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• W2134400731 abstract "Article25 November 2011Open Access The histone methyltransferase Setd8 acts in concert with c-Myc and is required to maintain skin Iwona Driskell Iwona Driskell Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Hisanobu Oda Hisanobu Oda Howard Hughes Medical Institute and Department of Biochemistry, New York University School of Medicine, New York, NY, USA National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan Search for more papers by this author Sandra Blanco Sandra Blanco Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Elisabete Nascimento Elisabete Nascimento Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Peter Humphreys Peter Humphreys Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Michaela Frye Corresponding Author Michaela Frye Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Iwona Driskell Iwona Driskell Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Hisanobu Oda Hisanobu Oda Howard Hughes Medical Institute and Department of Biochemistry, New York University School of Medicine, New York, NY, USA National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan Search for more papers by this author Sandra Blanco Sandra Blanco Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Elisabete Nascimento Elisabete Nascimento Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Peter Humphreys Peter Humphreys Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Michaela Frye Corresponding Author Michaela Frye Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Iwona Driskell1, Hisanobu Oda2,3, Sandra Blanco1, Elisabete Nascimento1, Peter Humphreys1 and Michaela Frye 1 1Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK 2Howard Hughes Medical Institute and Department of Biochemistry, New York University School of Medicine, New York, NY, USA 3National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan *Corresponding author. Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK. Tel.: +44 1223 760230; Fax: +44 1223 760241; E-mail: [email protected] The EMBO Journal (2012)31:616-629https://doi.org/10.1038/emboj.2011.421 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Setd8/PR-Set7/KMT5a-dependent mono-methylation of histone H4 at lysine 20 is essential for mitosis of cultured cells; yet, the functional roles of Setd8 in complex mammalian tissues are unknown. We use skin as a model system to explore how Setd8 may regulate cell division in vivo. Deletion of Setd8 in undifferentiated layers of the mouse epidermis impaired both proliferation and differentiation processes. Long-lived epidermal progenitor cells are lost in the absence of Setd8, leading to an irreversible loss of sebaceous glands and interfollicular epidermis. We show that Setd8 is a transcriptional target of c-Myc and an essential mediator of Myc-induced epidermal differentiation. Deletion of Setd8 in c-Myc-overexpressing skin blocks proliferation and differentiation and causes apoptosis. Increased apoptosis may be explained by our discovery that p63, an essential transcription factor for epidermal commitment is lost, while p53 is gained upon removal of Setd8. Both overexpression of p63 and deletion of p53 rescue Setd8-induced apoptosis. Thus, Setd8 is a crucial inhibitor of apoptosis in skin and its activity is essential for epidermal stem cell survival, proliferation and differentiation. Introduction Epigenetic modifications, such as DNA methylation and post-translational modifications of histones, play an important role in chromatin structure and promoter activity, and have been implicated in a wide range of biological processes including development, reprogramming, aging and cancer (Fraga and Esteller, 2007; Richly et al, 2010; Shafa et al, 2010). Although the functional roles of histone modifications, for instance methylation, have been extensively studied during embryonic stem cell differentiation and reprogramming, little is known about the importance of histone methylation in multipotent stem cells of adult mammalian tissues. Skin is the best-characterized mammalian tissue containing epithelial stem cells, and thus offers an ideal