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In vivo transcriptional governance of hair follicle stem cells by canonical Wnt regulators

Nature Cell Biology volume 16, pages 179–190 (2014)Cite this article

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Abstract

Hair follicle stem cells (HFSCs) regenerate hair in response to Wnt signalling. Here, we unfold genome-wide transcriptional and chromatin landscapes of β-catenin–TCF3/4–TLE circuitry, and genetically dissect their biological roles within the native HFSC niche. We show that during HFSC quiescence, TCF3, TCF4 and TLE (Groucho) bind coordinately and transcriptionally repress Wnt target genes. We also show that β-catenin is dispensable for HFSC viability, and if TCF3/4 levels are sufficiently reduced, it is dispensable for proliferation. However, β-catenin is essential to activate genes that launch hair follicle fate and suppress sebocyte fate determination. TCF3/4 deficiency mimics Wnt–β-catenin-dependent activation of these hair follicle fate targets; TCF3 overexpression parallels their TLE4-dependent suppression. Our studies unveil TCF3/4–TLE histone deacetylases as a repressive rheostat, whose action can be relieved by Wnt–β-catenin signalling. When TCF3/4 and TLE levels are high, HFSCs can maintain stemness, but remain quiescent. When these levels drop or when Wnt–β-catenin levels rise, this balance is shifted and hair regeneration initiates.

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Figure 1: HFSCs are Wnt-responsive, but do not require β-catenin to exist in a quiescent, undifferentiated state.
Figure 2: Bulge HFSCs can proliferate in vitro without β-catenin.
Figure 3: Engrafted β-catenin-deficient bulge HFSCs make only epidermis and not hair.
Figure 4: β-catenin-deficient bulge HFSCs can become activated in their native niche, but progress to make the wrong fate choice.
Figure 5: β-catenin influences expression of Wnt-responsive genes in bulge HFSCs that are first activated in the hair germ at anagen onset.
Figure 6: Co-occupancy of TCF3 and TCF4 on the bulge HFSC genome.
Figure 7: Within the stem cell niche, elevated TCF3 impairs bulge HFSC activation, while loss-of-TCF3/4 lowers the activation threshold.
Figure 8: TLE proteins act as co-repressors of TCF3/4 in quiescent bulge HFSCs.

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Acknowledgements

We thank S. Dewell for assistance in high-throughput sequencing (RU Genomics Resource Center), X. Guo for help in ChIP-seq analysis (Zheng laboratory), and S. Mazel, L. Li, S. Semova and S. Tadesse for FACS sorting (RU FACS facility). We also thank Fuchs’ laboratory members N. Stokes, D. Oristian and A. Aldeguer for assistance in mouse research (Fuchs laboratory); A. Rodriguez-Folgueras for assistance in confocal microscopy; and T. Chen, A. Rodriguez-Folgueras, S. Luo and B. Keyes for discussions and comments. W-H.L. was supported by a Harvey L. Karp Postdoctoral Fellowship and a Jane Coffin Child Fellowship. E.F. is an HHMI Investigator. This work was supported by a grant (to E.F.) from the NIH/NIAMS (R01AR31737) and partially by NIH/NIMH (R21MH099452, R01MH073164, D.Z.).

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  1. Wen-Hui Lien

    Present address: Present address: de Duve Institute and Université Catholique de Louvain, B-1200 Brussels, Belgium,

Authors and Affiliations

  1. Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology and Development, The Rockefeller UniversityNew York New York 10065 USA,

    Wen-Hui Lien, Lisa Polak, Kenneth Lay & Elaine Fuchs

  2. Department of Genetics, Albert Einstein College of MedicineBronx New York 10461 USA,

    Mingyan Lin & Deyou Zheng

  3. Department of Neurology, Albert Einstein College of MedicineBronx New York 10461 USA,

    Deyou Zheng

  4. Department of Neuroscience, Albert Einstein College of MedicineBronx New York 10461 USA,

    Deyou Zheng

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Contributions

W-H.L. and E.F. designed experiments, analysed data and wrote the paper. W-H.L. performed all experiments, collected all data and prepared the figures. L.P. carried out the grafting and lentiviral injection procedure. M.L. analysed RNA-seq data. D.Z. performed ChIP-seq and other bioinformatics analyses. K.L. assisted in performing some experiments during the revision. E.F. supervised the study.

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Correspondence to Elaine Fuchs.

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Integrated supplementary information

Supplementary Figure 1 Hair cycle.

