1. Chromosomes and Gene Expression
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NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

  1. Amy E Campbell  Is a corresponding author
  2. Sean C Shadle
  3. Sujatha Jagannathan
  4. Jong-Won Lim
  5. Rebecca Resnick
  6. Rabi Tawil
  7. Silvère M van der Maarel
  8. Stephen J Tapscott  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States
  2. University of Washington, United States
  3. University of Rochester Medical Center, United States
  4. Leiden University Medical Center, Netherlands
Research Article
Cite this article as: eLife 2018;7:e31023 doi: 10.7554/eLife.31023
6 figures, 2 tables, 1 data set and 5 additional files

Figures

Figure 1 with 1 supplement
NuRD complex components bind the D4Z4 macrosatellite repeat.

(A) Schematic summary of the enChIP procedure. A 3xFLAG-dCas9-HA-2xNLS fusion protein (FLAG-dCas9) consisting of an N-terminal triple FLAG (3xFLAG) epitope tag, catalytically inactive Cas9 endonuclease (dCas9), C-terminal human influenza hemagglutinin (HA) epitope tag and tandem nuclear localization signal (2xNLS) is expressed with one or more guide RNA (gRNA) in an appropriate cell context. Cells are crosslinked, chromatin is fragmented and complexes containing FLAG-dCas9 are immunoprecipitated with an anti-FLAG antibody. After reversing the crosslinks, molecules associated with the targeted genomic region are purified and identified by downstream analyses including mass spectrometry and next-generation sequencing. Adapted from Fujita et al. (2016). (B–E) ChIP-qPCR enrichment of NuRD complex components CHD4 (B), HDAC2 (C), MTA2 (D) and MBD2 (E) along the D4Z4 repeat in MB2401 control myoblasts. The Chr18q12 amplicon contains no CpG dinucleotides and serves as a negative control site, while the TMEM130 promoter is occupied by NuRD complex components in published ENCODE datasets (ENCODE Project Consortium, 2012) and functions as a positive control locus. Error bars denote the standard deviation from the mean of three biological replicates. Statistical significance was calculated by comparing the specific pulldown to the IgG control at each site using a two-tailed, two-sample Mann-Whitney U test. *, p≤0.05; ns, not significant, p>0.05. See also Figure 1—source data 1.

https://doi.org/10.7554/eLife.31023.003
Figure 1—source data 1

Source data for Figure 1.

This file contains the source data used to make the graphs presented in Figure 1 and Figure 1—figure supplement 1. GraphPad Prism was utilized to visually represent the quantitative data.

https://doi.org/10.7554/eLife.31023.005
Figure 1—figure supplement 1
Validation of myoblast cell lines used for enChIP-MS.

(A) Diagram showing the location of the three gRNAs targeting the D4Z4 unit and their relationship to the DUX4 open-reading frame. 1, gD4Z4-1; 2, gD4Z4-2; 3, gD4Z4-3. (B–D) MB135 control myoblasts stably expressing FLAG-dCas9 together with gRNA targeting the D4Z4 repeat (gD4Z4-1, gD4Z4-2, gD4Z4-3) or the MYOD1 distal regulatory region (DRR) (gMYOD1) were examined for FLAG-dCas9 expression level by immunoblot (B), subcellular localization by immunofluorescence (C) and chromatin occupancy by enChIP-qPCR (D). A + indicates that existing cell lines were super-infected to enhance FLAG-dCas9 levels and/or to co-express two D4Z4 gRNAs. The arrowhead in (B) indicates the expected size of full-length FLAG-dCas9. See also Figure 1—source data 1.

https://doi.org/10.7554/eLife.31023.004
Figure 2 with 6 supplements
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.

https://doi.org/10.7554/eLife.31023.007
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 16. GraphPad Prism was utilized to visually represent the quantitative data.

https://doi.org/10.7554/eLife.31023.014
Figure 2—figure supplement 1
Validation of HDAC1 and HDAC2 knockdown.

(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.

https://doi.org/10.7554/eLife.31023.008
Figure 2—figure supplement 2
Pharmacological inhibition of HDAC1/HDAC2.

(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.

https://doi.org/10.7554/eLife.31023.009
Figure 2—figure supplement 3
Validation of CHD4 knockdown.

