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Dissecting the Roles of Polycomb Repressive Complex 2 Subunits in the Control of Skin Development

  • Author Footnotes
    2 These authors contributed equally to this work.
    Katherine L. Dauber
    Footnotes
    2 These authors contributed equally to this work.
    Affiliations
    Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Author Footnotes
    2 These authors contributed equally to this work.
    Carolina N. Perdigoto
    Footnotes
    2 These authors contributed equally to this work.
    Affiliations
    Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Victor J. Valdes
    Affiliations
    Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Francis J. Santoriello
    Affiliations
    Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Idan Cohen
    Affiliations
    Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Elena Ezhkova
    Correspondence
    Correspondence: Elena Ezhkova, Icahn School of Medicine at Mount Sinai, One Gustave L Levy Place, Box 1020, New York, New York 10029, USA.
    Affiliations
    Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, USA
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  • Author Footnotes
    2 These authors contributed equally to this work.
Open ArchivePublished:March 16, 2016DOI:https://doi.org/10.1016/j.jid.2016.02.809
      Polycomb repressive complex 2 (PRC2) is an essential regulator of cell physiology. Although there have been numerous studies on PRC2 function in somatic tissue development and stem cell control, these have focused on the loss of a single PRC2 subunit. Recent studies, however, have shown that PRC2 subunits may function independently of the PRC2 complex. To investigate the function of PRC2 in the control of skin development, we generated and analyzed three conditional knockout mouse lines, in which the essential PRC2 subunits embryonic ectoderm development (EED), suppressor of zeste 12 homolog (Suz12), and enhancer of zeste homologs 1 and 2 (Ezh1/2) are conditionally ablated in the embryonic epidermal progenitors that give rise to the epidermis, hair follicles, and Merkel cells. Our studies showed that the observed loss-of-function phenotypes are shared between the three knockouts, indicating that in the skin epithelium, EED, Suz12, and Ezh1/2 function largely as subunits of the PRC2 complex. Interestingly, the absence of PRC2 results in dramatically different phenotypes across the different skin lineages: premature acquisition of a functional epidermal barrier, formation of ectopic Merkel cells, and defective postnatal development of hair follicles. The strikingly different roles of PRC2 in the formation of three lineages exemplify the complex outcomes that the lack of PRC2 can have in a somatic stem cell system.

      Abbreviations:

      FACS (fluorescence-activated cell-sorting), Krt (keratin), PRC2 (polycomb repressive complex 2), RT-qPCR (semiquantitative real-time PCR)

      Introduction

      Polycomb repressive complex (PRC) 2 is a major chromatin repressor (
      • Schwartz Y.B.
      • Pirrotta V.
      A new world of Polycombs: unexpected partnerships and emerging functions.
      ) that regulates tissue development and fate control, and is implicated in a number of diseases, including cancer (
      • Margueron R.
      • Reinberg D.
      The Polycomb complex PRC2 and its mark in life.
      ,
      • Perdigoto C.N.
      • Valdes V.J.
      • Bardot E.S.
      • Ezhkova E.
      Epigenetic regulation of epidermal differentiation.
      ,
      • Sauvageau M.
      • Sauvageau G.
      Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer.
      ,
      • Schwartz Y.B.
      • Pirrotta V.
      Polycomb complexes and epigenetic states.
      ,
      • Surface L.E.
      • Thornton S.R.
      • Boyer L.A.
      Polycomb group proteins set the stage for early lineage commitment.
      ). PRC2 consists of the core subunits embryonic ectoderm development (EED), suppressor of zeste 12 homolog (Suz12), and the histone methyltransferases enhancer of zeste homolog 1 (Ezh1) or enhancer of zeste homolog 2 (Ezh2), which catalyze the trimethylation of lysine 27 of histone H3, resulting in gene silencing by chromatin compaction (
      • Margueron R.
      • Li G.
      • Sarma K.
      • Blais A.
      • Zavadil J.
      • Woodcock C.L.
      • et al.
      Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms.
      ,
      • Schwartz Y.B.
      • Pirrotta V.
      A new world of Polycombs: unexpected partnerships and emerging functions.
      ,
      • Simon J.A.
      • Kingston R.E.
      Mechanisms of polycomb gene silencing: knowns and unknowns.
      ).
      Recent studies have suggested that PRC2 subunits might have PRC2-independent phenotypes, complicating the assessment of the functional significance of PRC2. Indeed, in prostate cancer, the catalytic subunit Ezh2 mediates the methylation of the androgen receptor to control its activity independently of the other PRC2 subunits (
      • Xu K.
      • Wu Z.J.
      • Groner A.C.
      • He H.H.
      • Cai C.
      • Lis R.T.
      • et al.
      EZH2 oncogenic activity in castration-resistant prostate cancer cells is polycomb-independent.
      ). Similarly, in breast cancer cells, Ezh2 is able to mediate the crosstalk between Wnt signaling and its effectors (
      • Shi B.
      • Liang J.
      • Yang X.
      • Wang Y.
      • Zhao Y.
      • Wu H.
      • et al.
      Integration of estrogen and Wnt signaling circuits by the polycomb group protein EZH2 in breast cancer cells.
      ), and to activate NF-κB target genes through the formation of a ternary complex, independently of the PRC2 complex (
      • Lee S.T.
      • Li Z.
      • Wu Z.
      • Aau M.
      • Guan P.
      • Karuturi R.K.
      • et al.
      Context-specific regulation of NF-kappaB target gene expression by EZH2 in breast cancers.
      ). Furthermore, recent loss-of-function studies in mice have shown that EED is required for proper hematopoiesis during development, whereas Ezh2 is dispensable (
      • Xie H.
      • Xu J.
      • Hsu J.H.
      • Nguyen M.
      • Fujiwara Y.
      • Peng C.
      • et al.
      Polycomb repressive complex 2 regulates normal hematopoietic stem cell function in a developmental-stage-specific manner.
      ). Thus, despite the vast number of studies reporting the results of the loss of a single PRC2 subunit in stem cells (
      • Aloia L.
      • Di Stefano B.
      • Di Croce L.
      Polycomb complexes in stem cells and embryonic development.
      ), it is still unclear which of the observed phenotypes are indeed due to the loss of PRC2 function.
      Ezh1/2 have been shown to play a critical role in skin development by restricting epidermal and Merkel cell differentiation programs and promoting hair follicle cell proliferation (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ,
      • Ezhkova E.
      • Pasolli H.A.
      • Parker J.S.
      • Stokes N.
      • Su I.H.
      • Hannon G.
      • et al.
      Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.
      ,
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). Because the loss of Ezh1/2 in the skin epithelium displayed strong yet different developmental alterations in these skin lineages (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ,
      • Ezhkova E.
      • Pasolli H.A.
      • Parker J.S.
      • Stokes N.
      • Su I.H.
      • Hannon G.
      • et al.
      Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.
      ,
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ), and considering that Ezh2 can have PRC2-independent roles, we wanted to determine the role of PRC2 in skin development. To this end, we conditionally ablated Ezh1/2, EED, or Suz12 from the embryonic epidermal progenitor cells and performed comparative analysis between the three different mutants to analyze the formation of the epidermis, hair follicles, and Merkel cells. We found that the loss of each subunit resulted in the same phenotypes, with the different mutants being indistinguishable from each other. Furthermore, our analysis revealed that loss of PRC2 resulted in distinct phenotypes for each of the epidermal lineages: premature formation of a functional epidermal barrier, an expansion in the number of Merkel cells, and defective postnatal hair follicle development. Our study illustrates how epigenetic regulation can have dramatically different functions in the development and differentiation of different cell lineages.

