The RNA-methyltransferase Misu (NSun2) poises epidermal stem cells to differentiate

Abstract

Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate.

Figure 1. Loss of Misu results in block of tRNA methylation and causes reduced body weight and partial alopecia.

(A) Gross phenotype of wild type, Misu +/− and −/− neonates. (B) Average weight of Misu −/− mice is reduced compared to controls. (C,D) Pelage of 10 months old wild-type (C) and Misu −/− mouse (D). Error bars represent standard deviation (SD). Wt: wild-type; +/−: heterozygous mice; −/−: homozygous mice. (E) Bisulfite sequencing of intron-containing tRNA^{Leu (CAA)} from wild-type and Misu −/− mouse skin. The diagram shows the methylation status of tRNA^{Leu (CAA)} transcribed from two different genes: tRNA-988^{Leu (CAA)} (top panel) and gene tRNA-1911^{Leu (CAA)} (bottom panel). Black boxes indicate methylated cytosine residues and white boxes indicate unmethylated cytosine residues. Numbers above boxes indicate the cytosine positions in the primary tRNA sequence amplified (nucleotides 29–36+intron sequence). Asterisk indicates the position of the expected methylated cytosine. Numbers+I indicate the cytosine positions in the intron sequence.

Figure 2. Expression of Misu during embryogenesis, skin morphogenesis, and in adult epidermis.

(A–F) LacZ staining (blue) in wild-type (wt) and Misu (+/−) embryos at E3.5 (A), E6.5 (B,B′), E9.5 (C), E13.5 (D) and E14.5 (E,F). HB: hindbrain; FB: forebrain; Br: branchial arch; H: heart; LB: limbs; YS: yolk sac. (B′) shows a section from the embryo whole mount in (B). (D,E) Arrows show LacZ staining in whiskers. Arrowheads indicate Misu expression in the eye. (G–I) LacZ staining of Misu (+/−) interfollicular epidermis at E15.5 (G), E16.5 (H) and E18.5 (I). (J,K) Misu-expression in the interfollicular epidermis after birth at P1 (J), P4 (K). (L,M) LacZ staining of hair follicles at P1 (L) and P4 (M). Dotted lines delineate the basement membrane in (J,K) and hair follicles in (L,M). Arrows in (J,K) indicate suprabasal layers of the interfollicular epidermis. Arrowhead in (M) indicate Misu-expression in the bulb of growing hair follicles. (N–R) Expression of Misu in adult skin is absent in telogen (N), low expressed in the bulge (Bu; arrows) and hair germ (HG; arrowhead) at early anagen (O) and high expressed in anagen hair follicles (P,Q). (R) mRNA levels of endogenous Misu during morphogenesis (M), catagen (C), telogen (T) and anagen (A). The grey box indicates anagen. Error bars indicate SEM (n = 2). Sections are counterstained with Eosin in (G–I, L, N–Q). Scale bars: 50 µm (G–Q).

Figure 3. Misu is a down-stream target gene of the Lef1/β-Catenin complex in adult skin.

(A–B) QPCR for c-Myc (A) and Lef1 (B) using total RNA isolated from back skin of wild-type mice during hair morphogenesis and the first hair cycle after birth. M; morphogenesis, C; catagen, T; telogen and A; anagen. The grey box marks anagen. Error bars represent SEM (n = 3). (C–E) Localisation of Lef1 protein (red) in the bulge (B, arrowheads) and hair germ (HG) at telogen (C), onset of anagen (D) and anagen (E). (F,G) Co-localisation of Misu (LacZ) and Lef1 (brown) in the lower bulge (B, arrowheads) and hair germ (HG) at the onset of anagen (F) and in the matrix later in anagen (G). DP indicates the dermal papilla. (H) Schematic overview of the Misu promoter indicating the putative Lef1-binding site, the TATA box and the transcriptional start site (TSS) for Misu. The red line indicates the PCR fragment amplified after ChIP. (I) Enrichment of promoters of Misu and as positive control Msx2 by ChIP using an antibody for Lef1. IgG and Gapdh served as negative controls. (J) Luciferase reporter assays using 2 kb of the mouse proximal promoter of Misu in Hela cells in the absence and presence of β-Catenin, Lef1 or ΔN63-Lef1. pLef7-fos-luc synthetic promoter was used as a positive control for Lef1/β-Catenin activity. Error bars represent SD (n = 3). (K) RNA levels of Misu in wild-type and K14-ΔLef1 skin. Nuclei are counterstained with DAPI (blue) (C–E). Scale bars: 20 µm (C–E). Scale bar: 25 µm (F,G).

Figure 4. Misu is expressed in bulge stem cells.