(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.

https://doi.org/10.7554/eLife.31023.010
Figure 2—figure supplement 4
CHD3 depletion in control and FSHD myoblasts.

(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.

https://doi.org/10.7554/eLife.31023.011
Figure 2—figure supplement 5
Validation of MBD2 knockdown.

(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.

https://doi.org/10.7554/eLife.31023.012
Figure 2—figure supplement 6
MBD3 depletion in control and FSHD myoblasts.

(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.

https://doi.org/10.7554/eLife.31023.013
Figure 3 with 7 supplements
MBD1/CAF-1 components repress the D4Z4 array.

(A) Schematic representation of the CAF-1 complex, with shading as in Figure 2A. All subunits have one line of evidence linking them to DUX4 regulation. (B–J) DUX4 and DUX4 target gene expression as determined by RT-qPCR following control (CTRL), CHAF1A/CHAF1B (B–D), MBD1 (E–G) or CHAF1A/CHD4 (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. See also Figure 3—source data 1.

https://doi.org/10.7554/eLife.31023.015
Figure 3—source data 1

Source data for Figure 3.

This file contains the source data used to make the graphs presented in Figure 3 and Figure 3—figure supplements 17. GraphPad Prism was utilized to visually represent the quantitative data.

https://doi.org/10.7554/eLife.31023.023
Figure 3—figure supplement 1
Validation of CHAFA1 and CHAF1B knockdown.

(A–C) CHAF1A and CHAF1B gene expression as determined by RT-qPCR following control (CTRL), CHAF1A or CHAF1B 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 3—source data 1.

https://doi.org/10.7554/eLife.31023.016
Figure 3—figure supplement 2
Validation of MBD1 knockdown.

(A–C) MBD1 gene expression as determined by RT-qPCR following control (CTRL) or MBD1 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 3—source data 1.

https://doi.org/10.7554/eLife.31023.017
Figure 3—figure supplement 3
Validation of CHAFA1 and CHD4 knockdown.

(A–C) CHAF1A and CHD4 gene expression as determined by RT-qPCR following control (CTRL), CHAF1A, CHD4 or simultaneous CHAF1A/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 3—source data 1.

https://doi.org/10.7554/eLife.31023.018
Figure 3—figure supplement 4
CHD4 depletion in additional FSHD cell lines.

(A–E) CHD4, DUX4, and DUX4 target gene expression as determined by RT-qPCR following control (CTRL) or CHD4 siRNA knockdown in 54–2 FSHD1 (A), 2305 FSHD2 (B), 2453 FSHD2 (C), 2338 FSHD2 (D) or 1881 FSHD2 (E) 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 and p was ≤0.05 unless otherwise specified as not significant (ns). See also Figure 3—source data 1.

https://doi.org/10.7554/eLife.31023.019
Figure 3—figure supplement 5
MBD2 depletion in additional FSHD cell lines.

(A–E) MBD2, DUX4, and DUX4 target gene expression as determined by RT-qPCR following control (CTRL) or MBD2 siRNA knockdown in 54–2 FSHD1 (A), 2305 FSHD2 (B), 2453 FSHD2 (C), 2338 FSHD2 (D) or 1881 FSHD2 (E) 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 and p was ≤0.05 unless otherwise specified as not significant (ns). See also Figure 3—source data 1.

https://doi.org/10.7554/eLife.31023.020
Figure 3—figure supplement 6
CHAF1A depletion in additional FSHD cell lines.

(A–E) CHAF1A, DUX4, and DUX4 target gene expression as determined by RT-qPCR following control (CTRL) or CHAF1A siRNA knockdown in 54–2 FSHD1 (A), 2305 FSHD2 (B), 2453 FSHD2 (C), 2338 FSHD2 (D) or 1881 FSHD2 (E) 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 and p was ≤0.05 unless otherwise specified as not significant (ns). See also Figure 3—source data 1.

https://doi.org/10.7554/eLife.31023.021
Figure 3—figure supplement 7
MBD1 depletion in additional FSHD cell lines.