      Results

      Loss of PRC2 leads to premature epidermal barrier formation

      To conditionally ablate EED and Suz12 in the skin epithelium (EEDcKO and Suz12cKO, respectively), we crossed EED floxed or Suz12 floxed mice with mice expressing Cre recombinase under the control of the keratin (Krt) 14 promoter, which is active in embryonic epidermal progenitors starting at embryonic day (E) 12 (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ,
      • Dassule H.R.
      • Lewis P.
      • Bei M.
      • Maas R.
      • McMahon A.P.
      Sonic hedgehog regulates growth and morphogenesis of the tooth.
      ). As in mice where Ezh1/2 were conditionally ablated in the epidermis (Ezh1/2 2KO) (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ,
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ), EED-null and Suz12-null epidermal cells lacked trimethylation of lysine 27 of histone H3 (Supplementary Figure S1a and b online), whereas dermal cells, which are not targeted by Krt14-Cre ablation, retained this histone mark (Supplementary Figure S1a). We next analyzed the formation of the interfollicular epidermis, the Merkel cells, and the hair follicles in EEDcKO and Suz12cKO mice compared with Ezh1/2 2KO to determine the common phenotypes, as these would likely be caused by a loss of PRC2 function, rather than PRC2-independent roles.
      As in Ezh1/2 2KO mice (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ), formation of the suprabasal epidermal layers was not grossly affected in new-born (P0) EEDcKO or Suz12cKO mice, as shown by histological analysis (Figure 1a). Immunofluorescence staining with antibodies against epidermal differentiation proteins of spinous (Krt10) (Figure 1b), granular (Loricrin) (Figure 1c), and stratum corneum (Filaggrin) layers (Figure 1d) confirmed proper formation of the different epidermal layers. Importantly, expression of Krt10, Loricrin, and Filaggrin was excluded from the Krt14-expressing basal layer, which contains epidermal progenitor cells (Figure 1b–d). A whole-mount dye-exclusion epidermal barrier assay (
      • Hardman M.J.
      • Sisi P.
      • Banbury D.N.
      • Byrne C.
      Patterned acquisition of skin barrier function during development.
      ) was performed on new-born EEDcKO and Suz12cKO mice and confirmed the formation of a functional epidermal barrier (Supplementary Figure S1c). Analysis of proliferation and apoptosis in PRC2-null basal and suprabasal cells did not reveal any abnormalities (Figure 1e; Supplementary Figure S1d).
      Figure 1
      Figure 1Loss of PRC2 leads to premature epidermis formation. (a) Hematoxylin and eosin staining showing similar epidermal structure in P0 WT and PRC2-null mice; quantification of epidermal thickness (n ≥ 2; P = 0.6475). (b–d) Immunofluorescence for Krt10 (K10) (b), Loricrin (Lor) (c), and Filaggrin (Flg) (d) showing that the differentiated suprabasal layers are unaffected in P0 PRC2-null mice. (e) Immunofluorescence for BrdU showing no changes in proliferation in the integrin β4 (β4)-labeled basal layer (BL) in P0 PRC2-null mice; quantification of percentage of BrdU(+) cells (n = 3; P = 0.1460). (f, g) Hematoxylin and eosin (H&E) staining (f) and immunofluorescence for Filaggrin (g) showing premature formation of the stratum corneum in E16 PRC2-null mice when compared with WT. Scale bars: 25 μm. Statistical significance: non significant (n.s.) P > 0.05. der, dermis; epi, epidermis; Krt, keratin; PRC2, polycomb repressive complex 2.
      During embryogenesis, Ezh2 represses epidermal differentiation and E16 Ezh2 skin conditional knockout (Ezh2cKO) embryos have premature formation of the stratum corneum (
      • Ezhkova E.
      • Pasolli H.A.
      • Parker J.S.
      • Stokes N.
      • Su I.H.
      • Hannon G.
      • et al.
      Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.
      ). If Ezh1/2, EED, and Suz12 are all functioning as part of the PRC2 complex to control epidermal barrier formation, we would expect that EEDcKO and Suz12cKO mice would also have premature stratum corneum development. We analyzed Suz12cKO and EEDcKO embryos at E16, when wild type (WT) embryos lack Filaggrin(+) stratum corneum layers. Both histological analysis and immunofluorescence staining for Filaggrin revealed premature acquisition of the stratum corneum in E16 Ezh1/2 2KO, Suz12cKO, and EEDcKO embryos (Figure 1f and g). Taken together, these data indicate that although the cornified layers of the interfollicular epidermis are acquired prematurely in PRC2-null mice, the neonate PRC2-null epidermis is fully functional and indistinguishable from WT epidermis.

