LSD1 controls a nuclear checkpoint in Wnt/β-Catenin signaling to regulate muscle stem cell self-renewal

Abstract

The Wnt/β-Catenin pathway plays a key role in cell fate determination during development and in adult tissue regeneration by stem cells. These processes involve profound gene expression and epigenome remodeling and linking Wnt/β-Catenin signaling to chromatin modifications has been a challenge over the past decades. Functional studies of the lysine demethylase LSD1/KDM1A converge to indicate that this epigenetic regulator is a key regulator of cell fate, although the extracellular cues controlling LSD1 action remain largely unknown. Here we show that β-Catenin is a substrate of LSD1. Demethylation by LSD1 prevents β-Catenin degradation thereby maintaining its nuclear levels. Consistently, in absence of LSD1, β-Catenin transcriptional activity is reduced in both MuSCs and ESCs. Moreover, inactivation of LSD1 in mouse muscle stem cells and embryonic stem cells shows that LSD1 promotes mitotic spindle orientation via β-Catenin protein stabilization. Altogether, by inscribing LSD1 and β-Catenin in the same molecular cascade linking extracellular factors to gene expression, our results provide a mechanistic explanation to the similarity of action of canonical Wnt/β-Catenin signaling and LSD1 on stem cell fate.

Graphical Abstract

Figure 1.

LSD1 regulates MuSCs self-renewal potential. (A) PAX7 and MyHC staining and percentage of PAX7+ cells after 48 h under myogenic differentiation conditions of CTRL SC and LSD1 SCiKO MuSCs. (B) PAX7 and MyHC staining and percentage of PAX7+ cells after 48 h under myogenic differentiation conditions of MuSCs treated with LSD1 inhibitors (OG-L002 and Pargyline). (C) Bodipy, PAX7, desmin and Oil Red O staining of cells after 48 h under myogenic differentiation conditions of CTRL SC and LSD1 SCiKO MuSCs. (D) CTX experimental setup. (E) Anti-Laminin staining on cryosections of regenerated TA muscles in CTRL SC and LSD1 SCiKO mice at 28 dpi. Quantification of the number of myofibers per mm2. (F) CSA distribution of muscle fibers in CTRL SC and LSD1 SCiKO mice TA cryosections at 28 dpi. (G) Anti-PAX7 and anti-Laminin staining on cryosections of regenerated TA muscles in CTRL SC and LSD1 SCiKO mice at 28 dpi. Quantification of the number of sublaminar PAX7+/Ki67- cells per mm2. (H) CTX experimental setup. (I) Anti-PAX7 and anti-Laminin staining on cryosections of regenerated TA muscles in Vehicle and OG-L002 treated mice at 21 dpi. Quantification of the number of sublaminar PAX7+/Ki67− cells per mm². (J) H&E and Oil Red O staining on cryosections of regenerated TA muscles in Vehicle and OG-L002 treated mice at 21 dpi. (K) Anti-Laminin staining on cryosections of regenerated TA muscles in Vehicle and OG-L002 treated mice at 21 dpi. Quantification of the number of myofibers per mm². Scale bars, 50 μm. n = 3 mice/genotype. n = 3 primary MuSC cultures/genotype. n = 3 primary MuSC cultures/treatments. Values are mean or percentage mean ± SEM. **P < 0.01 (Mann–Whitney–Wilcoxon test) ***P < 0.001 (Bonferroni test after one way-ANOVA).

Figure 2.

LSD1 SCiKO MuSCs maintain their regenerative potential after repeated injuries. (A) Repeated CTX experimental setup. (B) Laminin staining on cryosections of regenerated TA muscles in CTRL SC and LSD1 SCiKO mice at 7 and 28 dpiIII. Quantification of the number of myofibers per mm². (C) CSA distribution of muscle fibers in CTRL SC and LSD1 SCiKO mice TA cryosections at 28 dpiIII. (D) H&E and Oil Red O staining on cryosections of regenerated TA muscles in CTRL SC and LSD1 SCiKO mice at 28 dpiIII. (E) Anti-PAX7 and anti-Laminin staining on cryosections of regenerated TA muscles in CTRL SC and LSD1 SCiKO mice at 28 dpiIII. Quantification of the number of sublaminar PAX7 +/Ki67 – cells per mm². Scale bars, 50 μm. n = 5 mice/genotype. Values are mean ± SEM. *P < 0.05 (Mann–Whitney–Wilcoxon test).

Figure 3.

Lack of LSD1 increases MuSC pool upon injury. (A) EdU pulse labelling set up. (B) Cytometry-acquired quantification of EdU + MuSCs (ITGA7+) isolated from muscle 40 h post injury (hpi). (C) Percentage of PAX7 +/Ki67 + (violet), PAX7 +/Ki67 +/EdU + (red) and PAX7 +/Ki67 -/EdU – (green) MuSCs at 96 hpi was quantified per mm². Representative images of PAX7, Ki67 and EdU staining on cryosections of regenerated TA muscles. (D) Quantification of the total number of PAX7 + cells at 96 hpi per mm². Scale bars, 50 μm and 10 μm. n = 3 mice/genotype. Values are mean or percentage mean ± SEM. *P < 0.05 (Mann–Whitney–Wilcoxon test).