model to study the functional roles of histone methylation in vivo. The epidermis is a stratified epithelium that forms the outermost protective layer of the skin and comprises the interfollicular epidermis (IFE) and its appendages, including hair follicles (HFs) and sebaceous glands (SGs), all of which are maintained by resident skin stem cell pools (Blanpain and Fuchs, 2009; Watt and Jensen, 2009). During normal homeostasis, IFE and SGs are continuously regenerated and their overall proliferation rate is comparatively low. In contrast, HFs undergo cycles of growth, and their proliferation increases periodically in stages of anagen. Anagen replenishes terminally differentiated cells of the HF, and is followed by phases of regression (catagen) and rest (telogen) (Fuchs, 2009). Recent studies in skin showed that epigenetic factors controlling histone H3 methylation are involved in regulating epidermal stem cell survival, proliferation and differentiation (Sen et al, 2008; Eckert et al, 2011; Ezhkova et al, 2011). Epidermal stem cells have also been shown to undergo global changes in histone modifications during differentiation, and one such modification is the mono-methylation of histone H4 at lysine 20 (H4K20me1) (Frye et al, 2007). Although global changes in H4K20 methylation are a common hallmark of cancer (Fraga et al, 2005), the functional roles of methylated H4K20 in normal adult mammalian tissue are unknown. In mammalian cells, Setd8/PR-Set7/KMT5a is the sole enzyme required to catalyse the formation of H4K20me1 (Xiao et al, 2005). Embryonic deletion of Setd8 is lethal in flies and mice (Nishioka et al, 2002; Karachentsev et al, 2005; Oda et al, 2009). Thus, most functional studies on Setd8 have been carried out in vitro. Conditional deletion of Setd8 in embryonic stem cells results in cell-cycle arrest, DNA damage and genomic instability (Oda et al, 2009). Knockout approaches in transformed cells confirm an essential function of Setd8 in cell-cycle progression and replication (Jorgensen et al, 2007; Shi et al, 2007; Tardat et al, 2007, 2010; Houston et al, 2008; Huen et al, 2008). Both, levels of Setd8 and deposition of H4K20me1, are cell-cycle dependent and highest at G2/M phase of the cell cycle (Abbas et al, 2010; Centore et al, 2010; Oda et al, 2010). Unlike other epigenetic regulators, Setd8 is associated with mitotic chromosomes during cell division, which may represent a mechanism by which the H4K20-methyl mark is epigenetically transmitted (Rice et al, 2002). Although Setd8 is clearly essential for cellular survival in the early embryo and cultured cells, its function in less proliferative environments such as adult tissues is unknown. Here, we demonstrate for the first time that Setd8 is required for normal tissue homeostasis, in vivo. Inducible, conditional deletion of Setd8 in the undifferentiated layers of epidermis results in cell-cycle arrest and apoptosis of long-lived progenitor cells leading to irreversible loss of IFE and SGs. Setd8 is a target gene of c-Myc and required for Myc-induced epidermal differentiation. Setd8-depleted epidermal cells fail to express p63 but gain p53, and thus exhibit an impaired terminal differentiation programme and undergo apoptosis instead. Results Setd8 is weakly expressed in skin but upregulated with proliferation The histone methyltransferase Setd8 is specifically responsible for the mono-methylation of histone 4 at lysine 20 (H4K20me1). In skin, nuclei with high levels of H4K20me1 can be found in the basal undifferentiated layer of the IFE, the SG and in the growing anagen HF (Figure 1A; Frye et al, 2007). The accumulation of H4K20me1-positive nuclei in the bulb of HFs (Figure 1A, arrows) suggested that Setd8 activity might be highest in dividing skin progenitor cells. To confirm that Setd8 expression correlated with proliferation, we performed quantitative RT–PCR (QPCR) in skin after birth and during the first synchronized hair cycle. During morphogenesis (M), expression of Setd8 was highest at P9, when HFs are in anagen (Figure 1B). In adult skin, the first synchronized hair cycle begins with anagen (A) at P21. Setd8 RNA levels gradually increased from