Figure 9. MEF2D mediates Hdac9 de novo expression in the tKO.
(A) Hdac9 expression is notably induced in the sciatic nerves of the adult (P60) tKO mice (6.48 ± 0.53 × 10−6 au the tKO versus 1.46 ± 0.28 × 10−6 in the control; p < 0.0001). Only minor changes were observed in the cKO4 and cKO7. Four to eight mice per genotype were used. Unpaired t-test was used for comparations. (B) Hdac9 expression is increased from early postnatal development of the tKO nerve. At P2 we found 1.67 ± 0.13 × 10−6 au in the tKO versus 0.39 ± 0.03 × 10−6 in the controls (p < 0.0001) and at P8 we found 3.43 ± 0.52 × 10−6 au in the tKO versus 0.65 ± 0.09 × 10−6 in the controls (p < 0.0001). RT-qPCR with mouse-specific primers for Hdac9 was performed. The scatter plot, which include also the mean ± standard error (SE), shows the expression of Hdac9 normalized to the housekeeping 18S. Four to five mice per genotype were used. Data were analyzed with the unpaired t-test with Welch’s correlation. (C) ChIP-qPCR with anti-H3K9Ac of adult (P60) sciatic nerves of tKO and control mice. Three different experiments of four to five animals per genotype are shown. Data were normalized to the IgG value as shown as relative enrichment. Unpaired t-test was used for comparations. (D) Alignment of the reads of the RNA-seq from three individual sciatic nerves of control and three tKO mice, both uninjured and at 20 days post crush (20 dpi). Hdac9 gene is transcribed at detectable levels in the sciatic nerve of the uninjured tKO mice, whereas it is almost nondetectable in the control sciatic nerves. The tKO mice (but not the controls) increase additionally the expression of Hdac9 gene during remyelination (20 dpi). (E) mRNA levels of Hdac9 (as FPKMs) at 0, 1, 10, and 20 days post crush (dpi) in the RNA-seq experiment. Two-way analysis of variance (ANOVA) was used for statistical comparation. (F) Mef2d expression is increased early in development (P8) in tKO nerve (1.65 ± 0.18 in the tKO versus 0.97 ± 0.10 in controls; p = 0.025). RT-qPCR with mouse-specific primers for Mef2d was performed. The scatter plot, which include also the mean ± standard error (SE), shows the expression normalized to the housekeeping 18S. Four to five mice per genotype were used. Data were analyzed with the unpaired t-test with Welch’s correlation. (G) mRNA levels of Mef2d (as FPKMs) at 0, 1, 10, and 20 days post crush (dpi) in the RNA-seq experiment. (H) A representative WB of protein extracts from tKO, control, and wild-type nerves at 10 dpi is shown. In the quantification, MEF2D protein was increased in the tKO nerves (2.31 ± 0.19 au in the tKO versus 1.33 ± 0.19 in controls; p < 0.0069). (I) Same for 20 dpi (2.19 ± 0.03 au in the tKO versus 1.33 ± 0.11 in controls; p < 0.0073). Densitometric analysis was done for three to four WB from the same number of mice and normalized to the control 20 dpi. Data were analyzed with the unpaired t-test. (J) MEF2D colocalizes with the transcription factor SOX10+, suggesting that it is expressed by Schwann cells. P60 sciatic nerves were fixed and submitted to immunofluorescence with the indicated antibodies. Nuclei were counterstained with Hoechst. Representative confocal images of sections obtained from the sciatic nerves of control and tKO mice are shown. Scale bar: 20 μm. (K) MEF2D binds to the Hdac9 promoter in the tKO. ChIP-qPCR of 20 dpi nerves of tKO mice was performed using an anti-MEF2D-specific antibody. Five different experiments from four to five mice per genotype were performed. Data were analyzed with the unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ns: no significant). See source data file one online (graphs source data) for more details.
Figure 9—figure supplement 1. Hdac9 gene expression regulation.
