Zeb1 and Tle3 are trans-factors that differentially regulate the expression of myosin heavy chain-embryonic and skeletal muscle differentiation

Abstract

Myosin heavy chain-embryonic encoded by the Myh3 gene is a skeletal muscle-specific contractile protein expressed during mammalian development and regeneration, essential for proper myogenic differentiation and function. It is likely that multiple trans-factors are involved in this precise temporal regulation of Myh3 expression. We identify a 4230 bp promoter-enhancer region that drives Myh3 transcription in vitro during C2C12 myogenic differentiation and in vivo during muscle regeneration, including sequences both upstream and downstream of the Myh3 TATA-box that are necessary for complete Myh3 promoter activity. Using C2C12 mouse myogenic cells, we find that Zinc-finger E-box binding homeobox 1 (Zeb1) and Transducin-like Enhancer of Split 3 (Tle3) proteins are crucial trans-factors that interact and differentially regulate Myh3 expression. Loss of Zeb1 function results in earlier expression of myogenic differentiation genes and accelerated differentiation, whereas Tle3 depletion leads to reduced expression of myogenic differentiation genes and impaired differentiation. Tle3 knockdown resulted in downregulation of Zeb1, which could be mediated by increased expression of miR-200c, a microRNA that binds to Zeb1 transcript and degrades it. Tle3 functions upstream of Zeb1 in regulating myogenic differentiation since double knockdown of Zeb1 and Tle3 resulted in effects seen upon Tle3 depletion. We identify a novel E-box in the Myh3 distal promoter-enhancer region, where Zeb1 binds to repress Myh3 expression. In addition to regulation of myogenic differentiation at the transcriptional level, we uncover post-transcriptional regulation by Tle3 to regulate MyoG expression, mediated by the mRNA stabilizing Human antigen R (HuR) protein. Thus, Tle3 and Zeb1 are essential trans-factors that differentially regulate Myh3 expression and C2C12 cell myogenic differentiation in vitro.

Keywords: C2C12 cells; Tle3; Zeb1; mouse; myogenin; myosin heavy chain-embryonic; regeneration; skeletal muscle.

Figure 1. A 4.2 kb genomic region regulates Myh3 expression.

(A) Immunofluorescence staining for DAPI (blue), GFP (green; arrow heads), and MyHC-embryonic (red; arrows) in C2C12 cells at day 5 of myogenic differentiation; the rightmost panel is a merge of other channels; scale bar is 10 μm. (B) Schematic of the Myh3 FL, DC2, DC3, and DC8 promoter constructs; scale bar is 1.5 cm =500 bp. (C) In vitro promoter activity of constructs in (B) at day 5 of C2C12 myogenic differentiation. (D) In vivo promoter activity of constructs in (B) during mouse TA muscle regeneration post injury (n = 4 per construct). (E, F) Graphs showing the effect of MyoG (E), Zeb1, MyoD and Mitf (F) on Myh3-FL promoter activity in C2C12 cells. Graphs are presented as mean ±SEM of a minimum of three independent experiments; *p-value ≤.05 is considered significant.

Figure 2. Zeb1 knockdown leads to accelerated differentiation.

(A–D) Graphs showing transcript expression of Zeb1, MyoD, MyoG,and Myh3, quantified by qPCR in control and Zeb1 siRNA-treated cells at days 3, 5 and 7 of C2C12 differentiation. Control transcript levels are set to one (red dotted line in A–D). (E) Western blots for Zeb1, MyoD, MyoG, MyHC-embryonic, and β-actin protein levels and (F–I) their densitometric quantification normalized to β-actin (loading control), on protein lysates of control and Zeb1 siRNA-treated C2C12 cells at days 3, 5, and 7 of differentiation, respectively. (J) Graph quantifying the number of reserve cells per mm² in control siRNA and Zeb1 siRNA-treated C2C12 cells at day 5 of differentiation. (K) Immunofluorescence images of C2C12 cells treated with control (upper panel) or Zeb1 siRNA (lower panel), labeled for MyHCs (red), phalloidin (green), and DAPI (blue), at day 5 of differentiation; scale bar is 100 μm. (L) Graph showing fusion index quantifying the number of nuclei (two or more nuclei) per fiber as a fraction of the total number of nuclei in control and Zeb1 siRNA-treated cells, at day 5 of differentiation. (M) Immunoblots showing co-immunoprecipitation of Zeb1, Tle3 and MyoD in undifferentiated C2C12 cells with the Rabbit IgG used as control. Graphs are presented as mean ±SEM of a minimum of three independent experiments; *p-value ≤.05 is considered significant.

Figure 3. Tle3 knockdown leads to impaired differentiation.