(A–E) MBD1, DUX4, and DUX4 target gene expression as determined by RT-qPCR following control (CTRL) or MBD1 siRNA knockdown in 54–2 FSHD1 (A), 2305 FSHD2 (B), 2453 FSHD2 (C), 2338 FSHD2 (D) or 1881 FSHD2 (E) 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 and p was ≤0.05 unless otherwise specified as not significant (ns). See also Figure 3—source data 1.

https://doi.org/10.7554/eLife.31023.022
Figure 4 with 4 supplements
Additional transcriptional repressors silence the D4Z4 repeat.

(A–I) DUX4 and DUX4 target gene expression as determined by RT-qPCR following control (CTRL), TRIM28 (A–C), SETDB1 (D–F) or KDM1A (G–I) siRNA knockdown in MB2401 control (A,D,G), MB073 FSHD1 (B,E,H) or MB200 FSHD2 (C,F,I) 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 and p was ≤0.05 for all comparisons except those in (A) and (D). See also Figure 4—source data 1.

https://doi.org/10.7554/eLife.31023.024
Figure 4—source data 1

Source data for Figure 4.

This file contains the source data used to make the graphs presented in Figure 4 and Figure 4—figure supplements 14. GraphPad Prism was utilized to visually represent the quantitative data.

https://doi.org/10.7554/eLife.31023.029
Figure 4—figure supplement 1
Validation of TRIM28 knockdown.

(A–C) TRIM28 gene expression as determined by RT-qPCR following control (CTRL) or TRIM28 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 4—source data 1.

https://doi.org/10.7554/eLife.31023.025
Figure 4—figure supplement 2
Validation of SETDB1 knockdown.

(A–C) SETDB1 gene expression as determined by RT-qPCR following control (CTRL) or SETDB1 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 4—source data 1.

https://doi.org/10.7554/eLife.31023.026
Figure 4—figure supplement 3
Validation of KDM1A knockdown.

(A–C) KDM1A gene expression as determined by RT-qPCR following control (CTRL) or KDM1A 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 4—source data 1.

https://doi.org/10.7554/eLife.31023.027
Figure 4—figure supplement 4
SIN3A/SIN3B knockdown.

(A) Schematic representation of the SIN3 complex, with shading as in Figure 2A depicting the amount of experimental support linking each subunit to DUX4 regulation. (B–G) SIN3A, SIN3B, DUX4, and DUX4 target gene expression as determined by RT-qPCR following control (CTRL), SIN3A or SIN3B siRNA knockdown in MB2401 control (B–C), MB073 FSHD1 (D–E) or MB200 FSHD2 (F–G) myoblasts. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 4—source data 1.

https://doi.org/10.7554/eLife.31023.028
Figure 5 with 3 supplements
NuRD and CAF-1 complex components repress DUX4 in iPS cells.

(A–F) DUX4 gene expression as determined by RT-qPCR in human eMHF2 iPS cells following control (CTRL), HDAC1/HDAC2 (A), CHD4 (B), CHAF1A (C), SETDB1 (D), KDM1A (E) or SIN3B (F) siRNA knockdown. 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 and p was ≤0.05 unless otherwise specified as not significant (ns). See also Figure 5—source data 1.

https://doi.org/10.7554/eLife.31023.030
Figure 5—source data 1

Source data for Figure 5.

This file contains the source data used to make the graphs presented in Figure 5 and Figure 5—figure supplements 13. GraphPad Prism was utilized to visually represent the quantitative data.

https://doi.org/10.7554/eLife.31023.034
Figure 5—figure supplement 1
Validation of repressor protein knockdowns in eMHF2 iPS cells.

(A–F) Gene expression as determined by RT-qPCR in human eMHF2 iPS cells following control (CTRL), HDAC1/HDAC2 (A), CHD4 (B), CHAF1A (C), SETDB1 (D), KDM1A (E) or SIN3B (F) siRNA knockdown. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 5—source data 1.

https://doi.org/10.7554/eLife.31023.031
Figure 5—figure supplement 2
Validation of HFF3 iPS cell generation.

