by Ajoy Aloysius, Ramanuj DasGupta, and Jyotsna Dhawan The transcription factor Lef1 switches partners from β-catenin to Smad3 during muscle stem cell quiescence by Ajoy Aloysius, Ramanuj DasGupta, and Jyotsna Dhawan Sci. Signal. Volume 11(540):eaan3000 July 24, 2018 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

The Tcf/Lef transcriptional response is specific to G0 cells and does not require upstream components of canonical Wnt signaling. The Tcf/Lef transcriptional response is specific to G0 cells and does not require upstream components of canonical Wnt signaling. (A and B) Quantification of Axin2 and Lef1 mRNAs by quantitative real-time polymerase chain reaction (qRT-PCR) in proliferating C2C12 myoblasts (MB), quiescent (G0) cells, and differentiated myotubes (MT). n = 3 independent experiments. (C) Flow cytometric analysis of tRFP intensity in cells stably expressing the Tcf/Lef reporter TOPtRFP (wild-type Tcf/Lef binding sites) or FOPtRFP (mutated Tcf/Lef binding sites) during proliferation (MB), quiescence (G0), and re-entry. n = 3 independent experiments. (D) Western blot showing endogenous Wnt-activated phosphorylation of LRP6 at Ser1490 (pLRP6) and total LRP6 (Tot LRP6) in MB, G0 cells, and MT. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is a loading control. Molecular mass (in kilodaltons) markers are noted. Blot is representative of three independent experiments. (E) Densitometry analysis for Western blots of pLRP6 in MB, G0 cells, and MT normalized to the total Lrp6 in each cell type. n = 3 independent experiments. a.u., arbitrary units. (F) Western blot showing activated (not phosphorylated at Ser37 and Thr41) β-catenin (Act β-catenin) and total β-catenin (Tot β-catenin) in MB, G0 cells, and MT. Blot is representative of four independent experiments. (G) Densitometry analysis for Western blots of active β-catenin in MB, G0 cells, and MT normalized to the total β-catenin in each cell type. n = 4 independent experiments. (H) Immunofluorescence showing activated β-catenin in MB, G0 cells, and MT. The boxed regions indicate the areas magnified in the high-magnification images (Zoom) in which the nuclei are outlined. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Image is representative of three independent experiments. Scale bars, 10 μm. (I) Box plot showing image intensity analysis of active β-catenin immunostaining in the nuclei of MB, G0 cells, and MT. Quantification is based on measurements of >100 nuclei from randomly selected fields of three independent experiments. (J) Western blot showing β-catenin phosphorylated (pβ-catenin) at Tyr654 and total β-catenin in MB, G0 cells, and MT. Blot of representative of three independent experiments. (K) Densitometry analysis for Western blots of phosphorylated β-catenin (at Tyr654) in MB, G0 cells, and MT normalized to the total β-catenin in each cell type. n = 3 independent experiments. (L and M) ChIP–quantitative PCR (qPCR) analysis for β-catenin occupancy on the WRE region of the Axin2 (L) and Lef1 (M) promoters in MB, G0 cells, and MT. Immunoglobulin G (IgG) is a negative control. n = 3 independent experiments. Error bars represent mean ± SEM. Significance was determined by Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Ajoy Aloysius et al., Sci. Signal. 2018;11:eaan3000 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Interaction with β-catenin is not required for Tcf/Lef-mediated transcriptional activation in G0 cells. Interaction with β-catenin is not required for Tcf/Lef-mediated transcriptional activation in G0 cells. (A) Reporter activity in G0-arrested C2C12 cells stably transfected with the TOPflash Tcf/Lef reporter and transfected with siRNA-targeting β-catenin [siβ-cat(1) or siβ-cat(2)] or control siRNA (Scr). Values are presented as relative light units (RLU) normalized to the total protein (RLU per microgram protein). β-cat KD, β-catenin knockdown. (B) Quantification of Axin2 and Lef1 mRNAs by qRT-PCR in β-catenin knockdown and control siRNA in G0 cells. (C) Colony formation assay for β-catenin knockdown and control siRNA in G0 cells. (D) Tcf/Lef reporter activity in G0 cells transfected with siRNA targeting Lef1 [siLef1(1) or siLef1(2)] or control siRNA. (E) Quantification of Axin2 and Lef1 mRNAs by qRT-PCR in Lef1 knockdown and control siRNA in G0 cells. (F) Colony formation assay for Lef1 knockdown and control siRNA in G0 cells. (G) Tcf/Lef reporter activity in G0 cells expressing DNLef1, which lacks the β-catenin binding domain and untreated (UT). (H) Tcf/Lef reporter activity in G0 cells treated with iCRT3, which disrupts the interaction between Tcf and β-catenin or dimethyl sulfoxide [DMSO; control (Con)]. (I) Quantification of Axin2 and Lef1 mRNAs by qRT-PCR in G0 cells treated with iCRT3 or DMSO (control). (J) Colony formation assay in G0 cells treated with iCRT3 or DMSO (control). For all panels, n = 3 independent experiments. Error bars represent mean ± SEM. Significance was determined by Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Ajoy Aloysius et al., Sci. Signal. 