P21 until P33 and dropped at P36, when the destructive phase (catagen; C) of the hair cycle begins (Figure 1B). Figure 1.Endogenous expression of Setd8 in skin correlates with proliferation. (A) Detection of H4K20me1 (red)-positive nuclei in the interfollicular epidermis (IFE), sebaceous glands (SGs) and hair follicles (HFs) in a skin whole mount. Arrows indicate anagen hair follicles. (B) QPCR for Setd8 RNA during morphogenesis (M) and the first adult hair cycle of catagen (C), telogen (T) and anagen (A) at indicated postnatal days (P). Grey indicates anagen stages. (C–E) Co-labelling for H4K20me1-positive nuclei (red) with BrdU (green) in HF (C), SG (D) and IFE (E). Arrows indicate BrdU-positive nuclei lacking H4K20me1. Nuclei are counterstained with DAPI (blue) (A–E). (F–K) Detection of β-galactosidase in wild-type and LacZ-reporter mice (RRB075). Arrows indicate the HF (F, I), SG (G, J), IFE (H, K) and arrowhead marks unspecific staining in SG (G, J). (L–N) Whole mount LacZ staining of wild-type (wt) and RRB075 (RB) mouse embryos at indicated embryonic days (E). (N) Higher magnification of boxed area in (M). Arrow indicates hair follicles. Scale bars: 100 μm (A, F, G, I, J); 50 μm (C–E, H, K). Download figure Download PowerPoint To investigate whether H4K20me1 generally marked dividing cells, we labelled mouse skin with BrdU and co-stained the nuclei for H4K20me1 (Figure 1C–E). BrdU labelling requires cells to be in S phase at the time of pulse; and we found that BrdU and H4K20me1 labelling was mutually exclusive in the HF, SGs and IFE (Figure 1C–E; arrows). Thus, in line with recent studies showing that Setd8 protein is degraded in S phase (Oda et al, 2010), H4K20me1 was also absent in S phase of the cell cycle. However, labelling for H4K20me1 and BrdU overlapped in the region of the bulb of anagen HFs where committed progenitor cells reside (Figure 1C). Detection of endogenous Setd8 protein in tissues is hampered by the lack of suitable antibodies. To localize Setd8 in vivo, we generated a reporter mouse carrying the β-galactosidase gene as a GeneTrap in intron 3 (RRB075) (Huen et al, 2008). Only in RRB075 mice, we detected high levels of β-galactosidase in the bulb of anagen HFs (Figure 1F and I). Whereas the base of the SGs stained unspecific for LacZ in wild-type and RRB075 mice (Figure 1G and J; arrowheads), the lower part of the SGs exhibited β-galactosidase activity only in RRB075 mice (Figure 1G and J; arrows). Expression of LacZ was weak in the IFE but we detected a patchy β-galactosidase activity in the reporter mice (Figure 1H and K; arrows). Staining for LacZ during late embryonic development at E13.5 and E15.5 demonstrated that Setd8 was highly expressed throughout the developing epidermis and HFs (Figure 1L–N). In conclusion, we found a widespread but weak expression of Setd8 in SGs, IFE and the HF that correlated well with the occurrence of H4K20me1-positive nuclei, and increased with proliferative phases of the skin. Skin cannot develop or be maintained in the absence of Setd8 The expression pattern of Setd8 during morphogenesis indicated that Setd8 might be required for skin development. To test this hypothesis, we conditionally deleted Setd8 in the basal, undifferentiated layers of the developing epidermis (K14Setd8Δ/Δ) (Materials and methods; Supplementary Figure S1A). Mice with deleted Setd8 from E14.5, when the keratin 14 (K14) promoter is active died shortly after birth. To follow the fate of Setd8-depleted epidermal cells during development, we crossed the K14Setd8Δ/Δ mice with a green fluorescent protein (GFP)-reporter line for Cre-recombinase (Materials and methods; Figure 2A; Kawamoto et al, 2000). As soon as Setd8 was deleted at E14.5, we noted the disappearance of GFP-positive epidermal cells (Figure 2A). Further analyses of Setd8-depleted embryos at E18.5 showed that limb development was impaired and the skin was indeed absent (Figure 2B and C). Histological analysis of section obtained from embryos at E15.5 confirmed the lack of a developing epidermis (Figure 2D and E). When we labelled embryonic skin for markers of undifferentiated epidermis, we found that some areas in E14.5 