      Loss of PRC2 results in ectopic formation of Merkel cells

      Recent studies have shown that embryonic epidermal progenitors give rise to Merkel cells (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ,
      • Morrison K.M.
      • Miesegaes G.R.
      • Lumpkin E.A.
      • Maricich S.M.
      Mammalian Merkel cells are descended from the epidermal lineage.
      ,
      • Van Keymeulen A.
      • Mascre G.
      • Youseff K.K.
      • Harel I.
      • Michaux C.
      • De Geest N.
      • et al.
      Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis.
      ), but the mechanisms controlling Merkel cell lineage specification and differentiation are largely unknown. Merkel cell differentiation is a temporally regulated process during which early specified Merkel cells start to express the transcription factors atonal bHLH transcription factor 1 (Atoh1), SRY-box 2 (Sox2) and ISL LIM homeobox 1 (Isl1), essential for Merkel differentiation, cytoskeletal proteins (Krt8, Krt18, and Krt20), and components of the synaptic machinery, and undergo innervation by sensory neurons expressing neurofilament NF200 (
      • Haeberle H.
      • Fujiwara M.
      • Chuang J.
      • Medina M.M.
      • Panditrao M.V.
      • Bechstedt S.
      • et al.
      Molecular profiling reveals synaptic release machinery in Merkel cells.
      ,
      • Owens D.M.
      • Lumpkin E.A.
      Diversification and specialization of touch receptors in skin.
      ,
      • Perdigoto C.N.
      • Bardot E.S.
      • Valdes V.J.
      • Santoriello F.J.
      • Ezhkova E.
      Embryonic maturation of epidermal Merkel cells is controlled by a redundant transcription factor network.
      ,
      • Vielkind U.
      • Sebzda M.K.
      • Gibson I.R.
      • Hardy M.H.
      Dynamics of Merkel cell patterns in developing hair follicles in the dorsal skin of mice, demonstrated by a monoclonal antibody to mouse keratin 8.
      ).
      Our previous studies have shown that in P0 Ezh1/2-null skin, there is an increase in the number of Merkel cells due to the derepression of essential Merkel cell-specific genes and differentiation of Ezh1/2-null epidermal progenitors toward the Merkel cell lineage (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ). We analyzed P0 Suz12-null and EED-null epidermis and observed a similar increase in the number of Merkel cells. Indeed, immunofluorescence analysis with antibodies against Krt20 and Krt18 revealed a significant increase in the number of Krt20(+) (Figure 2a) and Krt18(+) (Supplementary Figure S2a online) cells in the back skin of P0 Ezh1/2 2KO, EEDcKO, and Suz12cKO mice compared with WT. An increase in the number of Merkel cells was also observed in other Merkel cell-rich areas, such as the whisker interfollicular epidermis (Supplementary Figure S2d and e) and the paws (Supplementary Figure S2f and g), whereas no increase in the number of Merkel cells was observed in the whisker follicles (Supplementary Figure S2b and c) of EEDcKO and Suz12cKO mice compared with WT. This is similar to what was previously seen in Ezh1/2 2KO mice (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ).
      Figure 2
      Figure 2Loss of PRC2 results in Merkel cell expansion. (a) Immunofluorescence for Krt20 (K20) showing a significant increase in the number of Merkel cells (MC) in P0 PRC2-null epidermis, compared with WT; quantification of number of MC (n ≥ 3; P < 0.0001). (b, c) Immunofluorescence for MC markers Krt20 (b), Isl1 (b), Krt18 (K18) (c), and Sox2 (c) showing coexpression of markers in PRC2-null MC. (d) Immunofluorescence for Krt20 and NF200 showing that the PRC2-null MC are innervated. (e) Ki67 staining showing that MC are not proliferating in P0 PRC2-null epidermis. (f) RT-qPCR in FACS-purified interfollicular epidermis (IFE) cells showing upregulation of Merkel genes Isl1 and Sox2 in P14 gWT and gPRC-null skin (mean ± SD; n = 3; all significant, P < 0.05). Scale bars: (a) 100 μm; (b–e) 25 μm. Statistical significance: significant *P = 0.01–0.05; very significant **P = 0.01–0.001; extremely significant ***P < 0.001. FACS, fluorescence-activated cell-sorting; Krt, keratin; PRC2, polycomb repressive complex 2; RT-qPCR, semiquantitative real-time PCR.
      Characterization of the EED-null and Suz12-null Merkel cells confirmed that they express key Merkel cell regulatory proteins such as Isl1 and Sox2 (Figure 2b and c) and are innervated by NF200(+) sensory neurons (Figure 2d). As with Ezh1/2-null epidermis, the increase in the number of Merkel cells was not due to their aberrant proliferation, as analysis of the proliferation marker Ki67 in P0 WT, EEDcKO, and Suz12cKO mice showed that, as in WT mice, the PRC2-null Merkel cells were Ki67-negative (Figure 2e). Finally, we confirmed that apoptosis was not altered in the Merkel cells of P0 WT, Ezh1/2 2KO, EEDcKO, or Suz12cKO skin (Supplementary Figure S2h).
      In Ezh1/2 2KO mice, Merkel cell expansion is due to the derepression of key Merkel cell differentiation genes, Isl1 and Sox2, in epidermal progenitors (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ). We purified Suz12-null and EED-null epidermal progenitors by fluorescence-activated cell-sorting (FACS), performed semiquantitative real-time PCR (RT-qPCR), and confirmed increased transcription of Isl1 and Sox2 in knockout cells (Figure 2f). Therefore, we concluded that PRC2 represses the Merkel cell differentiation program in epidermal progenitors.