(A,B) Co-localisation of LacZ and keratin 15 (K15) (A) and P-cadherin (PCad) (B) in Misu +/− hair follicles. Arrows in (A) indicate K15/LacZ double positive cells. (C–E) Expression of Misu protein (green) in bulge (arrows) and hair germ (HG; arrowheads) at telogen (C), onset of anagen (D) and anagen (E). Bulge cells are labelled for K15 (red) and nuclei are counter stained with DAPI (blue) (C–E). (F–K) Percentage of FDG^+ve sorted epidermal cells in the bulge (Itgα6^high/CD34^+ve) (F–H) and the hair germ (Itgα6^low/P-cadherin^high) (I–K) at P21 in Misu +/− mice. (F,I) Cells were first gated by side scatter (SSLin) versus high expression of CD34 (F) or P-cadherin (I). The cells were further gated for Itgα6^high/FDG^+ve (G,H) and Itgα6^low/FDG^+ve (J,K). The percentage of Itgα6^high/CD34^+ve/FDG^+ve and Itgα6^low/P-cadherin^high/FDG^+ve cells is marked by the red squares (G,H,J,K). Wild-type cells were negative for FDG (G,J). Errors represent SEM (n = 3). (L–P) QPCR using FDG^+ve and FDG^−ve epidermal cells sorted from Misu +/− mice at P21 as indicated in Figure S6 for Misu (L), CD34 (M), NFATc1 (N), Lgr5 (O), and Lef1 (P). Error bars represent SEM (n = 3). Scale bars: 50 µm (A–E).

Figure 5. Misu delays cell cycle entry of bulge stem cells at the onset of anagen.

(A) Schematic representation of the first postnatal hair cycle after morphogenesis (M) including catagen (C), telogen (T) and anagen (A). Postnatal days (PD) are indicated on the right. Red marks the transition of telogen to anagen at P21. (B–I) Cell cycle profiles (B,D,F,H) and quantification (C,E,G,I) of whole epidermal preparations (B,C), Itgα6^high/CD34^+ve bulge stem cells (D,E), Itgα6^low/P-cadherin^high hair germ cells (F,G) and Itgα6^high/CD34^+ve bulge stem cells at P24 (H,I) using a minimum of three wild-type and three Misu −/− mice. Error bars represent SEM.

Figure 6. Deletion of Misu increases dormancy of bulge stem cell.

(A,B) Whole mount staining of wild-type and Misu −/− tail skin for LRC (green), keratin 14 (K14) (red) and DAPI (blue) after 4 months chase period. (C) Automated quantification of LRC in tail outer hair follicles in (A,B). (D) Frequency distributions of the intensity of the BrdU-label in LRC in (A,B). Error bars represent SEM (n = 10) from at least 3 independent experiments. (E,F) Flow cytometry for Itgα6 and CD34 in epidermis in telogen (P49) (E) and catagen (P40) (F). The percentage of double positive cells in each group is shown ± SEM (n = 3). The red lines in (E) indicate a cell population that increases in Misu −/− skin in telogen. (G,H) Epidermal cells sorted for CD34^+ve and low (L) and high (H) levels of Itgα6 were subjected to QPCR for bulge and hair germ markers as indicated (G). RNA levels were measured relative to GAPDH and presented as fold enrichment in Misu −/− mice versus wild-type controls. Error bars represent SEM (n = 3). (I) BrdU labelling and chasing regime to measure migration of LCR from bulge to hair germ at telogen at P47 (arrow). (J,K) Detection of LRC (green), Ki67 (red) and DAPI (blue) in high and low part of the bulge (straight line) and hair germ (HG) in hair follicles, indicated by dotted line, from wild-type (wt) (J) and Misu −/− mice (K). (L) Automated quantification of LRC in the whole hair follicle (total), high and low bulge region and the hair germ. Error bars indicate SEM (n = 5). (M) Percentages of hair follicles without LRC in the lower bulge region. Scale bars: 250 µm (A,B); 50 µm (J,K).

Figure 7. Lack of Misu affects self-renewal of epidermal stem cells.

(A,B) Colony forming efficiency of flow-sorted bulge stem cells (A) and 5×10³ or 10⁴ whole epidermal cells isolated from wild-type (wt) and Misu −/− back skin (B). Error bars represent SD (n = 3). (C) Representative culture dishes from (B). (D) Confocal images of wild-type and Misu −/− primary keratinocytes in culture stained for Misu (green) and DAPI (blue). (E–J) Ki67 staining (E,H) and Haematoxylin and Eosin staining (F,G,I,J) of epidermal sections taken at them same regions of dorsal skin at indicated postnatal days obtained from anagen in Misu −/− and wild-type mice. (K–M) Percentages of male wild-type and Misu-depleted males in the first adult anagen (K), the second adult anagen (L), and the first anagen in K14Misu^Δ/Δ and Misu^f/f mice (M) at time points indicated. Detailed numbers of animals are summarized in Table S1. (N–U) Delayed expression of signalling molecules in growing hair follicles of Misu −/− mice compared to wild-type littermates at P25: Lef1 (green) (N,O), hair keratin 72 (green) and Dlx3 (red) (P,Q), phospho-Smad1/3/5 (green) (R,S) and hair keratin 31 (green) and Gata3 (red) (T,U). Nuclei are counter stained with DAPI (blue). Insert in (O) shows higher magnification of matrix cells. Arrows in (I) mark dermal papilla and arrows in (N–U) indicate matrix cells. Scale bars: 50 µm (D), 200 µm (E–J) and 100 µm (N–U).