pGL3-basic vector containing Fst promoter and 10 ng pRL-SV40 vector as control using lipofectamine 2000 (Invitrogen) according to manufacturer's instruction. 24 hours after transfection, ES cells were lysed with 1×PLB (positive lysis buffer, Promega), shaken for 15 min, and then centrifuged at 13000 rpm for 10 min at 4˚C. The supernatants were collected and analyzed for luciferase activity by dual reporter assay according to manufacturer's instructions.

Chromosome XMP13 FISH and telomere FISH
XMP13 probe (D-1413, Metasystems) specific for mouse Chromosome 13 was used for chromosome identification. FISH on chromosome spread was performed according to manufacturer's instructions. The probe was added and coverslip placed, sealed with rubber cement, denatured by heating slide at 75˚C for 2 min, and incubated in humidified chamber at 37˚C overnight. Slides were washed and stained with 0.5 μg/ml DAPI in VectaShield antifade medium. Digital images were captured using a CCD camera on a Zeiss Imager Z2 microscope. The coordinates of the chromosome were recorded with the venire scale along the top and side of the microscope stage. The slides were washed and performed with telomere FISH as described above. After staining with DAPI again, fluorescence from chromosomes and telomeres was digitally imaged using the same microscope according to the recorded coordinates. The telomeres of chromosome 13 were revealed by comparison of the images from the same coordinates.

Statistical analysis
The data from multiple groups were analyzed by ANOVA, and means were compared by Fisher's protected least significant difference (PLSD) using the StatView software from SAS Institute. T-test was used to analyze statistical significance of the two-paired groups. Significant differences were defined as p < 0.05, 0.01, or lower.