      Loss of PRC2 leads to defective postnatal development of hair follicles due to decreased proliferation and increased apoptosis

      So far, our analysis has revealed that the loss of PRC2 from embryonic epidermal progenitors leads to premature epidermal development and ectopic formation of Merkel cells. During development, embryonic epidermal progenitors also give rise to hair follicles. Interestingly, and in contrast to the epidermal and Merkel cell lineage phenotypes, the hair follicles of Ezh1/2 2KO mice never reached their full length (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). This striking difference between the roles of PRC2 in control of the epidermal/Merkel cell lineages and the hair follicle lineage led us to question whether defective formation of Ezh1/2-null hair follicles is indeed due to the loss of PRC2 or PRC2-independent functions of Ezh1/2. Therefore, we analyzed hair follicle development in Suz12-null and EED-null skins at a time point when WT hair follicles are fully formed.
      In P0 mice, when hair follicle formation is still ongoing, no alterations in hair follicle formation were observed in Ezh1/2 2KO, EEDcKO, and Suz12cKO mice compared with WT (Supplementary Figure S3a online). Like Ezh1/2 2KO (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ), EEDcKO and Suz12cKO mice were born alive, but were unable to eat and died soon after birth. To analyze hair follicle development, which is completed postnatally, we performed full-thickness grafting of P0 skins onto immunocompromised nude mice (Supplementary Figure S3b), as was done for the analysis of Ezh1/2-null hair follicles (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). Whenever possible, male donor skins were grafted onto female nude hosts, and fluorescence in situ hybridization for the Y-chromosome was used to detect the grafted male donor skins (Supplementary Figure S3c), as previously described (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ,
      • Nowak J.A.
      • Polak L.
      • Pasolli H.A.
      • Fuchs E.
      Hair follicle stem cells are specified and function in early skin morphogenesis.
      ).
      The hair follicles in grafted Suz12cKO and EEDcKO (gSuz12cKO and gEEDcKO) skin were significantly shorter than those in grafted WT (gWT) skin and lacked hair shafts 14 days after grafting (P14) (Figure 3a). Incomplete formation of the gSuz12-null and gEED-null hair follicles was confirmed by immunofluorescence analysis of Krt6 protein, which is expressed in the differentiated layers of the hair follicle. Although in both grafted gPRC2-null and gWT skin the hair follicles contained the early, Krt6(+), differentiated companion layer, the terminally differentiated medulla layer located at the center of the hair shaft was missing in the PRC2-null follicles (Supplementary Figure S3d). Additional characterization revealed that PRC2-null hair follicles were not cycling properly. By analyzing a later time point after grafting (P56), we observed that the gWT hair follicles had undergone one hair cycle, as shown by the presence of club hairs (Supplementary Figure S3g). In contrast, gEED-null hair follicles were arrested, and lacked club hairs (Supplementary Figure S3g), as was previously observed in the Ezh1/2 2KO (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). These data further confirm the inability of PRC2-null hair follicles to fully develop.
      Figure 3
      Figure 3Loss of PRC2 causes reduced proliferation and increased apoptosis in hair follicles. (a) Hematoxylin and eosin showing that P14 gPRC2-null mice have shorter hair follicles (HFs) than gWT; quantification of HF length (n ≥ 2; P < 0.0001). (b) BrdU staining showing reduced proliferation in the matrix (Mx) of P14 gPRC2-null HFs; quantification of percentage of BrdU(+) matrix cells (n = 3; gEEDcKO, P = 0.0083; gSuz12cKO, P < 0.0001). AE13 marks matrix limit. (c) Activated Caspase 3 (Casp3) staining showing increased apoptosis in P14 gPRC2-null HFs, labeled with E-Cadherin (Ecad); quantification of percentage of Casp3(+) HFs (n ≥ 2; gEEDcKO, P = 0.0055; gSuz12cKO, P = 0.0055). (d) RT-qPCR of FACS-purified outer root sheath (ORS) cells showing p15 (INK4B), p16 (INK4A), and p19 (ARF) upregulation in P14 gPRC2-null mice (mean ± SD; n = 3; all significant, P < 0.05). Scale bars: (a) 100 μm; (b, c) 25 μm. Statistical significance: significant *P = 0.01–0.05; very significant **P = 0.01–0.001; extremely significant ***P < 0.001. FACS, fluorescence-activated cell-sorting; PRC2, polycomb repressive complex 2; RT-qPCR, semiquantitative real-time PCR; SD, standard deviation.