Figure 4.

Lack of LSD1 stimulates symmetric division. (A) Representative images of MuSC doublets. Anti MYF5 (yellow) and PAX7 (red) staining on EDL myofibers isolated from CTRL SC and LSD1 SCiKO mice and cultured for 42 h. Percentage of doublets of CTRL SC or LSD1 SCiKO MuSCs. Asymmetric determination = PAX7+/MYF5+; Symmetric determination = PAX7+/MYF5–. n = 6 mouse/genotype. Values are percentage mean ± SEM. **P < 0.001 (Mann–Whitney–Wilcoxon test). (B) Representative images of two MuSCs dividing with Wnt3a-beads at angles of ∼10° and 90°. Orthogonal orange lines depict major and minor axis, orange line points to Wnt3a-bead and shows the angle measured. Wnt3a-bead is highlighted with orange dashed circle. Scale bar, 10 μm. Rose plots illustrating the distribution of mitotic spindle angles in CTRL SC MuSCs (C) or LSD1 SCiKO MuSCs (D), dividing with Wnt3a-beads. n = number of cells analyzed. (E) Statistical comparison of the distribution of mitotic spindle angles in LSD1 SCiKO MuSCs to the one observed in CTRL SC MuSCs. ***P < 0.001 (Kolmogorov–Smirnov test).

Figure 5.

LSD1 demethylates β-catenin protein. (A) BCL-9 and β-catenin protein-protein interactions evaluated using in situ proximity ligation assay (PLA). Complexes visualized as red dots. Scale bar, 1 μm. Quantification of BCL-9/ β-catenin PLA assay on CTRL SC and LSD1 SCiKO cells after 6 h of Wnt3A treatment. Red dots were quantified in nucleus from at least 100 cells per condition. (B) Localization of β-catenin at the Core Enhancer region (CER) of MyoD gene locus after 72 h in myogenic differentiation medium (MDM). ChIP analysis was performed on shSCRA and shLSD1 cells with an anti-β-catenin antibody. Enrichment values were shown as fold difference relative to the NEG region. (C) LSD1 knockdown accelerated the turnover rate of endogenous β-catenin in shSCRA and shLSD1 cells after 72 h in MDM, in a time-course CHX treatment. (D) Loss of LSD1 function caused an increase in methylated β-catenin protein in the nucleus after 72 h in MDM. (E) Demethylation assay using methylated and non-methylated lysine 180 β-catenin peptides as substrate. LSD1 WT, LSD1 K661A and LSD1 K661A/W754A/Y761S and commercial recombinant LSD1 were incubated with β-CAT K180Me and β-CAT K180 and analyzed by western blot with anti KpanMe antibody. The streptavidin antibody was used to detect the β-catenin peptides, which were conjugated to biotin. Values are mean of at least three experiments. ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Bonferroni test after one way-ANOVA).

Figure 6.

β-Catenin is required for LSD1-mediated Wnt3A-spindle orientation. Rose plots depicting the distribution of mitotic spindle angle orientations in CTRL SC transfected with (A) shRNA control (shSCRA) and (B) shRNA against Ctnnb1 (shCTNNB1). (C) ***P< 0.001 in boxes indicate statistical significance calculated by multiple Kolmogorov–Smirnov tests against CTRL SC shSCRA or LSD1 SCiKO cells dividing with a Wnt3a-beads. Rose plots depicting the distribution of mitotic spindle angle orientations in LSD1 SCiKO transfected with (D) empty vector or (E) overexpressing wild-type β-catenin fused to mCherry (WT-β-cat-mCherry). (F) ***P< 0.001 in boxes indicate statistical significance calculated by multiple Kolmogorov–Smirnov tests against LSD1 SCiKO empty vector or CTRL SC cells dividing with a Wnt3a-beads. n = number of cells.

Figure 7.

LSD1 regulates β-catenin turnover in ESCs. (A) Western blot analysis of nuclear β-catenin protein in CTRL WT and 4 different LSD1 KO ESCs clones. (B) The TCF transcriptional activity of CTRL WT, β-Cat KO and LSD1 KO ESCs is shown as a ratio of TOP-FLASH to FOP-FLASH luciferase-mediated signals, when cultured for 6 h in the presence of Wnt3A. (C) Upon Wnt3A treatment, LSD1 KO ESCs displayed an accelerated turnover rate of nuclear β-catenin compared to CTRL WT ESC, in a time-course CHX treatment. Values are mean of at least three experiments. ± SEM. **P < 0.01, ***P < 0.001 (Bonferroni test after one way-ANOVA). Rose plots depicting the distribution of mitotic spindle angle orientations in (D) CTRL WT or (E) LSD1 KO ESCs. n = number of cells. (F) ***P< 0.001 in box indicates statistical significance calculated by multiple Kolmogorov-Smirnov tests against CTRL WT ESC dividing with a Wnt3a-beads.