(a) During the resting phase (telogen), HFSCs residing in the bulge (Bu) remain in quiescence as the terminally differentiated inner bulge cells express high levels of inhibitory signals (e.g. BMP, FGF18). At the onset of the regenerative phase (anagen), activated HFSCs located in hair germ (HG) proliferate and initiate HF regeneration in response to the activating cues (e.g. BMP inhibitors and Wnts) produced from crosstalk with the underlying mesenchymal stimulus, referred to as the dermal papilla (DP). Soon after, HFSCs in the bulge are also activated. Some activated HFSCs move downward from the bulge along the outer layer of HFs (ORS), creating an inverse gradient of proliferative cells that fuel the continued production of most proliferative, transient amplifying matrix cells (Mx) at the base of full anagen HFs. In response to high levels of Wnt signaling, matrix progenitors in the pre-cortex region (Pre-co) terminally differentiate to form the hair shaft (HS). At the end of anagen, HFs enter a destructive phase (catagen) and the matrix and much of the lower part of the HF undergo apoptosis. As the epithelial strand regresses, DP is drawn upward towards the bulge/HG and the HF re-enters telogen. IFE, interfollicular epidermis; SG, sebaceous gland.

Supplementary Figure 2 Wnt-responsive Bu-HFSCs resemble HG HFSCs that first get activated and proliferate at the transition.

(a) FACS-Gal sorting scheme to purify early anagen Bu-HFSCs. After sorting for Integrin α6 and CD34 surface expression, Bu-HFSCs were further fractionated into Wnt-responsive (LacZ+) and non-responsive (LacZ-) populations. (b) EdU and immunolabeling show that HG cells at the bulge base and in closest proximity to the dermal papilla (DP) stimulus, are the first to proliferate at the telogen→anagen transition. They expand and give rise to the transit-amplifying (TA) matrix; several days later, cells within the bulge begin to proliferate (arrow). Scale bar represents 50 μm.

Supplementary Figure 3 YFP(+) β-cat cKO HFSCs fail to activate at anagen onset, but remain expressing HFSC stemness genes.

(a) Immunolabelling shows that at telogen→anagen when the first sign of proliferation (arrow) is seen in YFP(+) control hair germ cells, YFP(+) β-cat cKO HFSCs remain quiescent. Quantifications of EdU-labeling experiments are at bottom. Scale bar represents 50 μm. Data are reported as average + SD. n=3; **, p<0.01. (b) FACS analysis for YFP(+)/CD34(+) Bu-HFSCs reveals comparable percentages of control (β-cat Het) and β-cat cKO (β-cat cKO) Bu-HFSCs among HFs from eight different pairs of littermates. n=8; NS, not significant. (c) Real-time PCR analysis for HFSC stemness genes. FACS-purified telogen-phase Bu-HFSCs from Het and β-cat cKO mice (10d post RU486-induced ablation). Values are normalized to β-cat Het Bu-HFSC mRNAs. Data are reported as average + SD. n=3. Error bars for qPCR calculated from technical triplicates. Similar results were reproduced in 3 independent experiments.

Supplementary Figure 4 Genes that are differentially expressed depending upon β-catenin status.

(a) qPCR of telogen-phase Bu-HFSCs confirms RNA-seq data from Telo→Ana (see Fig. 5) showing that upon β-catenin ablation, a cohort of genes are also downregulated in quiescent Bu-HFSCs lacking β-catenin. (b) qPCR confirms that upon β-catenin ablation, a smaller subset of genes are upregulated at Telo→Ana within Bu-HFSCs. Note that fatty acid metabolism genes are elevated in β-cat cKO Bu-HFSCs. (c) Depilation-activated β-cat cKO HFSCs show similar gene changes to those seen in Fig. 5b and 4b. Values in (a)-(c) are normalized to β-cat Het Bu-HFSC mRNAs. Data are reported as average + SD. n=3; *, p<0.05. Error bars for qPCR calculated from technical triplicates. Similar results were reproduced in (a) 3, (b) 3 and (c) 2 independent experiments.

Supplementary Figure 5 TCF3 and TCF4 co-target genes.