embryos still had a single layer of epithelial cells, which was largely lost at E15.5 (Figure 2F and G; Supplementary Figure S1B and C). In rare cases, we found single epidermal cells at E15.5 in K14Setd8Δ/Δ mice, these cells lacked nuclear p63 but expressed keratin 8 (K8) (Figure 2G; Supplementary Figure S1B and C). Since, we detected p53-positive cells in Setd8-depleted epidermis at E15.5 (Figure 2H), we concluded that loss of Setd8 caused apoptosis rather than inhibiting cell differentiation. Figure 2.Embryonic and adult skin is disrupted in the absence of Setd8. (A) Visualization of GFP as a reporter for Cre-activity in control (left hand panels) and K14CreSetd8Δ/Δ (right hand panels) embryos at the indicated embryonic days (E). (B, C) Mouse embryos at E18.5 with normal expression of Setd8 (control) (B) and deleted Setd8 (K14CreSetd8Δ/Δ) in skin (C). (D, E) Haematoxylin & Eosin staining of skin sections from control (D) and K14CreSetd8Δ/Δ (E) littermates at E15.5. (F–H) Skin sections of control (upper panels) and K14CreSetd8Δ/Δ (lower panels) littermates at E14.5 (F) and E15.5 (G, H) labelled with antibodies to Itga6 (green) and keratin 14 (K14; red) (F) or p63 (red) (G) or p53 (brown) (H). Arrows in (F) indicate the loss of an epithelial cell layers starting from E14.5. (I–L) Histological sections of adult skin expressing inducible Cre-recombinase under control of the K14 promoter (K14CreER) crossed with wild-type mice (Setd8wt/wt) (I), or animals heterozygous (Setd8Δ/wt) (K), homozygous (Setd8Δ/Δ) (J) for the floxed Setd8 allele, or mice carrying one floxed and one null allele for Setd8 (Setd8Δ/null) (L). (M, N) Labelling for FAS and Fabp5 (green) in the presence (K14CreERSetd8wt/wt) (left hand panels) or absence of Setd8 (K14CreERSetd8Δ/Δ) (right hand panels). Nuclei are counterstained with DAPI (blue) in (F, G, M, N). Arrows in (I–N) indicate sebaceous glands and arrowheads (I–L) point to IFE. Scale bars: 100 μm (F–N). Download figure Download PowerPoint Because the deletion of Setd8 during development was lethal, we generated an inducible conditional knockout mouse model for Setd8 in skin (K14CreERSetd8Δ/Δ) (Materials and methods). Setd8 RNA expression decreased (Supplementary Figure S1D) and mono-methylation of H4K20 was lost when Setd8 was deleted (Supplementary Figure S1E and F). To confirm that Cre-recombinase was uniformly activated in the epidermis we crossed the K14CreER mice to a GFP-reporter mouse (Supplementary Figure S2A and B; Kawamoto et al, 2000). Whereas we detected homogenous Cre-recombinase activity in the IFE and SGs, the HFs showed only a patchy staining for GFP (Supplementary Figure S2A). A time course of treatment with 4-hydroxytmaoxifen (4-OHT) on the GFP-reporter mice further revealed that efficient recombination was only achieved by 9 days in tail skin (Supplementary Figure S2B), we therefore treated the mice a minimum of 14 days with 4-OHT. Control mice carrying the wild-type Setd8 alleles or being heterozygous for Setd8 deletion did not show any phenotype when treated with 4-OHT (Figure 2I and K). In contrast, when both alleles of Setd8 were deleted, either by breeding the mice to homozygosity for the floxed Setd8 alleles or by crossing the floxed mice with mice carrying one deleted allele (null) for Setd8 the epidermis was severely disrupted. The cellularity of the IFE was reduced (Figure 2I–L; arrowheads). Treatment of the tissue sections with DOPA showed that the black granules in the IFE of transgenic mice were melanin (Figure 2L, arrowhead; data not shown), indicating that melanocytes were recruited to damaged areas of the skin. Furthermore, SGs (arrows) were absent when Setd8 was deleted (Figure 2J and L). We confirmed the lack of functional SGs by labelling sections of back skin for fatty acid synthase (FAS) and the fatty acid binding protein (Fabp5) (Figure 2M and N; arrows), which are prominent markers for SGs (Collins and Watt, 2008; Lo Celso et al, 2008). The morphology of HFs was not affected by deletion of Setd8 (Figure 2I–L). However, GFP-reporter mice demonstrated that Setd8 might not be efficiently deleted in the HFs (Supplementary Figure S2A and B) and we