2018;11:eaan3000 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Expression of distinct Tcf/Lef family members affects Wnt-Tcf transcriptional activation in G0. Expression of distinct Tcf/Lef family members affects Wnt-Tcf transcriptional activation in G0. (A) Western blot showing the transcriptional activator Lef1 in C2C12 MB, G0 cells, and MT. Gapdh is a loading control. Migration of molecular mass markers is noted (in kilodaltons). Blots are representative of four independent experiments. (B) Densitometry analysis for Western blots of Lef1 in different cellular states normalized to Gapdh in each cell type. n = 4 independent experiments. (C) Western blot showing the transcriptional repressor Tcf7l1 in MB, G0 cells, and MT. Two short isoforms of Tcf7l1 present in MT are indicated with arrows. Blots are representative of four independent experiments. (D) Densitometry analysis for Western blots of Tcf7l1, both upper (75 kDa) and lower (55 kDa) bands normalized with Gapdh in MB, G0 cells, and MT. n = 4 independent experiments. (E) Immunofluorescence showing Lef1 and Tcf7l1 in MB, G0 cells, and MT. The boxed regions indicate the areas magnified in the high-magnification (Zoom) images showing nuclear localization of Lef1 (white arrows) and Tcf7l1 (yellow arrows). Nuclei are outlined with dotted lines. Images are representative of three independent experiments. Scale bars, 10 μm. (F) Quantification of image intensity of Lef1 and Tcf7l1 immunostaining in nuclei of MB, G0 cells, and MT. Quantification is based on measurements of >100 cells from randomly selected fields of three independent experiments. (G to J) ChIP-qPCR analysis showing Lef1 (E and F) and Tcf7l1 (G and H) occupancy on the WRE region of the Axin2 and Lef1 genes in MB, G0 cells, and MT. IgG is a negative control. n = 3 independent experiments. Error bars represent mean ± SEM. Significance was determined by Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Ajoy Aloysius et al., Sci. Signal. 2018;11:eaan3000 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

The TGF-β pathway plays a role in G0-specific Tcf/Lef-mediated transcriptional activation. The TGF-β pathway plays a role in G0-specific Tcf/Lef-mediated transcriptional activation. (A) Tcf/Lef reporter (TOPflash) activity in G0 cells treated with the TGF-β receptor inhibitor SB431542 (SB) or DMSO (control). Values are presented as RLU normalized to total protein (RLU per microgram protein). n = 3 independent experiments. (B) Colony formation assay of G0 cells treated with the indicated concentrations of SB or DMSO (control). n = 3 independent experiments. (C) Quantification of Axin2 and Lef1 mRNAs by qRT-PCR in G0 cells treated with SB or DMSO (control). n = 3 independent experiments. (D) Western blot showing Smad3 phosphorylated at Ser423 and Ser425 (pSmad3) and total Smad3 (Tot Smad3) in G0 cells treated with SB or DMSO (control). Gapdh is a loading control. (E) Densitometry analysis for Western blots of pSmad3 normalized to total Smad3 in SB-treated G0 cells. n = 3 independent experiments. (F) Western blot showing Smad2 phosphorylated at Ser462 (pSmad2) and total Smad2 (Tot Smad2) in G0 cells treated with SB or DMSO (control). (G) Densitometry analysis for Western blots of pSmad2 normalized to total Smad2 in SB-treated G0 cells. n = 3 independent experiments. (H) Western blot showing pSmad3 and total Smad3 in MB, G0 cells, and MT. Blot is representative of three independent experiments. (I) Densitometry analysis for Western blots of pSmad3 in MB, G0 cells, and MT normalized to the total Smad3. n = 3 independent experiments. (J) Immunofluorescence showing pSmad3 in MB, G0 cells, and MT. The boxed regions are magnified in the images on the right (Zoom), with nuclei outlined. Images are representative of three independent experiments. Scale bars, 10 μm. (K) Box plot showing image intensity analysis of pSmad3 immunostaining of nuclei in MB, G0 cells, and MT. Quantification is based on measurements of >100 nuclei from randomly selected fields of three independent experiments. Error bars represent mean ± SEM. Significance was determined by Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Ajoy Aloysius et al., Sci. Signal. 2018;11:eaan3000 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

