Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
- Tokyo Medical and Dental University, Japan
- Chiba University, Japan
- INSERM U955 IMRB-E10 UPEC, ENVA, EFS, France
Figures
Figure 1 with 1 supplement
Klf5 is upregulated during myogenesis.
(A) Relative mRNA expression of Klf5, Pax7, Myod1, Myog and Myh3 (embryonic myosin heavy chain) in intact and regenerating TA muscles. The animals were sacrificed on day 4 after CTX injection. Data …
Figure 1—figure supplement 1
Preparation of SC primary cultures.
(A) Isolated EDL myofibers with their associated SCs were immediately fixed and immunostained for Pax7 and Klf5. (B) SCs were prepared from EDL muscles from wild-type C57/B6 mice and stained for …
Figure 2 with 1 supplement
Klf5 is required for muscle regeneration in vivo.
CTX was injected into the TA muscles of SC-specific Klf5 knockout mice (Pax7CE/+;Klf5flox/flox) and control Pax7+/+;Klf5flox/flox mice. The animals were then sacrificed on day 4 (A), 7 (B–D) or 28 (E…
Figure 2—figure supplement 1
Tamoxifen treatment does not affect TA muscle morphology.
TA muscles were obtained from Klf5 knockout mice (Pax7CE/+; Klf5flox/flox) and control Pax7+/+; Klf5flox/flox mice on day 28 after tamoxifen injection. Muscle morphology was assessed by H–E staining.
Figure 3 with 3 supplements
Klf5 is essential for muscle differentiation.
(A) Establishment of Klf5 knockout (KO) myoblasts using a CRISPR-Cas9 system. Klf5 KO C2C12 cells or Control cells were immunostained for MyHC and Klf5 or myogenin. Klf5 KO cells do not express Klf5 …
Figure 3—figure supplement 1
Klf5 knockdown impairs myoblast differentiation.
(A) Klf5 expression during C2C12 differentiation. Bars depict means ± SEM. n = 3, biological replicates. GM: growth medium, DM: differentiation medium. (B) Induction of Klf5 expression during C2C12 …
Figure 3—figure supplement 2
Establishment of Klf5 knockout C2C12 cells.
(A) C2C12 cells were transfected with pSpCas9-Klf5 or GFP-targeted control vector. After puromycin selection, the cells were cloned using the limiting dilution method. Three Klf5 knockout (KO) …
Figure 3—figure supplement 3
Phenotype of Klf5 knockdown cells.
(A–C) Klf5, myogenin (Myog), and MyHC expression in C2C12 cells after siRNA treatment. C2C12 cells were treated with either siKlf5 or siControl (siCtrl). After siRNA treatment in GM for 24hr, the …
Figure 4
Transient Klf5 knockdown results in a repression of the myogenic program.
(A) Hierarchical clustering and heatmap of the expression levels (log2 normalized counts) of genes involved in the Hallmark Myogenesis (hallmark gene sets M5909) in C2C12 myoblasts transfected with …
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Figure 4—source data 1
Gene ontology analysis on the genes upregulated by Klf5 knockdown.
Functional annotations associated with genes that were induced >1.5-fold with Klf5 knockdown at day 3 post differentiation.
- https://doi.org/10.7554/eLife.17462.011
Figure 5 with 3 supplements
Klf5 regulates myogenesis-related target genes in concert with MyoD.
(A) UCSC genome browser images illustrating normalized tag counts for Klf5 (post-differentiation day 0, 2, 5), MyoD (myotube, differentiated for 2 days) and Mef2D (myotube, differentiated for 5 …
Figure 5—figure supplement 1
Klf5, MyoD and Mef2D are colocalized in the myogenesis-related gene loci.
UCSC genome browser images illustrating normalized tag counts for Klf5 (post differentiation day 0, 2, 5), MyoD (myotubes, differentiated for 2 days) and Mef2D (myotubes, differentiated for 5 days) …
Figure 5—figure supplement 2
MyoD and Klf5 are recruited to the E-box-containing enhancer of the Myog locus.
Recruitment of MyoD (A), Klf5 (B) and Mef2 (C) to the Myog promoter was monitored using ChIP assays with chromatin prepared from C2C12 myoblasts after differentiation for 0, 2 or 5 days. The Gapdh …
Figure 5—figure supplement 3
Klf5 binding regions in undifferentiated myoblasts.
Heatmap for binding of Klf5 (post-differentiation day 0, 2, and 5), MyoD (myotubes, differentiated for 2 days) and Mef2D (myotubes, differentiated for 5 days) within 2 kb around the center of the …
Figure 6 with 2 supplements
MyoD function is inhibited in Klf5-null C2C12 myoblasts.
(A) UCSC genome browser images illustrating normalized tag counts for MyoD at Myod1, Myog, Myl4 and Mybph gene loci in GFP-targeted control (orange) or Klf5-null (red) C2C12 myotubes differentiated …
Figure 6—figure supplement 1
Levels of MyoD and Mef2 protein were not altered by Klf5 deletion.
Levels of MyoD, Mef2, Klf5 and Myog protein were analyzed by Western blotting using GFP-targeted control or Klf5 knockout myoblasts differentiated for 5 days. Representative data from four …
Figure 6—figure supplement 2
MyoD function is inhibited in Klf5-null C2C12 myoblasts -analysis in the another set of control and Klf5-null cells.
(A) UCSC genome browser images illustrating normalized tag counts for MyoD at Myog, Myl4, Mybph and Myod1 gene loci in GFP-targeted control or Klf5-null C2C12 myotubes differentiated for 3 days. (B) …
Figure 7
Klf5 is required for satellite cell differentiation.
(A) Isolated SCs from Klf5 knockout mice (Pax7CE/+;Klf5flox/flox) were cultured in the presence or absence of 4OH-Tmx in growth medium for 2 days. Purified protein derived from primary myotubes …
Author response image 1
(A) Histograms of MyoD tag densities around MyoD binding peaks proximal to MyoD target genes that were upregulated or downregulated (>1.5-fold) by Klf5 deletion. (B) MA plot of normalized tag counts …
Author response image 2
(A) Histograms of MyoD tag densities at MyoD binding regions proximal to MyoD target genes that were upregulated or downregulated (>1.5-fold) by Klf5 deletion. (B) Klf5 binding to MyoD-binding …
Author response image 3
Author response image 4
Additional files
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Supplementary file 1
Table 1: list of oligonucleotides, Table 2: list of antibodies
- https://doi.org/10.7554/eLife.17462.020