therefore focused our further studies on the IFE and SGs. Proliferation and differentiation are impaired by loss of Setd8 Since epidermal morphology varies at different body sites we chose to measure the effect of Setd8 deletion on mouse tail and back skin. We first confirmed by whole mount staining for keratin 14 (K14) that deletion of Setd8 in tail skin resulted in the same phenotype as back skin; SGs were degenerated or lost and the integrity of the IFE was severely impaired (Supplementary Figure S3A and B; arrows). We next investigated markers for the IFE in back and tail skin (Figure 3). We began by testing the proliferative capacity of cells in the IFE. BrdU incorporation was four-fold reduced in the IFE in back and tail skin in the absence of Setd8 (Figure 3A and B, arrows; Supplementary Figure S3C). Itga6, a marker for basal, undifferentiated epidermal cells, was reduced or lost in some areas of the IFE when Setd8 was ablated (Figure 3C and D; arrows). Deposition of collagen IV remained unchanged when Setd8 was deleted (Supplementary Figure S3D and E). GFP-reporter mice (Materials and methods) confirmed that Setd8-deleted epidermis exhibited lower expression of Itga6 and incorporated BrdU to a lesser extent (Supplementary Figure S4A and B). Figure 3.Deletion of Setd8 impairs epidermal proliferation and differentiation. (A–J) Immunofluorescence labelling for K14 (red) and BrdU (green) (A, B) and Itga6 (green) (C, D) as markers for undifferentiated skin, Involucrin (green) (E, F) and K10 (green) (G, H) as markers for differentiated skin, and p63 (green) (I) red (J) in sections (A, C, E, G, I) and whole mounts (B, D, F, H, J) from control and K14CreERSetd8Δ/Δ mice. Arrows in (B–E, G, I) point to IFE and arrowheads in (H, J) indicate a parakeratotic scale. Inserts in (J) are a higher magnification of the IFE. Nuclei are counterstained with DAPI. Scale bars: 100 μm (A, C, E, G, I) and 250 μm (B, D, F, H, J). Download figure Download PowerPoint To test whether the low number of proliferating cells in Setd8-depleted skin impaired differentiation processes, we labelled skin for the terminal differentiation markers keratin 10 (K10) and Involucrin (Ivl) (Figure 3E–H). Similarly to Itga6, we detected areas with disrupted expression of both differentiation markers in the back skin (Figure 3E and G). A mis-localization of differentiation markers was even more apparent in tail skin (Figure 3F and H). Skin of the mouse tail exhibits a regular, ordered pattern of parakeratotic scale epidermis that alternates with orthokeratotic interscale epidermis (Schweizer et al, 1987). Parakeratotic scales, which are negative for K10 but express high levels of p63 in wild-type mice (Figure 3H and J; left hand panels; arrowheads), were lost when S was depleted (Figure 3H and J; right hand panels). Since undifferentiated and proliferating cell populations were compromised in the epidermis, the reduction of expression of differentiation markers might be an indirect effect of Setd8 deletion. The transcription factor p63 is one of the most important regulators of skin homeostasis and, similar to conditional deletion of Setd8 in E18.5 embryos (Figure 2A–H), embryos lacking p63 fail to undergo epidermal commitment (Koster, 2010). We asked whether impaired proliferation and differentiation processes in the IFE might be due to altered p63 levels. In the absence of Setd8, p63-positive nuclei were lost in the IFE of both back and tail epidermis (Figure 3I, arrows; Figure 3J, insert). QPCR confirmed that p63 RNA levels decreased in Setd8-ablated skin (Supplementary Figure S4C). We concluded that Setd8's essential function for epidermal stratification might at least in part be mediated by p63. Stem/progenitor cells of IFE and SGs are lost in Setd8-depleted skin The depletion of SGs and IFE in the absence of Setd8 indicated that epidermal stem cells might require Setd8 for cell division and survival. We speculated that if ablation of Setd8 eliminated resident stem cell pools, the phenotype should be irreversible and SGs and IFE should not regenerate, even when we stopped treatment with 4-OHT. To test our hypothesis, we treated the back skin of