The TGFβ effector Smad3 is a key player in Tcf/Lef-mediated transcriptional activation in G0 cells. The TGFβ effector Smad3 is a key player in Tcf/Lef-mediated transcriptional activation in G0 cells. (A) Tcf/Lef reporter (TOPflash) activity in G0 C1C12 cells treated with the indicated concentrations of the Smad3-specific inhibitor SIS3 or DMSO (control). Values presented as RLU normalized to total protein (RLU per microgram protein). n = 3 independent experiments. (B) Colony formation assay for G0 cells treated with SIS3 or DMSO (control). n = 3 independent experiments. (C) Quantification of Axin2 and Lef1 mRNAs by qRT-PCR in G0 cells treated with SIS3 or DMSO (control). n = 3 independent experiments. (D) Tcf/Lef reporter (TOPflash) activity in G0 cells transfected with siRNA targeting Smad3 [siSmad3(1) or siSmad3(2)] or control siRNA. n = 3 independent experiments. (E) Colony formation assay for Smad3 knockdowns G0 cells. n = 3 independent experiments. (F) Quantification of Axin2, Lef1, and Smad3 mRNAs by qRT-PCR in Smad3 knockdown G0 cells. n = 3 independent experiments. (G and H) ChIP-qPCR analysis for pSmad3 occupancy on the WRE region of Axin2 (G) and Lef1 (H) in MB, G0 cells, and MT. n = 3 independent experiments. (I) Western blot (WB) showing total and active β-catenin, pSmad3, and Lef1 in pSmad3 and Lef1 immunoprecipitates (IP) from nuclear lysates of MB and G0 cells. Immunoprecipitation with IgG is a negative control. n = 3 β-catenin. (J to M) ChIP-qPCR analysis for pSmad3 (J and L) and Lef1 (K and M) occupancy on the WRE region of Axin2 and Lef1 in G0 cells treated with siRNA targeting Lef1 (siLef1) or a control siRNA. n = 3 independent experiments. (N to Q) ChIP-qPCR analysis for Lef1 (N and P) and pSmad3 (O and Q) occupancy on the WRE region of Axin2 and Lef1 in G0 cells treated with siRNA targeting Smad3 (siSmad3) or a control siRNA. n = 3 independent experiments. Error bars represent mean ± SEM. Significance was determined by Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Ajoy Aloysius et al., Sci. Signal. 2018;11:eaan3000 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Lef1-Smad3 may interact in quiescent MuSCs (satellite cells) in vivo. Lef1-Smad3 may interact in quiescent MuSCs (satellite cells) in vivo. (A to D) Immunofluorescence showing active β-catenin (A), Tcf7l1 (B), Lef1 (C), and pSmad3 (D) in MuSCs (arrow) and myonuclei (asterisks) of a single muscle fiber from the EDL of adult Pax7-nGFP mice, which express nuclear GFP in MuSCs. Images are representative of three animals. Scale bars, 20 μm. (E) Immunofluorescence showing active β-catenin, Lef1, and pSmad3 in MuSCs (white arrowheads) of single prequiescent (1-week postnatal), quiescent (8-week postnatal), and activated (8-week postnatal muscle cultured for 24 and 48 hours) EDL muscle fibers from Pax7-nGFP mice. Activated muscle fibers were cultured in the presence of EdU to label proliferating cells. Insets show the MuSC nuclei (DAPI, blue), Pax7 (green), and EdU (yellow) in the same field of view as the large image. n = 3 independent experiments, each using four mice to obtain sufficient fibers for each experiment. Scale bars, 5 μm. (F) Box plot showing fluorescence intensity analysis of active β-catenin, Lef1, and pSmad3 in nuclei of prequiescent (PreQ), quiescent (0H), and activated (24H and 48H) MuSCs from experiment in (E). Fifty to 100 cells were analyzed in each sample from three biological replicates. (G) Percentage of MuSCs on single EDL fibers that were Pax7+MyoD−EdU after 3 days of culture in proliferation medium, followed by treatment with the Smad3-specific inhibitor SIS3, the TGF-β receptor inhibitor SB, the Tcf–β-catenin interaction disruptor iCRT3, or DMSO (control). n = 3 independent experiments. Three mice were used to obtain sufficient fibers for each experiment. More than 200 cells from fibers were analyzed in each treatment condition. Each experiment was normalized to the number of MuSCs scoring positive in control samples. More than 200 cells were analyzed per condition. Error bars represent mean ± SEM. Significance was determined by Student’s t test. *P < 0.05. n = 3 independent experiments. (H) Western blot showing total and active β-catenin, pSmad3, and Lef1 in Lef1 immunoprecipitates from nuclear lysates of C2C12 MB treated with iCRT3 or DMSO (control). Blot is representative of three independent experiments. (I) Model illustrates Lef1 switching partners from β-catenin to pSmad3 during the switch from the proliferative to the quiescent state. APC, adenomatous polyposis coli; CK1, casein kinase 1; TGF-βR, transforming growth factor–β receptor. Ajoy Aloysius et al., Sci. Signal. 2018;11:eaan3000 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works