      To determine the mechanisms involved in the arrested postnatal hair follicle phenotype, we first sought to analyze whether there were defects in the hair follicle stem cells. During normal postnatal development, hair follicle stem cells express critical transcriptional regulators Sox9 and Lhx2 (
      • Blanpain C.
      • Fuchs E.
      Epidermal homeostasis: a balancing act of stem cells in the skin.
      ,
      • Mardaryev A.N.
      • Meier N.
      • Poterlowicz K.
      • Sharov A.A.
      • Sharova T.Y.
      • Ahmed M.I.
      • et al.
      Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote epidermal regeneration after injury.
      ,
      • Nowak J.A.
      • Polak L.
      • Pasolli H.A.
      • Fuchs E.
      Hair follicle stem cells are specified and function in early skin morphogenesis.
      ,
      • Rhee H.
      • Polak L.
      • Fuchs E.
      Lhx2 maintains stem cell character in hair follicles.
      ,
      • Vidal V.P.
      • Chaboissier M.C.
      • Lutzkendorf S.
      • Cotsarelis G.
      • Mill P.
      • Hui C.C.
      • et al.
      Sox9 is essential for outer root sheath differentiation and the formation of the hair stem cell compartment.
      ). Immunofluorescence analysis revealed that both gWT and gPRC2-null hair follicles contained Sox9(+) and Lhx2(+) cells (Supplementary Figure S3e and f), suggesting that the hair follicle stem cells are not lost in the P14 gPRC2-null skin. We next analyzed the proliferation and apoptosis in hair follicle cells. Analysis of gSuz12-null and gEED-null hair follicles compared with the corresponding WT follicles revealed a drastic reduction in the percentage of BrdU(+) cells in the matrix, the lower portion of the hair follicle, which typically contains highly proliferative cells (Figure 3b). In addition, we observed an increased percentage of activated caspase 3(+) hair follicles in gSuz12-null and gEED-null back skin (Figure 3c), as was previously observed in the gEzh1/2-null skin (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). We concluded that the defective formation of PRC2-null hair follicles is due to decreased proliferation and increased apoptosis.
      RT-qPCR analyses of RNAs purified from FACS-isolated P14 gWT and gPRC-null hair follicle progenitors, localized in the outer root sheath, revealed strong upregulation of the INK4A/ARF/INK4B locus in knockout hair follicles (Figure 3d). This locus encodes the critical G1-S cell cycle inhibitors p15 (INK4b) and p16 (INK4a), and the apoptosis regulator p19 (ARF) (
      • Sherr C.J.
      Ink4-Arf locus in cancer and aging.
      ); it is also a direct target of PRC2 repression (
      • Bracken A.P.
      • Kleine-Kohlbrecher D.
      • Dietrich N.
      • Pasini D.
      • Gargiulo G.
      • Beekman C.
      • et al.
      The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells.
      ,
      • Chen H.
      • Gu X.
      • Su I.H.
      • Bottino R.
      • Contreras J.L.
      • Tarakhovsky A.
      • et al.
      Polycomb protein Ezh2 regulates pancreatic beta-cell Ink4a/Arf expression and regeneration in diabetes mellitus.
      ). Immunofluorescence staining for p19 confirmed its expression in gEED-null and gSuz12-null hair follicles (Supplementary Figure S3h). Importantly, these findings are consistent with the previously identified role for this locus in inducing defective proliferation and apoptosis in gEzh1/2-null hair follicles (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). In this study, proliferation defects in Ezh1/2-null hair follicle progenitor cells could be rescued, in vitro, by knocking down the INK4A/ARF/INK4B locus, suggesting that the derepression of this locus was responsible for the defective proliferation in vivo (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). We therefore concluded that PRC2 is required for proper hair follicle development because of its regulation of proliferation and apoptosis in the developing hair follicles. This regulation is carried out, at least in part, by repression of the INK4A/ARF/INK4B locus.