K14CreERSetd8Δ/Δ and control mice with 4-OHT for 14 days and let the mice recover for 3 weeks. Skin samples were taken at four time points: the end of the treatment and after 1, 2 and 3 weeks of recovery (Figure 4A). Figure 4.Long-lived progenitors of IFE and sebaceous glands are irreversibly lost in K14CreERSetd8Δ/Δ mice and recovered skin derives from hair follicles (HFs). (A) Schematic overview of the 4-OHT-treatment regime. (B, C) Haematoxylin & Eosin (H&E) staining of skin section from control (B) and K14CreERSetd8Δ/Δ (C) mice at the end of treatment with 4-OHT and after 7, 14 and 21 days of recovery. Arrows point to IFE and arrowheads to sebaceous glands. (D–G) Co-labelling of control (D, F) and K14CreERSetd8Δ/Δ epidermis (E, G) for K14 (green) and GFP (red) (D, E) or GFP (red) only (F, G) after treatment with 4-OHT (end treatment) or after 7 and 21 days of recovery. (H) BrdU incorporation (BrdU; green) and labelling for K14 (red) in the IFE, sebaceous glands (SGs) and HF bulges/bulbs (Bu/Bb) in control (upper panels) and K14CreERSetd8Δ/Δ (Setd8Δ/Δ; lower panels) epidermis. (I) Quantification of BrdU-positive cells from (H). (J) Detection of quiescent label-retaining cells (LRCs) (BrdU; green) in bulges of control and K14CreERSetd8Δ/Δ (Setd8Δ/Δ) mice. (K) Quantification of (J). KO: K14CreERSetd8Δ/Δ. Nuclei are counterstained with DAPI (blue) in (D–H, J). Scale bars: 250 μm (A–G); 50 μm (H) and 75 μm (J). Download figure Download PowerPoint Skin of control mice was not affected by treatment with 4-OHT during the course of the experiment and also skin of K14CreERSetd8Δ/Δ mice recovered both epidermal compartments, the IFE (arrows) and SGs (arrowhead) (Figure 4B and C). However, two aspects of recovered Setd8-depleted skin were unusual. First, skin of K14CreERSetd8Δ/Δ mice never exited anagen (Figure 4C). Second, dividing cells at 7 days of recovery mainly localized to the upper part of the HF (Supplementary Figure S5A and B, 7 days; arrow and line). One explanation for these observations was that deletion of Setd8 induced a wound healing reaction, in which HF stem cells contribute to formation of IFE, a process that can be associated with prolonged or induced anagen (Ito et al, 2005; Ansell et al, 2011). Thus, K14CreERSetd8Δ/Δ skin may be recovered by HF stem cells that escaped Setd8 deletion (Supplementary Figure S2A). To determine the epidermal population responsible for the recovery of IFE and SGs in the absence of Setd8, we repeated the experiment using control- and K14CreERSetd8Δ/Δ-GFP-reporter mice (Figure 4D–G). After 21 days of recovery, control skin still expressed GFP, demonstrating that we successfully labelled long-lived progenitor cells of the IFE and SGs (Figure 4D and F; 21 days). In contrast, we did not detect any GFP-positive epidermal cell in skin of K14CreERSetd8Δ/Δ-GFP-reporter mice (Figure 4E and G; 21 days). These results indicated that the newly formed IFE and SGs were derived from HFs. Our hypothesis that newly formed IFE might derive from upwards migrating HF cells was supported by the observation that proliferation in Setd8-depleted skin was only decreased in the IFE but increased in HFs (Bu/Bb) (Figure 4H and I). We next asked whether the recovery of IFE in the absence of Setd8 required the activation of quiescent HF bulge stem cells. One characteristic of bulge stem cells is that if they incorporate BrdU in neonatal epidermis they retain the label in adulthood; such cells are known as label-retaining cells (LRCs; Bickenbach and Chism, 1998; Braun et al, 2003). However, the number of LRC in the bulge of K14CreERSetd8Δ/Δ and control skin remained unchanged (Figure 4J and K), indicating that the recovered IFE in the absence of Setd8 does not require the activation of quiescent bulge stem cells. Together, our data demonstrated that long-lived progenitor cells of the IFE and the SGs required Setd8 for survival. Setd8 mediates functions of c-Myc in skin So far our data showed that the loss of long-lived progenitor cells in Setd8-depleted skin was the likely result of a failure to undergo cell divisions. c-Myc is one important transcription factor in skin that induces epidermal stem cells to