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. 2018 Mar 13;7:e31023. doi: 10.7554/eLife.31023

Figure 2. The MBD2/NuRD complex represses the D4Z4 array.

(A) Schematic representation of the NuRD complex. Subunits colored darkest grey have the most lines of evidence linking them to DUX4 regulation (e.g. enChIP, siRNA and ChIP data), while more lightly colored subunits have progressively less experimental support. Adapted from Hota and Bruneau (2016). (B–J) DUX4 and DUX4 target gene expression as determined by RT-qPCR following control (CTRL), HDAC1/HDAC2 (B–D), CHD4 (E–G) or MBD2 (H–J) siRNA knockdown in MB2401 control (B,E,H), MB073 FSHD1 (C,F,I) or MB200 FSHD2 (D,G,J) myoblasts. Error bars denote the standard deviation from the mean of three biological replicates. Statistical significance was calculated by comparing the specific knockdown to the control knockdown for each gene using a two-tailed, two-sample Mann-Whitney U test. *, p≤0.05.See also Figure 2—source data 1.

Figure 2—source data 1. Source data for Figure 2.
This file contains the source data used to make the graphs presented in Figure 2 and Figure 2—figure supplements 1–6. GraphPad Prism was utilized to visually represent the quantitative data.
elife-31023-fig2-data1.xlsx (72.1KB, xlsx)
DOI: 10.7554/eLife.31023.014

Figure 2.

Figure 2—figure supplement 1. Validation of HDAC1 and HDAC2 knockdown.

Figure 2—figure supplement 1.

(A–C) HDAC1 and HDAC2 gene expression as determined by RT-qPCR following control (CTRL), HDAC1, HDAC2 or simultaneous HDAC1/HDAC2 siRNA knockdown in MB2401 control (A), MB073 FSHD1 (B) or MB200 FSHD2 (C) myoblasts. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 2—source data 1.
Figure 2—figure supplement 2. Pharmacological inhibition of HDAC1/HDAC2.

Figure 2—figure supplement 2.

(A) DUX4 and DUX4 target gene expression as determined by RT-qPCR in MB200 FSHD2 myoblasts treated with 2.5 µM MS-275 for the indicated times. Statistical significance was calculated by comparing the mRNA level at each time point to that at 0 hr using a two-tailed, two-sample Mann-Whitney U test. (B) ChIP-qPCR enrichment of histone H4 acetylation (H4Ac) along the D4Z4 repeat in MB200 FSHD2 myoblasts treated with 2.5 µM MS-275 for 24 hr. Statistical significance was calculated by comparing the H4Ac signal in untreated versus MS-275-treated cells at each site using a one-tailed, one-sample Wilcoxon signed-rank test. *, p≤0.05; ns, not significant, p>0.05. Error bars denote the standard deviation from the mean of three (or six, for the 0 hr and 12 hr time points in (A)) biological replicates. See also Figure 2—source data 1.
Figure 2—figure supplement 3. Validation of CHD4 knockdown.

Figure 2—figure supplement 3.

(A–C) CHD4 gene expression as determined by RT-qPCR following control (CTRL) or CHD4 siRNA knockdown in MB2401 control (A), MB073 FSHD1 (B) or MB200 FSHD2 (C) myoblasts. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 2—source data 1.
Figure 2—figure supplement 4. CHD3 depletion in control and FSHD myoblasts.

Figure 2—figure supplement 4.

(A–C) DUX4 and CHD3 gene expression as determined by RT-qPCR following control (CTRL) or CHD3 siRNA knockdown in MB2401 control (A), MB073 FSHD1 (B) or MB200 FSHD2 (C) myoblasts. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 2—source data 1.
Figure 2—figure supplement 5. Validation of MBD2 knockdown.

Figure 2—figure supplement 5.

(A–C) TMEM130 and MBD2 gene expression as determined by RT-qPCR following control (CTRL) or MBD2 siRNA knockdown in MB2401 control (A), MB073 FSHD1 (B) or MB200 FSHD2 (C) myoblasts. Error bars denote the standard deviation from the mean of three biological replicates. Statistical significance was calculated by comparing the specific knockdown to the control knockdown for each gene using a two-tailed, two-sample Mann-Whitney U test. *, p≤0.05. See also Figure 2—source data 1.
Figure 2—figure supplement 6. MBD3 depletion in control and FSHD myoblasts.

Figure 2—figure supplement 6.

(A–C) DUX4 and MBD3 gene expression as determined by RT-qPCR following control (CTRL) or MBD3 siRNA knockdown in MB2401 control (A), MB073 FSHD1 (B) or MB200 FSHD2 (C) myoblasts. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 2—source data 1.