(A–D) Graphs showing transcript expression of Tle3, MyoD, MyoG, and Myh3, quantified by qPCR in control and Tle3 siRNA-treated cells at days 3 and 5 of C2C12 myogenic differentiation. Control transcript levels are set to one (red dotted line in A–D). (E) Western blots for Tle3, MyoD, MyoG, MyHC-embryonic, and β-actin protein levels and (F–I) their densitometric quantification normalized to β-actin (loading control), on protein lysates of control and Tle3 siRNA-treated C2C12 cells at days 3, and 5 of differentiation, respectively. (J) Western blots for Tle3, MyoG, MyHC-embryonic and β-actin (loading control) on C2C12 protein lysates overexpressing pCMV-3Tag3b-FLAG-Tle3 or control pCMV-3Tag3b-FLAG plasmids, and (K, L) the densitometric quantification of MyoG and MyHC-embryonic at day 5 of differentiation. (M) Immunofluorescence images of C2C12 cells treated with control (upper panel) or Tle3 siRNA (lower panel), labeled for MyHCs (red), phalloidin (green), and DAPI (blue), at day 5 of differentiation; scale bar is 100 μm. (N, O) Graphs showing fusion and differentiation indices, respectively, quantifying the number of nuclei (two or more nuclei for fusion index) per fiber as a fraction of the total number of nuclei in control and Tle3 siRNA-treated cells, at day 5 of differentiation. Graphs are presented as mean ±SEM of a minimum of three independent experiments; *p-value ≤.05 is considered significant.

Figure 4. Tle3 knockdown leads to downregulation of Zeb1 expression.

(A) Western blots for Tle3, Zeb1, KMT1A and β-actin (loading control), on protein lysates of control and Tle3 siRNA-treated C2C12 cells at days 3 and 5 of differentiation, respectively. (B, E) Graphs showing densitometric quantification of Zeb1 and KMT1A protein levels normalized to β-actin following Tle3 knockdown at days 3 and 5 of differentiation, respectively. (C, D) Graphs showing transcript levels of Zeb1 and miR-200c following Tle3 knockdown at days 3 and 5 of differentiation. (F) Western blots for Zeb1, Tle3, KMT1A and β-actin (loading control), on protein lysates of control and Zeb1 siRNA-treated C2C12 cells at days 3, 5, and 7 of differentiation, respectively, and (G, I) densitometric quantification of Tle3 and KMT1A protein levels normalized to β-actin. (H) Graph showing transcript levels of miR-200c following Zeb1 knockdown at days 3, 5, and 7 of differentiation. Control transcript levels are set to one (red dotted line in C, D and H). Graphs are presented as mean ±SEM of a minimum of three independent experiments; *p-value ≤.05 is considered significant.

Figure 5. Double knockdown of Tle3 and Zeb1 leads to inhibition of muscle differentiation.

(A) Western blots for Tle3, Zeb1, MyoG, MyHC-embryonic, and β-actin (loading control), respectively, on protein lysates of control, and Tle3+Zeb1 siRNA-treated C2C12 cells at day 5 of differentiation. (B-E) Graphs showing densitometric quantification of Tle3, Zeb1, MyoG, and MyHC-embryonic protein levels normalized to β-actin following the combined knockdown of Tle3 and Zeb1 at day 5 of differentiation. Graphs are presented as mean ±SEM of a minimum of three independent experiments; *p-value ≤.05 is considered significant.

Figure 6. Zeb1 represses Myh3 promoter activity via a novel binding site, whereas Tle3 regulates muscle differentiation post-transcriptionally.

(A) Schematic representation of the Myh3 promoter-enhancer depicting the predicted Zeb1 binding E-boxes at –3436 bp (distal region) and at –580 bp (intron 2, upstream of the ATG start site in exon 3), respectively. (B, C) Chromatin immunoprecipitation using Zeb1 and control IgG antibodies on C2C12 myoblasts for the E-boxes starting at –3436 and –580 bp in the promoter-enhancer of Myh3 and a control region on the same chromosome. (D) Schematic depicting the Zeb1 full length (top) or N- and C-terminal zinc-finger domain deleted Zeb1-Mut (bottom) protein. (E) Graph showing relative luciferase activity of Myh3-FL promoter construct alone, Myh3-FL with Zeb1-Mut, Myh3-Z1-Mut alone, Myh3-Z1-Mut with Zeb1, Myh3-Z1-Mut with Zeb1-Mut, Myh3-Z1-Mut with MyoD, and Myh3-Z1-Mut with Mitf constructs, respectively (left to right), following transfection into C2C12 cells in vitro, at day 5 of differentiation. (F, G) Graphs showing luciferase activity of Myh3-FL promoter construct transfected into C2C12 cells in vitro following Zeb1 and Tle3 knockdown respectively, at day 5 of differentiation. (H, I) Graphs showing luciferase activity of MyoG 3’UTR luciferase construct in vitro, following Tle3 and Zeb1 knockdown, respectively, at day 5 of C2C12 differentiation. (J) Graph showing transcript expression of HuR quantified by qPCR in control and Tle3 siRNA-treated cells at days 3 and 5 of C2C12 differentiation; control transcript levels are set to one (red dotted line in J). Graphs are presented as mean ±SEM of a minimum of three independent experiments; *p-value ≤.05 is considered significant.