Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells

Abstract

While the Polycomb complex is known to regulate cell identity in ES cells, its role in controlling tissue-specific stem cells is not well understood. Here we show that removal of Ezh1 and Ezh2, key Polycomb subunits, from mouse skin results in a marked change in fate determination in epidermal progenitor cells, leading to an increase in the number of lineage-committed Merkel cells, a specialized subtype of skin cells involved in mechanotransduction. By dissecting the genetic mechanism, we showed that the Polycomb complex restricts differentiation of epidermal progenitor cells by repressing the transcription factor Sox2. Ablation of Sox2 results in a dramatic loss of Merkel cells, indicating that Sox2 is a critical regulator of Merkel cell specification. We show that Sox2 directly activates Atoh1, the obligate regulator of Merkel cell differentiation. Concordantly, ablation of Sox2 attenuated the Ezh1/2-null phenotype, confirming the importance of Polycomb-mediated repression of Sox2 in maintaining the epidermal progenitor cell state. Together, these findings define a novel regulatory network by which the Polycomb complex maintains the progenitor cell state and governs differentiation in vivo.

Figure 1

Number of Merkel cells is increased in Ezh1/2 2KO epidermis. (A, B) Immunofluorescent (IF) staining of WT and Ezh1/2 2KO skin with antibodies against Merkel markers K18 (A) or K20 (B) shows increase in the number of K18(+) and K20(+) cells. Arrows indicate positively stained K18(+) and K20(+) cells. Skins counterstained with basal layer marker K5. Quantifications are provided at the right and show statistically significant increase in the number of Merkel cells (P0 WT/2KO K18 P<0.0001, n=127/150 mm, 310/1285 cells; P90 WT/2KO K18 P=0.0026, n=84/83 mm, 2/95 cells; P0 WT/2KO K20 P=0.0003, n=72/52 mm, 94/298 cells; P90 WT/2KO K20 P=0.0017, n=81/39mm, 5/33 cells). (C) IF studies with K18 and K20 antibodies show overlap of the two markers. (D) Confocal analysis of K20-labeled cells showing lack of overlap with anti-K5-labeled cells. (E, F) IF studies show overlap of K18 with Merkel cell protein Rab3c (E), and innervation of K18(+) cells by neurons, as labeled by NF200 (F). Numbers in (F) show similar levels of innervation in WT and Ezh1/2 2KO skin (WT/2KO P=0.8497, n=78/220 cells). (G) TEM imaging shows fully differentiated Merkel cells in the whisker area of P0 WT and Ezh1/2 2KO mice. Dotted lines outline Merkel cells. Arrows indicate characteristic dense core neuroendocrine granules, magnified at the right. TEM quantifications are shown at the right (WT/2KO P=0.0009, n=9/8.7 mm, 37/120 cells) and confirm increase in the number of Merkel cells in Ezh1/2 2KO skin. Scale bars for (A–F) are 50 μm. Scale bars for (G) are 1 μm.

Figure 2

Loss of Ezh1/2 leads to accelerated differentiation of epidermal progenitors to Merkel cells. (A) IF studies of P0 WT and Ezh1/2 2KO skin using antibodies against K18 and H3K27me3 show that K18(+) Merkel cells lack H3K27me3 signal. Fluorescence intensity quantification of H3K27me3 signal (arbitrary units) is shown at the right (WT versus 2KO epi P<0.0001, n=20/20 cells; WT versus 2KO K18 P<0.0001, n=13/14 cells). (B) IF studies of P0 WT and Ezh1/2 2KO mice expressing K14-GFP-H2B show that K18(+) cells are GFP(+). Relative fluorescence intensity quantification of K14-GFP-H2B signal is shown at the right and normalized to the average basal layer signal (WT K18 versus der P<0.0001, n=7; 2KO K18 versus der P<0.0001, n=6). (C, D) IF analysis of E16 (C) or P0 (D) WT and Ezh1/2 2KO skin using antibodies against K18 and the proliferation marker Ki67 show that Merkel cells are postmitotic. (E, F) IF studies of E16 WT and Ezh1/2 2KO skin using antibodies against K5 and either K18 (E) or K20 (F) showing the premature formation of Merkel cells in Ezh1/2 2KO skin. In all panels, epi: epidermis; der: dermis; HF: hair follicle; BL: basal layer; MC: Merkel cell. All scale bars are 50 μm.