      Discussion

      Although PRC2 was first identified several decades ago, the role of this complex in the regulation of stem cell fate and differentiation of somatic tissues in vivo is still not well understood. Understanding how this complex functions in stem cells in vivo is of paramount importance, as a wide variety of human genomic studies have revealed the importance of the Polycomb proteins for different human diseases (
      • Perdigoto C.N.
      • Valdes V.J.
      • Bardot E.S.
      • Ezhkova E.
      Epigenetic regulation of epidermal differentiation.
      ,
      • Sauvageau M.
      • Sauvageau G.
      Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer.
      ).
      To define the role of PRC2 in the skin epithelium, we provided a systematic study comparing the function of all core PRC2 subunits in a mammalian somatic stem cell system. Our analysis revealed that the absence of any of the PRC2 components resulted in a complete loss of trimethylation of lysine 27 of histone H3 in the epidermis. This indicates that all core PRC2 subunits are essential for the establishment of this histone mark and is in accordance with previous observations in embryonic stem cells (
      • Leeb M.
      • Pasini D.
      • Novatchkova M.
      • Jaritz M.
      • Helin K.
      • Wutz A.
      Polycomb complexes act redundantly to repress genomic repeats and genes.
      ,
      • Pasini D.
      • Bracken A.P.
      • Hansen J.B.
      • Capillo M.
      • Helin K.
      The polycomb group protein Suz12 is required for embryonic stem cell differentiation.
      ). Our data further show that the conditional knockouts for any core PRC2 subunits are indistinguishable from each other, indicating that, EED, Suz12, and Ezh1/2 all function as PRC2 subunits in the skin epithelium. Finally, we show that the loss of PRC2 results in strikingly different outcomes across different skin lineages: premature acquisition of epidermal stratum corneum, ectopic Merkel cell formation, and defective postnatal hair follicle development (Figure 4). In addition, although the epidermal and Merkel cell lineage phenotypes are evident during embryogenesis and at birth, alterations in hair follicle development are observed postnatally, showing a difference in the timing of phenotype onset. The drastically different roles of PRC2 in the formation of the three lineages exemplify the complex outcomes that the lack of PRC2 can have in any given stem cell system.
      Figure 4
      Figure 4Loss of polycomb repressive complex 2 (PRC2) results in three distinct skin phenotypes. Schematic diagram showing how loss of PRC2 affects the development of different skin lineages. Embryonic epidermal progenitors give rise to the epidermis, Merkel cells, and hair follicles. In mice in which either Ezh1/2, EED, or Suz12 are deleted from the epidermal progenitors, there is premature formation of the stratum corneum, an increase in the number of Merkel cells, and defective postnatal hair follicle development due to decreased matrix proliferation and increased apoptosis of the hair follicles.
      Although ablation of the core PRC2 subunits in embryonic stem cells does not compromise their ability to self-renew (
      • Chamberlain S.J.
      • Yee D.
      • Magnuson T.
      Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency.
      ,
      • Leeb M.
      • Pasini D.
      • Novatchkova M.
      • Jaritz M.
      • Helin K.
      • Wutz A.
      Polycomb complexes act redundantly to repress genomic repeats and genes.
      ,
      • Pasini D.
      • Bracken A.P.
      • Hansen J.B.
      • Capillo M.
      • Helin K.
      The polycomb group protein Suz12 is required for embryonic stem cell differentiation.
      ), the vast majority of in vivo phenotypes resulting from the lack of PRC2 subunits in somatic stem cells are associated with inhibited proliferation. For example, conditional ablation of Ezh2 from embryonic cardiomyocytes results in lethal congenital heart malformations due to cardiac hypoplasia (
      • He A.
      • Ma Q.
      • Cao J.
      • von Gise A.
      • Zhou P.
      • Xie H.
      • et al.
      Polycomb repressive complex 2 regulates normal development of the mouse heart.
      ). Similarly, in hepatic progenitors, islet β-cells, hematopoietic stem cells, and astrocytes in the subventricular zone, loss of Polycomb repression results in decreased proliferation and an inability of the somatic stem cells to self-renew (
      • Aoki R.
      • Chiba T.
      • Miyagi S.
      • Negishi M.
      • Konuma T.
      • Taniguchi H.
      • et al.
      The polycomb group gene product Ezh2 regulates proliferation and differentiation of murine hepatic stem/progenitor cells.
      ,
      • Chen H.
      • Gu X.
      • Su I.H.
      • Bottino R.
      • Contreras J.L.
      • Tarakhovsky A.
      • et al.
      Polycomb protein Ezh2 regulates pancreatic beta-cell Ink4a/Arf expression and regeneration in diabetes mellitus.
      ,
      • Hidalgo I.
      • Herrera-Merchan A.
      • Ligos J.M.
      • Carramolino L.
      • Nunez J.
      • Martinez F.
      • et al.
      Ezh1 is required for hematopoietic stem cell maintenance and prevents senescence-like cell cycle arrest.
      ,
      • Hwang W.W.
      • Salinas R.D.
      • Siu J.J.
      • Kelley K.W.
      • Delgado R.N.
      • Paredes M.F.
      • et al.
      Distinct and separable roles for EZH2 in neurogenic astroglia.
      ). In all cases, these in vivo phenotypes are associated with the activation of the INK4A/ARF/INK4B locus, which triggers cell death and apoptosis in the PRC2-null cells. Our transcriptional profiling of FACS-purified cells from PRC2-null mice revealed upregulation of the cell cycle inhibitor INK4A/ARF/INK4B locus in the hair follicle progenitors, which resulted in cell cycle arrest and apoptosis. These data underline the importance of PRC2 in proper tissue homeostasis as a regulator of proliferation and apoptosis via the repression of the INK4A/ARF/INK4B locus. Importantly, alterations of this locus are a common cytogenic alteration in human cancers, whereas its upregulation has been associated with aging (
      • Kim W.Y.
      • Sharpless N.E.
      The regulation of INK4/ARF in cancer and aging.
      ). Therefore, it will be critical to better understand how PRC2 regulates the INK4A/ARF/INK4B locus in somatic stem cells.
      In addition, transcriptional profiling of PRC2-null epidermal cells revealed upregulation of key Merkel cell signature genes Isl1 and Sox2. These Merkel cell signature genes and the INK4A/ARF/INK4B locus are normal targets of PRC2 repression in wild type cells. However, the Merkel cell and the hair follicle phenotypes become evident at different developmental time points. It will be very interesting to further understand how the different cell signaling events and transcriptional programs specific to each lineage interact with PRC2-dependent regulation of gene repression to ensure proper cell fate specification during development.
      Not only does PRC2 have essential functions in stem cells and during development, but alterations in PRC2 function have been found in multiple types of cancer (
      • Perdigoto C.N.
      • Valdes V.J.
      • Bardot E.S.
      • Ezhkova E.
      Epigenetic regulation of epidermal differentiation.
      ,
      • Sauvageau M.
      • Sauvageau G.
      Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer.
      ). Understanding how PRC2 functions in the different systems and how a complex that regulates expression of a wide number of genes can be misregulated in different cancers will likely be extremely important to further understanding tumorigenesis and for the development of novel therapeutic approaches.

