The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor

Abstract

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.

Keywords: MyoD; TLE; basic helix-loop-helix (bHLH) transcription factor; mesenchymal stem cells (MSCs); myogenesis; skeletal muscle.

Figure 1.

TLE3 is expressed in activated and proliferating satellite cells of skeletal muscles. A, isolated EDL myofibers with their associated satellite cells were either immediately fixed (Day 0) or cultured in plating medium for 2 days before fixation and immunostaining for TLE3, Pax7, MyoD, or Ki67. Arrowheads indicate satellite cells. B, immunocytochemistry on satellite cells plated on Matrigel confirmed that TLE3 was expressed in activated and Ki67-positive proliferating satellite cells maintained in growth medium (GM) for 7 days (7d) after isolation. C–F, mice were sacrificed 0 (Intact), 1, 3, 5, 7, 10, or 14 days after CTX injection. Gene expression of TLE3 (C), Cyclin A2 (D), and Myogenin (E) was analyzed by qPCR. Protein levels of TLE3, Myogenin, Cyclin A2, and β-actin were assessed by Western blotting (F). G, mice were sacrificed 3.5 days after CTX injection (CTX 3.5d) and tibial anterior muscle cryosections were analyzed by immunostaining for Pax7, MyoD, and TLE3. The data in A, B, F, and G are representative of at least three individual mice. Scale bars = 20 μm (A and G) and 40 μm (B). The data in C–E are expressed as the mean ± S.D. (n = 3). **, p < 0.01 versus intact.

Figure 2.

Knockdown of TLE3 expression stimulates myoblast differentiation and reduces proliferation of plated satellite cells. A–F, plated satellite cells were transfected with control shRNAs (Mock) or shRNAs against TLE3 (shTLE3) and then cultured in either growth medium (GM) (A–C) or differentiation medium (DM) (D–F) for 2 days, and gene expression was analyzed by qPCR. Myog, Myogenin. G–K, plated satellite cells were transfected with control siRNAs (Mock) or siRNA against TLE3 (siTLE3) and then cultured in GM (G–I) or DM (J and K) for 2 days, and gene expression was analyzed by qPCR. L–O, plated satellite cells were transfected with control siRNA (Mock) or siRNA against TLE3 (siTLE3) and cultured in GM for 2 days (L, M, and O) or DM for 3.5 days (L and N) after transfection. Immunocytochemistry analysis was performed using anti-TLE3, anti-Cyclin D1, or anti-MHC antibodies. Scale bars = 100 μm (L). M and N, protein levels of Cylin D1, Cyclin A2, MHC, TLE3, and β-actin were assessed by Western blotting. Proliferation of plated satellite cells following TLE3 knockdown was assessed using Cell Counting Kit 8 (O). Similar results were obtained in three independent experiments (L and M). The data are expressed as mean ± S.D. (n = 3). **, p < 0.01; *, p < 0.05 versus control shRNAs (Mock) or siRNA-transfected cells (A–K and O).

Figure 3.

TLE3 regulates myoblast differentiation in C2C12 cells. A, C2C12 myoblasts were immunostained with anti-TLE3 antibody or normal IgG together with phalloidin and DAPI. Scale bar = 20 μm. B–D, C2C12 cells were transfected with control shRNA (Mock) or shRNA against TLE3 (shTLE3) and then cultured in myogenic medium. Western blot analysis with anti-TLE3 antibody showed reduction of endogenous TLE3 by shTLE3 along with increased Myogenin on day 2 (B). C and D, C2C12 cells transfected with control (Mock) or shRNA against TLE3 (shTLE3) were cultured in myogenic medium for 5 days and immunostained with anti-MHC antibody. Scale bar = 50 μm (C). The number of MHC-positive fibers (Fusion Index) was quantified on day 5 (D). E–G, cells were transfected with control siRNA (Mock) or siRNA against TLE3 (siTLE3) and cultured in myogenic medium. The mRNA levels of TLE3 (E), Myogenin (F), and Myh14 (G) were assessed by qPCR analysis on day 2. H, the protein levels of MHC, TLE3, Myogenin, and β-actin were assessed by Western blotting analysis on day 3. Similar results were obtained in three independent experiments (A–C and H). The data are expressed as mean ± S.D. (n = 3). **, p < 0.01 versus mock-transfected cells (D and E–G).

Figure 4.