Figure 3

The Merkel signature gene Sox2 is directly regulated by Ezh1/2-mediated repression in epidermal stem cells. (A) Venn diagram of Merkel cell signature genes with genes that are upregulated in Ezh1/2 2KO epidermal progenitors shows overlap of 78 genes. (B) Of those 78 genes, 55 (70.5%) are direct targets of H3K27me3 repression, and Sox2 is among the most upregulated. (C) qPCR confirmation of microarray data shows that Sox2 is 26-fold upregulated in Ezh1/2 2KO progenitors. (D) In vivo ChIP-qPCR confirmation of ChIP-seq data showing that Sox2 is directly targeted by the Ezh1/2-dependent H3K27me3 histone mark, and that the H3K27me3 ChIP signal is drastically decreased in total skin cells isolated from P0 Ezh1/2 2KO mice. Please note that total skin contained both epithelial and a small fraction of non-epithelial cells (melanocytes, immune cells, etc.) that were not targeted by the K14-Cre-mediated ablation of Ezh1/2. Thus, a small residual level of H3K27me3 at Sox2 is observed in 2KO cells. (E) IF studies of E16 and E17 WT skin using antibodies against K20 and Sox2 reveal that Sox2 is expressed prior to other Merkel cell markers. Quantifications are provided at the right and show statistically significant decrease in the number of Sox2+K20− cells throughout development (E15 versus E17 P=0.0002; E17 versus P0 P=0.0036; E15 n=109mm/0 Sox2(+)K20(+) cells/19 Sox2+K20− cells; E17 n=97/99/151; P0 n=68/74/26). (F) IF analysis of E15 WT and Ezh1/2 2KO skin using antibodies against Sox2 and ß4-integrin shows that Sox2 is expressed prematurely in Ezh1/2 2KO skin. Quantifications are provided at the right and show statistically significant increase in the number of Sox2(+) cells in 2KO versus WT (WT versus 2KO P=0.0013, n=90/83mm, 14/45 cells). All scale bars are 50 μm.

Figure 4

Sox2 is required for proper Merkel cell development. (A, B) IF analysis using antibodies against Sox2 and either K18 (A) or ß4-integrin (B) shows loss of Sox2 from Merkel cells but not from the dermal papilla. (C, D) IF using antibodies against either K18 (C) or K20 (D) shows dramatic reduction of Merkel cells from Sox2cKO skin. Quantifications are shown at right (WT versus Sox2cKO K20 P<0.0001, n=82/80mm, 156/16 cells; K18 P=0.0002, n=73/80mm, 112/5 cells). (E) TEM imaging (left) shows that few Merkel cells present are fully developed. Dotted lines outline Merkel cells. Arrows indicate characteristic dense core neuroendocrine granules, magnified at right. TEM quantifications are shown at right and confirm drastic decrease in the number of fully differentiated Merkel cells present in the whisker area of P0 Sox2cKO mice (WT versus Sox2cKO P=0.0003, n=9/9.5mm, 37/3 cells). (F) Decrease in the number of fully differentiated Merkel cells in Sox2cKO persists into adulthood as shown by IF analysis of K20. Quantifications are provided at right (WT versus Sox2cKO P=0.0163, n=99/95mm 15/2 cells). (G) Analysis of apoptosis using antibodies against Act-Casp3 and K20 shows no difference between WT and Sox2cKO skin. All scale bars are 50 μm.

Figure 5

Sox2 regulates expression of Atoh1, a critical regulator of Merkel cell specification. (A) Expression of the obligate Merkel cell transcription factor Atoh1 is reduced in total skin isolated from P0 Sox2cKO mice as measured by RT-qPCR. (B) Overexpression of Sox2 in WT epidermal stem cells was confirmed by western blot. Histone H3 serves as a loading control. (C) Sox2-overexpressing cells had a dramatic increase in the level of Atoh1 mRNA and a slight upregulation of K20 mRNA, while expression of other Merkel cells markers remained unchanged. (D) ChIP studies on Sox2-overexpressing cells showed Sox2 binding at the Atoh1 enhancer region and the known Sox2 autoregulatory site. ChIP-qPCR signal is normalized to experimental control cells infected with an empty vector. (E) Concomitant expression of Sox2 and Atoh1 is observed throughout Markel cell development, as shown by IF studies of Sox2 on Atoh1-GFP back skin samples. (F) As with Sox2, Atoh1 is an early marker of Merkel cell differentiation as shown by lack of co-staining between Atoh1-GFP and K20 at E16. (G) IF studies show that the presence of Atoh1-GFP(+) cells is dependent on Sox2, as shown by decrease in the number of Atoh1-GFP+ cells at P0 and their absence at P35. Quantifications are provided at right (P0 WT versus Sox2cKO P=0.0283, n=103/86mm 226/144 cells. P35 WT versus Sox2cKO n=103/92mm 16/0 cells). All scale bars are 50 μm. Source data for this figure is available on the online supplementary information page.

Figure 6

Loss of Sox2 attenuates the Ezh1/2 2KO Merkel cell phenotype. (A) TEM imaging showing fully developed Merkel cells in Ezh1/2 Sox2 3KO skin. Dotted lines outline Merkel cells. Arrow indicates dense core neuroendocrine granules shown in the magnified region at right. TEM quantifications shown at the right indicate the reduction in Merkel cells in Ezh1/2 Sox2 3KO skin relative to Ezh1/2 2KO (WT/3KO P=0.0003, 3KO n=9/5.8mm 37/13 cells). (B, C) IF analysis of Ezh1/2 Sox2 3KO skin using antibodies against either K20 (B) or Rab3c (C) confirms TEM data showing the reduction in Merkel cell number relative to Ezh1/2 2KO skin. Quantifications are shown at the right (K20 2KO versus 3KO P=0.0033, n=24/53mm 119/5 cells; Rab3c 2KO versus 3KO P=0.0002, n=56/64mm 128/10 cells). (D) Model. The Polycomb complex is responsible for the direct repression of Sox2 and Sox2-regulated Merkel cell genes (Atoh1, Isl1, others) to repress the Merkel cell differentiation program in epidermal stem cells. Scale bars for (A) are 1 μm, all others are 50 μm. WT: wild type; 2KO: Ezh1/2 2KO; 3KO: Ezh1/2 Sox2 3KO.