      Materials and Methods

      Mice

      All mice were housed and cared for according to the Icahn School of Medicine at Mount Sinai's Institutional Animal Care and Use Committee approved protocols. At least two animals were used for each analysis. Ezh1/2 2KO mice were previously reported (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). EED floxed and Suz12 floxed mice were generously provided by Weipeng Mu and Terry Magnuson (
      • Mu W.
      • Starmer J.
      • Fedoriw A.M.
      • Yee D.
      • Magnuson T.
      Repression of the soma-specific transcriptome by Polycomb-repressive complex 2 promotes male germ cell development.
      ). As previously described with Ezh1/2 2KO mice, mice null for EED or Suz12 die shortly after birth, and all analysis of these mice after P0 was performed on grafted skin obtained using the previously described full-thickness grafting protocol (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). Krt14-Cre and immunocompromised nude mice were obtained from The Jackson Laboratories. Mice were genotyped by PCR using DNA extracted from tail skin. BrdU was administered as previously reported (
      • Ezhkova E.
      • Pasolli H.A.
      • Parker J.S.
      • Stokes N.
      • Su I.H.
      • Hannon G.
      • et al.
      Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.
      ). Briefly, BrdU was administered (50 μg BrdU per 1 g mouse weight) to mice 3–5 hours before killing.

      Immunofluorescence and Y-chromosome florescence in situ hybridization

      Tissues were collected from mice, embedded fresh into Optimal Cutting Temperature (OCT) compound (Tissue-Tek, Torrance, CA), and subsequently cut into 5 μm or 10 μm sections. Slides were then fixed for 10 minutes in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA) and blocked for 1 hour or overnight in blocking solution (phosphate buffered saline-Triton with 1% bovine serum albumin, 0.25% normal goat serum and 0.25% normal donkey serum). Primary antibodies were diluted in blocking solution and incubations were carried out for 1 hour or overnight, followed by incubation in secondary antibodies for 1 hour at room temperature. Slides were then counterstained with 4′,6-diamino-2-phenylindole (DAPI) and mounted using antifade mounting media. Y-chromosome florescence in situ hybridization (FISH) analysis was performed as previously described (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ,
      • Nowak J.A.
      • Polak L.
      • Pasolli H.A.
      • Fuchs E.
      Hair follicle stem cells are specified and function in early skin morphogenesis.
      ) on OCT sections using a Cy3 Star*FISH detection kit (Cambio, Cambridge, UK).

      Barrier assay

      A whole-mount dye-exclusion epidermal barrier assay was performed as described (
      • Ezhkova E.
      • Pasolli H.A.
      • Parker J.S.
      • Stokes N.
      • Su I.H.
      • Hannon G.
      • et al.
      Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells.
      ,
      • Hardman M.J.
      • Sisi P.
      • Banbury D.N.
      • Byrne C.
      Patterned acquisition of skin barrier function during development.
      ). Briefly, unfixed, untreated, freshly isolated P0 mice were immersed in standard X-gal solution (1 mg/ml Xgal substrates in phosphate buffered saline with 100 mM NaPO4,1.3 mM MgCl2, 3 mM K3Fe(CN)6, and 3 mM K4Fe(CN)6; pH adjusted to 4.5) overnight, rotating, at 37°C.

      Microscopy and quantification

      Slides were imaged using a Leica DM5500 upright microscope and either ×10, ×20, or ×40 objectives or a Zeiss Axioplan2 microscope and ×40 or ×63 objectives. For each analysis, at least two mice per genotype were used for quantification (n ≥ 2). Fluorescence intensity was calculated from at least three raw, single-channel grayscale images per condition using the Leica LAS AF software. Fluorescence intensity was normalized to nonnuclear background. Quantifications of epidermal thickness were measured from the bottom of the basal layer to the top of the stratum corneum using the Leica LAS AF software. Quantifications of Merkel cells per mm of skin were performed as described (
      • Bardot E.S.
      • Valdes V.J.
      • Zhang J.
      • Perdigoto C.N.
      • Nicolis S.
      • Hearn S.A.
      • et al.
      Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells.
      ). Quantifications of hair follicle length were measured using the Leica LAS AF software; the numbers of hair follicles quantified were as follows: WT = 91; Ezh1/2 2KO = 24; EEDcKO = 46; Suz12cKO = 56. BrdU cells were quantified using the Leica LAS AF software or ImageJ; nuclear DAPI staining was used to quantify the total number of cells and the percentage of BrdU(+) cells was quantified. For the quantification of BrdU(+) matrix cells, the numbers of hair follicles quantified were as follows: EED WT = 16; EEDcKO = 17; Suz12 WT = 19; Suz12cKO = 18. The quantification of activated caspase 3(+) and (−) hair follicles was performed using a Leica DM5500 upright slide microscope, and positive hair follicles were presented as a percentage of the total counted hair follicles; the numbers of hair follicles quantified were as follows: EED WT = 466; EEDcKO = 1052; Suz12 WT = 454; Suz12cKO = 395.

      Fluorescence-activated cell sorting analysis

      FACS analysis was performed as previously described (
      • Ezhkova E.
      • Lien W.H.
      • Stokes N.
      • Pasolli H.A.
      • Silva J.M.
      • Fuchs E.
      EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair.
      ). Briefly, for gWT, gEzh1/2 2KO, gEEDcKO, and gSuz12cKO analysis, grafts were collected and subjected to 0.25% collagenase digestion for 90 minutes at 37°C, followed by 0.25% trypsin for 14 minutes at 37°C with moderate shaking. Cell suspension was passed through a cell strainer, and 2 million cells were then stained with Eprhin-B1 (1:20) followed by secondary SAV-FITC (1:1000), EpCAM-APC (1:200), α6-integrin-PE (1:100), and Sca1-PreCP-Cy5.5 (1:200). Interfollicular epidermis was sorted as EpCAM(+), Sca1(+), α6-integrin(+); outer root sheath was sorted as EpCAM(+), Sca1(−), α6-integrin(+), Ephrin(−). The dead cell population was excluded by DAPI staining. Sorting was performed on DB-FACS Aria II at 35 psi with a 100-μm nozzle, and 50,000 cells were collected directly into RLT+ buffer (Qiagen, Valencia, CA) with β-mercaptoethanol.

