Polycomb repressive complex 2 in adult hair follicle stem cells is dispensable for hair regeneration

Abstract

Hair follicle stem cells (HFSCs) are multipotent cells that cycle through quiescence and activation to continuously fuel the production of hair follicles. Prior genome mapping studies had shown that tri-methylation of histone H3 at lysine 27 (H3K27me3), the chromatin mark mediated by Polycomb Repressive Complex 2 (PRC2), is dynamic between quiescent and activated HFSCs, suggesting that transcriptional changes associated with H3K27me3 might be critical for proper HFSC function. However, functional in vivo studies elucidating the role of PRC2 in adult HFSCs are lacking. In this study, by using in vivo loss-of-function studies we show that, surprisingly, PRC2 plays a non-instructive role in adult HFSCs and loss of PRC2 in HFSCs does not lead to loss of HFSC quiescence or changes in cell identity. Interestingly, RNA-seq and immunofluorescence analyses of PRC2-null quiescent HFSCs revealed upregulation of genes associated with activated state of HFSCs. Altogether, our findings show that transcriptional program under PRC2 regulation is dispensable for maintaining HFSC quiescence and hair regeneration.

Fig 1

HFSCs are maintained upon PRC2 loss (A) Schematic illustrating expected tdtomato expression in the HF bulge of K15-CrePGR; Rosa^CAG-tdtomato mice. (B) Schematic showing the experimental strategy to induce K15-CrePGR activity in telogen II (P50) HFs. (C) IF analyses of tdtomato (red) and DAPI (blue) in K15-CrePGR; Rosa^CAG-tdtomato mice showing efficient targeting of the HF bulge. (D) IF analysis of H3K27me3 (red), CD34 (green) and DAPI (blue) in HFSCs of P70 control and Eed iKO mice. H3K27me3 single channel is shown in gray. Bulge (Bu) and hair germ (HG) regions of the follicle has been outlined in yellow. (E) Fluorescence intensity quantification of H3K27me3 signal (arbitrary units) in control and Eed-null HFSCs P<0.00001, n = 120 cells from 15 HF sections from three independent biological replicates for each group. (F-J) IF analyses of (F) SOX9 (red) and DAPI (blue), (G) NFATC1 (red) and DAPI (blue), (H) activated-CASPASE3 (red), CD34 (green) and DAPI (blue), (I) Ki67 (red), CD34 (green) and DAPI (blue) and (J) LEF1 (red), CD34 (green) and DAPI (blue) in HFs of P70 control and Eed iKO mice. LEF1 single channel is shown in gray. All IF experiments in this figure was conducted on skin sections collected from three animals for each group from two independent litters. Scale bar: 10μm.

Fig 2

Loss of PRC2 leads to expression of a subset of anagen enriched genes (A) FACS strategy for isolating HFSCs from the back skin of mice. (B) RT-qPCR analysis of Eed mRNA in HFSCs isolated from control and Eed iKO mice. n = 3. ***p-value < 0.001; Data are mean ± SE. Three biological replicates for each group were used from at least two independent litters. (C) Volcano Plot depicting the differentially expressed genes in FACS-purified Eed iKO HFSCs vs corresponding control. Genes with absolute fold change ≥ 1.5 and adjusted p-value < 0.05 were considered significantly upregulated or downregulated for further analysis. RNA-seq analysis was done on three biological replicates for each group from at least two independent litters. (D) Graph depicting RNA-seq analysis of H3K27me3 demarcated HF-TACs genes in Eed iKO HFSCs. Fold change of these genes was not significant. (E) Gene set enrichment analysis of genes differentially expressed in Eed iKO HFSCs with anagen or telogen enriched genes. There was significant enrichment with anagen enriched gene set with p-value <0.05. Anagen and telogen enriched gene dataset was obtained by conducting RNA-seq on FACS purified HFSCs of six P28-30 and P50-52 animals, respectively. (F) RT-qPCR analysis of anagen enriched genes and H3K27me3 targets that are upregulated in HFSCs of Eed iKO mice. Three biological replicates for each group were used from at least two separate litters. ***p-value < 0.001, **p-value < 0.01; Data are mean ± SE. (G) Schematic showing the experimental strategy to ablate Eed in quiescent HFSCs in telogen II (P50) HFs. (H) Images of back skin of control and Eed iKO mice on P50 (day 1 of treatment) and P70 (day 20 post treatment). (I) Hematoxylin and Eosin (H&E) analysis of back skins from P70 control and Eed iKO mice. Analysis was done on three independent biological replicates for each group from two separate litters. Scale bar for H&E: 50μm.

Fig 3

HFSCs lacking PRC2 progress normally through hair cycle and form new HF bulge (A) Schematic showing the experimental strategy to ablate Eed in quiescent HFSCs in telogen I HFs. (B) Images of back skin of control and Eed iKO mice on P19 (day 1 of treatment) and P52 (day 27 post treatment). (C) H&E analysis of P52 control and Eed iKO back skin. Club hairs are indicated by arrows. (D) IF analysis of H3K27me3 (red), CD34 (green) and DAPI (blue) in HFSCs of P52 control and Eed iKO mice. H3K27me3 single channel is shown in gray. (E) Fluorescence intensity quantification of H3K27me3 signal (arbitrary units) in control and Eed-null HFSCs P<0.0001, n = 100 cells from 10–12 HFs from at least three independent biological replicates. (F) IF analyses of SOX9 (red) and DAPI (blue) in HFSCs of P52 control and Eed iKO mice. (G) Schematic showing the experimental strategy to ablate Eed in telogen I (P19) HFSCs followed by waxing (depilation) at P50 to induce regeneration. (H) Images of waxed back skin of control and Eed iKO mice on day 0, day 10, and day 22 after waxing. (I) H&E analysis of skins collected 22 days post waxing from control and Eed iKO back skin. (J) IF analysis of H3K27me3 (red), CD34 (green) and DAPI (blue) in HFSCs of 22 days post waxed skin from control and Eed iKO mice. H3K27me3 single channel is shown in gray. (K) IF analyses of SOX9 (red) and DAPI (blue) in HFSCs of P72 control and Eed iKO mice. All reported experiments in this figure was conducted on three biological replicates from two separate litters. Scale bar for H&E: 50μm, Scale bar for IF: 10μm.