(a) LEF1 and TCF1 are expressed and nuclear in adult anagen HFs. Mx, matrix; Pre-Co, pre-cortex. Scale bar represents 50 μm. (b) The specificity and quality of TCF3 and TCF4 antibodies were validated by their pull-down ability in HFSC lysates relative to those pull-down with Tcf7l1-null or Tcf7l2-null skin cells, and to IgG controls. Immunoblotting analyses of immunoprecipitations reveal that sufficient amounts of TCF3 and TCF4 proteins in HFSC lysates were pulled down relative to IgG controls. (c) Genomic distribution of TCF3/4 ChIP-seq co-peaks relative to RefSeq transcripts. Promoter: within ±2kb of transcription start site. Downstream: within ±2kb of transcription end site. Gene proximal: within upstream 2-50kb of transcription start site. Gene distal: beyond upstream 50kb of transcription start site. (d) Comparison of the observed TCF3/4-bound peak distribution to the predicted distribution based on the genomic representation of each compartment. (e) Putative transcription factor binding motifs were discovered from de novo motif analysis using MEME-ChIP software59. The percentages of motif occurrences within TCF3/4-bound peaks versus random genomic sequences are indicated for each motif as determined by the program MAST (p<0.0005). p-values are determined by compared to the percentage observed in random sequences. In addition to Lef1/TCF motif, NFATc1 was the top known motif found at TCF3/4-bound peaks. (f) Most enriched gene ontology for the overlapping targets (n=1,126) of TCF3 targets in mESCs (n=3,591; green)28, and TCF3/4 targets in HFSCs (n=3,387; blue).

Supplementary Figure 6 Gain- and loss-of-function for TCF3 and TCF4.

(a) TCF3 overexpression. qPCR (left) and immunoblotting (right) analyses showing elevated TCF3 mRNA and protein in Bu-HFSCs following Doxy induction of K14rtTA/TRE-mycTCF3 mice. Values are normalized to K14rtTA Bu-HFSC mRNAs. Rps16, mRNA control; α-tubulin; protein loading control. (b) Depletion of TCF3 and TCF4 in HFSCs. E18.5 back skins of K15CrePGR/Tcf7l1fl/fl/Tcf7l2−/−/Rosa26−Y FPfl/stop/fl embryos were grafted onto nude mice, and 7 weeks later, when HFs were in telogen, RU486 was administered to generate double knockout (dKO) HFSCs. qPCR analysis with FACS-isolated Bu-HFSCs from RU486-treated K15CrePGR/Tcf7l1+/fl/Tcf7l2+/−/Rosa26−Y FPfl/stop/fl (TCF3/4 Het) and K15CrePGR/Tcf7l1fl/fl/Tcf7l2−/−/Rosa26−Y FPfl/stop/fl (TCF3/4 dKO) grafted skins. (Left) qPCR analysis demonstrates Tcf3 and TCF4 depletion. (Right) Immunostaining of grafts demonstrates Tcf4 depletion and Cre activation (YFP+). (c) Elevated TCF3 prohibits depilation-induced HFSC activation. Doxy was given throughout. One day after depilation, EdU was administered for 24 hours to identify proliferating cells. (Left) Immunostaining for EdU and Bu-HFSC marker CD34. (Right) Quantifications of EdU-labeling experiments. Note that HFs with increased TCF3 display reduced HFSC activation. Scale bars in (b) and (c) represent 50 μm. Data in (a)-(c) are reported as average + SD. n=3; **, p<0.01. Error bars for qPCR calculated from technical triplicates. Similar results were reproduced in (a) 5 and (b) 3 independent experiments.

Supplementary Figure 7 Co-occupancy of TCF3, TCF4 and TLE in quiescent bulge HFSCs.

(a) TLE occupies bulge HFSC DNA locations that are also bound by TCF3 and TCF4. Density maps of TCF3 (blue), TCF4 (orange), TLE (purple) ChIP-seq reads (x-axis, centered on TCF3 peak summits) at TCF3-bound peaks (Y-axis), broken into peaks in promoter and enhancer regions. (b) Overexpression of TLE4 in HFSCs. (Left) Lentiviral construct carrying Doxy-inducible TRE-Tle4 and H2BRFP genes was introduced to E9.5 K14rtTA embryos by in utero infection52, and at adulthood, Doxy was administered according to schematic. (Middle) A transduced (RFP+) HF. Scale bar represents 50 μm. (Right) qPCR analysis of FACS-isolated RFP(+) Bu-HFSCs from Doxy-induced mice. Note elevated Tle4 mRNA levels relative to control. Data are reported as average + SD. n=3; *, p<0.05. Error bars for qPCR calculated from technical triplicates. Similar results were reproduced in 3 independent experiments.

Supplementary Figure 8 Full scan of immunoblots.

Full scan of cropped immunoblot images in Figs, 2b, 8a, 5b and 6a are shown.

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Lien, WH., Polak, L., Lin, M. et al. In vivo transcriptional governance of hair follicle stem cells by canonical Wnt regulators. Nat Cell Biol 16, 179–190 (2014). https://doi.org/10.1038/ncb2903

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