      RNA purification and RT-qPCR

      Sorted cells were lysed in RLT+ buffer (Qiagen, Valencia, CA) with β-mercaptoethanol, and RNA was isolated using the Qiagen RNeasy Mini kit with DNaseI treatment. Reverse transcription was performed using qScript (Quanta, Gaithersburg, MD) Superscript Supermix. All RT-qPCR was performed using Roche SYBR green reagents (Roche, Indianapolis, IN) and a Lightcycler480 machine. All primers are listed in Supplementary Table S1 online.

      Statistics

      In RT-qPCR column bar graphs, mean value ± standard deviation is presented. Comparisons were made between three cKO and at least three Cre(−) WT siblings, using Student’s t-test (GraphPad Prism 5). Box-and-whisker plots show first to third quartiles around the median, with whiskers showing the 5–95% range and outliers presented as individual data points. All quantifications were performed on multiple cell populations from at least two different animals (n ≥ 2). To determine the significance between WT and EEDcKO or WT and Suz12cKO in the quantifications of BrdU(+) matrix cells (Figure 3b) and caspase 3(+) hair follicles (Figure 3c), comparisons were made using Student’s t-test (GraphPad Prism 5). To determine the significance between groups in all other quantification experiments (as indicated in the figures by parentheses), comparisons were made using one-way ANOVA with the Bonferroni correction (GraphPad Prism 5). For all statistical tests, the P < 0.05 level of confidence was accepted for statistical significance.

      Antibodies

      Antibodies were used as follows: Krt14 (generous gift of Dr Segre, National Human Genome Research Institute, MD, USA, 1:20,000); H3 (abcam, ab1791, 1:10,000); trimethylation of lysine 27 of histone H3 (Millipore, 07-449, 1:300); Krt18 (abcam, ab668, 1:100); Krt20 (Dako, M7019, 1:70); Sox2 (Stemgent, 09-0024, 1:150); Isl1 (abcam, ab109517, 1:250); NF200 (abcam, ab8135, 1:1,000); Krt6 (generous gift of Dr Fuchs, The Rockefeller University, NY, USA, 1:250); Lhx2 (generous gift of Dr Fuchs, 1:5,000); Sox9 (generous gift of Dr Fuchs, 1:1,000); BrdU (abcam, ab6326, 1:250; abcam, ab1893, 1:250); AcCasp3 (R&D, AF835, 1:250); Krt10 (Covance, PRB-159P, 1:500); Loricrin (Covance, PRB-145P, 1:250); Filaggrin (generous gift of Dr Segre, 1:500); Integrin β4/CD104 (BD Biosciences, 553745, 1:500); Ki67 (Novocastra, NCL-L-Ki67-MM1, 1:250); AE13 (Abcam, ab16113, 1:100); E-Cadherin (Invitrogen, 131900, 1/2,000); p19/Arf (Abcam, Ab80, 1/200). For immunofluorescence, secondary Abs coupled to FITC, Alexa488, 549, 649, RRX, or Cy5 were from Jackson ImmunoResearch Laboratories (1:1,000). For FACS: anti-mEphrin-B1 (BAF473 R&D, 1:20), FITC-Streptavidin (554061 BD, 1:1000), Ep-CAM-APC (118214, BioLegend, 1:200), Ly-6A/E Sca1PerCP-Cy5.5 (45-5981-82, eBioscience, 1:200), CD49f-α6 integrin-PE (12-0495-83, eBioscience, 1:100). For western blot, TrueBlot AntiRabbit IgG horseradish peroxidase (Rockland, 18-8816-33, 1:10,000) was used as a secondary Ab.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We would like to thank Evan Bardot, Jose Silva, Julie Segre, and Elaine Fuchs for help, advice, critical suggestions, experimental input, and reagents,. We would also like to thank Weipeng Mu and Terry Magnuson for their generous gift of the Suz12 floxed and EED floxed mice (
      • Mu W.
      • Starmer J.
      • Fedoriw A.M.
      • Yee D.
      • Magnuson T.
      Repression of the soma-specific transcriptome by Polycomb-repressive complex 2 promotes male germ cell development.
      ). The Ezh1 mutant mice were generated at the Research Institute of Molecular Pathology (IMP, Vienna, Austria) by Donal O'Carroll (laboratory of Thomas Jenuwein) with the help of Maria Sibilia (laboratory of Erwin Wagner). We would like to thank Alexander Tarakhovsky for previously providing us with Ezh2 floxed mice. Microscopy was performed at the Microscopy CORE at the Icahn School of Medicine at Mount Sinai. KLD is a trainee of the NIDCR-Interdisciplinary Training Program in Systems and Developmental Biology and Birth Defects T32HD075735. CNP was supported by EMBO fellowship ALTF 552-2012. VJV is a Pew Latin American Fellow in the Biomedical Sciences, supported by The Pew Charitable Trusts. EE is an Ellison Medical Foundation New Scholar in Aging. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number R01 AR063724 and R01 AR069078. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by a New York State Stem Cell Science (NYSTEM) IDEA grant through New York State Department of Health (N11G-152).

      Author Contributions

      KLD, CNP, and EE designed the study. KLD, CNP, VJV, FJS, and IC performed the experiments. KLD, CNP, VJV, FJS, IC, and EE analyzed the data. KLD, CNP, and EE wrote the manuscript with input from all other authors.

      Supplementary Material

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