TLE3 suppresses myoblast differentiation in C3H10T1/2 cells. A, whole-cell lysates from C2C12 cells were immunoprecipitated (IP) using anti-MyoD antibody and immunoblotted (IB) with an anti-TLE3 antibody. C3H10T1/2 cells were co-transfected with FLAG-tagged MyoD and either a mock vector or Myc-tagged TLE3. B, whole-cell lysates were immunoprecipitated using FLAG-tagged magnetic agarose beads and immunoblotted with anti-Myc or anti-FLAG. C–H, C3H10T1/2 cells were co-transfected with empty vector (Mock) or TLE3 along with MyoD. The mRNA levels of Myogenin (C), MCK (D), or Myh14 (E) were determined by qPCR on day 2. Western blot analysis was performed using anti-Myogenin antibody, anti-FLAG-antibody, anti-Myc-antibody, and anti-β-actin antibody on day 2 (F). G and H, immunocytochemical analysis was performed using anti-MHC antibody. Scale bar = 50 μm (G), and MHC-positive cells were counted on day 3 (H). I and J, C3H10T1/2 cells were transfected with TLE3 or empty vector (Mock) along with MyoD and MG185-luc (I) or MCK0.8-luc (J) reporter plasmid. Cells were transfected with FLAG-tagged MyoD together with or without Myc-tagged TLE3. K, chromatin immunoprecipitation analysis with anti-FLAG antibody and PCR primers for the Myogenin promoter. Similar results were obtained in three independent experiments (A, B, F, G, and K). The data are expressed as mean ± S.D. (n = 3). **, p < 0.01; *, p < 0.05 versus mock-transfected cells (C–E, H, I, and J).

Figure 5.

Both the Q and SP domain of TLE3 are essential for MyoD interaction and repression. A, schematic of the C-terminally truncated forms of the Myc-tagged TLE3 constructs used in these experiments. GP, glycine/proline-rich domain. B, C-terminally truncated forms of TLE3 or empty vector (Mock) were co-transfected with the MG185-luciferase reporter along with MyoD in C3H10T1/2 cells. Luciferase activity was determined on day 1. C3H10T1/2 cells were co-transfected with the FLAG-tagged MyoD and empty vector (Mock) or C-terminally truncated forms of Myc-tagged TLE3. C, whole-cell lysates were immunoprecipitated (IP) using anti-FLAG magnetic beads and immunoblotted (IB) with an anti-Myc antibody or anti-FLAG antibody. D, schematic of the N-terminally truncated forms of the Myc-tagged TLE3 constructs. C3H10T1/2 cells were co-transfected with the FLAG-tagged MyoD and empty vector (Mock) or N-terminally truncated forms of Myc-tagged TLE3. E, whole-cell lysates were immunoprecipitated using FLAG-tagged magnetic agarose and immunoblotted with an anti-Myc antibody or anti-FLAG antibody. Empty vector (Mock) or N-terminally truncated forms of TLE3 were transfected with MG185-luciferase reporter along with MyoD in C3H10T1/2 cells. F, luciferase activity was determined on day 1. Similar results were obtained in three independent experiments (C and E). The data are expressed as mean ± S.D. (n = 3). **, p < 0.01 versus mock-transfected cells (B and F).

Figure 6.

TLE3 interacts with the bHLH domain of MyoD. A, schematic of the C-terminally truncated forms of the Myc-tagged MyoD constructs used in these experiments. N-TAD, N-terminal transactivation domain; C-TAD; C-terminal transactivation domain. B, Empty vector (Mock) or C-terminally truncated forms of MyoD were transfected with MG185-luciferase reporter with or without TLE3 in C3H10T1/2 cells. Luciferase activity was determined on day 1. The data are expressed as mean ± S.D. (n = 3). **, p < 0.01 versus mock-transfected cells. C3H10T1/2 cells were co-transfected with the FLAG-tagged TLE3 and empty vector (Mock) or C-terminally truncated forms of Myc-tagged MyoD. C, whole-cell lysates were immunoprecipitated (IP) using anti-FLAG magnetic beads and immunoblotted (IB) with an anti-Myc antibody or anti-FLAG antibody. Similar results were obtained in three independent experiments.

Figure 7.

The suppressive mechanism of TLE3 on MyoD involves E protein. A, C3H10T1/2 cells were transfected with MG185-luc along with the indicated plasmids, and luciferase activity was measured on day 1. B, cells were co-transfected with FLAG-tagged E12 along with empty vector (Mock), Myc-tagged MyoD, or Myc-tagged TLE3. Whole-cell lysates were immunoprecipitated (IP) with anti-FLAG magnetic beads and immunoblotted (IB) with an anti-Myc or anti-FLAG antibody. C, cells were co-transfected with FLAG-tagged MyoD and empty vector (Mock) or Myc-tagged E12 together with or without V5-tagged TLE3. The whole-cell lysates were immunoprecipitated using FLAG-tagged magnetic agarose and immunoblotted with anti-Myc, anti-FLAG, or anti-V5 antibodies. The data are expressed as mean ± S.D. (n = 3). *, p < 0.05 versus mock-transfected cells (A). Similar results were obtained in three independent experiments (B and C). D, a model for TLE3 regulation of MyoD transcriptional activity.