exit the stem cell compartment and stimulates their proliferation as progenitors (Arnold and Watt, 2001; Frye et al, 2003). Furthermore, Myc-induced exit from the epidermal stem cell niche is accompanied by transient accumulation of nuclei with high levels of H4K20me1 (Frye et al, 2007). Thus, we asked whether Setd8 might mediate c-Myc's function on epidermal stem cells. We confirmed that expression of Setd8 RNA was increased in skin when c-Myc was overexpressed (K14MycER) (Figure 5A and B). In K14MycER transgenic mice, c-Myc can be activated by topical application of 4-OHT, and we found a time-dependent upregulation of Setd8 RNA over 6 days of treatment in K14MycER skin relative to wild-type epidermis (Figure 5B). We detected several putative Myc-binding sites in the Setd8 promoter using TESS (Transcription Element Search System) (Figure 5C, insert; Schug, 2008). Chromatin immunoprecipitations (ChIPs) using a c-Myc antibody confirmed a 15-fold enrichment of the Setd8 promoter in K14MycER epidermis compared with wild-type skin (Figure 5C). Nucleolin (Ncl1) is direct target gene of c-Myc and served as positive control (Figure 5C; Greasley et al, 2000). ChIP on chip experiments further confirmed a transcriptional regulation of Setd8 by c-Myc (Supplementary Figure S6A). Figure 5.Setd8 is transcriptionally regulated by c-Myc and required to mediate skin-specific functions of c-Myc. (A) Relative expression of Setd8 RNA in wild-type (WT) and K14MycER transgenic (TG) mice measured by QPCR. (B) Log2 fold-change (FC) of Setd8 RNA expression in K14MycER epidermis relative to wild-type skin in time course of treatment with 4-OHT for the days indicated. Data are obtained from gene expression arrays. (C) ChIP for Setd8 and Nucleolin (Ncl1) promoters using a c-Myc antibody in skin of WT and K14MycER TG animals using two different sets of primers (Setd8.1; Setd8.2). Ctr indicates the negative control. The insert is a graphical overview of the Setd8 promoter and the putative c-Myc binding sites. Red lines indicate putative c-Myc binding sites amplified by QPCR. (D, E) Measuring proliferation, using Ki67 as a marker (D) or BrdU incorporation (E) in control, K14CrERSetd8Δ/Δ, K14MycER animals and K14MycER mice with deleted Setd8 alleles (K14MycER-Setd8Δ/Δ). K14 (red) was used as counterstain in (E). Lines in (D, E: K14MycER-Setd8Δ/Δ) indicate loss of cycling cells. Areas outside the lines may have not deleted Setd8 due to short treatment with 4-OHT (6 days) (Supplementary Figure S2C and D). (F–K) Labelling for K10 (F), p63 (G), K8 (H), 14-3-3σ (I) (red) and p53 (J) (brown), or TUNEL (green) (K). Arrows in (F) mark cells of the basal layer and arrows in (H) indicate K8-positive cells in the IFE. Nuclei are counterstained with DAPI (blue) in (E–I, K). Scale bars: 100 μm (D, E) and 50 μm (F–K). (L) QPCR for p63 RNA levels in the indicated transgenic lines. (M) Quantification of (J) as percentage of p53-positive cells in the IFE. (N) Colour code for transgenic lines used in (L–N) and survival of epidermal cells in culture for the indicated hours. Y axis shows relative survival of the 4-OHT-treated lines to their untreated controls. Download figure Download PowerPoint To test whether Setd8 was an essential functional mediator of c-Myc in skin, we deleted Setd8 in K14MycER transgenic mice (Figure 5D–N). Ki67-labelling and BrdU incorporation assays showed that c-Myc's promoting effect on cellular proliferation was reduced or absent when Setd8 was deleted (Figure 5D and E, lines; Supplementary Figure S6B). The decrease of proliferation in K14MycER skin" @default.
• W2134400731 created "2016-06-24" @default.
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• W2134400731 creator A5027485491 @default.
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• W2134400731 date "2011-11-25" @default.
• W2134400731 modified "2023-09-27" @default.
• W2134400731 title "The histone methyltransferase Setd8 acts in concert with c-Myc and is required to maintain skin" @default.
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• W2134400731 doi "https://doi.org/10.1038/emboj.2011.421" @default.
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