(A–E) Gene expression as determined by RT-qPCR in HFF3 fibroblasts (FB), three isogenic iPS cell lines generated by reprogramming the fibroblast line, and embryoid bodies (EB) derived from the iPS cells. OCT4 (A) is a pluripotency gene, while NCAM1 (B), KDR (C), AFP (D), and CDX2 (E) are highly expressed in ectoderm, mesoderm, endoderm, and trophoblast, respectively. See also Figure 5—source data 1.

https://doi.org/10.7554/eLife.31023.032
Figure 5—figure supplement 3
NuRD and CAF-1 knockdown in HFF3 fibroblasts and iPS cells.

(A–D) Gene expression as determined by RT-qPCR following control (CTRL), CHD4 (A–B) or CHAF1A (C–D) siRNA knockdown in human HFF3 fibroblasts (FB) and three isogenic iPS cell lines generated by reprogramming the fibroblast line. DUX4 data were generated with 4qAL primers (see Materials and methods). Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 5—source data 1.

https://doi.org/10.7554/eLife.31023.033
Figure 6 with 2 supplements
MBD3L2 expression de-represses the D4Z4 array.

(A–C) DUX4 and DUX4 target gene expression as determined by RT-qPCR in MB2401 control (A), MB073 FSHD1 (B) or MB200 FSHD2 (C) myoblasts without (-) or with (+) doxycycline (Dox) treatment for 48 hr to induce MBD3L2 transgene expression in clonal cell lines. (D–E) DUX4-positive nuclei upon overexpression of MBD3L2 in MB200 FSHD2 myoblasts as in (C) were detected by immunofluorescence (D) and quantified by counting three fields representing >125 nuclei (E). (F–G) DUX4 and DUX4 target gene expression as determined by RT-qPCR following control (CTRL) or MBD3L family gene shRNA knockdown in MB073 FSHD1 (F) or MB200 FSHD2 (G) myotubes. 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 and p was ≤0.05 for all comparisons except in (A). See also Figure 6—source data 1.

https://doi.org/10.7554/eLife.31023.035
Figure 6—source data 1

Source data for Figure 6.

This file contains the source data used to make the graphs presented in Figure 6 and Figure 6—figure supplements 12. GraphPad Prism was utilized to visually represent the quantitative data.

https://doi.org/10.7554/eLife.31023.038
Figure 6—figure supplement 1
Validation of MBD3L2 overexpression and depletion.

(A–E) The ectopic (A–C) or endogenous (D–E) expression of MBD3L2 as determined by RT-qPCR in MB2401 control (A), MB073 FSHD1 (B) or MB200 FSHD2 (C) myoblasts cultured without (-) or with (+) doxycycline (Dox) for 48 hr, or in MB073 FSHD1 (D) or MB200 FSHD2 (E) myotubes expressing control (CTRL) or MBD3L gene shRNAs. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 6—source data 1.

https://doi.org/10.7554/eLife.31023.036
Figure 6—figure supplement 2
Additional MBD3L knockdown experiments.

(A–D) MBD3L2, DUX4, and DUX4 target gene expression as determined by RT-qPCR in two additional independent experiments with control (CTRL) or MBD3L shRNA-expressing MB073 FSHD1 (A–B) or MB200 FSHD2 (C–D) muscle cell lines differentiated into myotubes. Error bars denote the standard deviation from the mean of three biological replicates. See also Figure 6—source data 1.

https://doi.org/10.7554/eLife.31023.037

Tables

Table 1
Examples of proteins identified by enChIP-MS.
https://doi.org/10.7554/eLife.31023.006
Gene nameSampleCategory
gD4Z4gMYOD1
# peptides*% coverage# peptides*% coverage
CBX3/HP1γ4.715.70.00.0Known D4Z4-associated proteins
NCL47.422.334.014.2
PDS5B2.011.20.00.0
RAD211.82.90.00.0
SMC1A7.05.82.01.8
SMC317.06.71.01.0
SMCHD11.62.40.00.0
CHD48.33.10.00.0NuRD complex components
HDAC22.55.63.06.8
MTA21.22.21.01.5
RBBP44.57.54.06.9
  1. *Average number of peptides recovered from each sample type, combining like (gD4Z4 or gMYOD1) immunoprecipitations.

    Average percentage of each protein covered by the identified peptides from each sample type, combining like (gD4Z4 or gMYOD1) immunoprecipitations.

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line (H. sapiens)1881 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Cell line (H. sapiens)2305 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Cell line (H. sapiens)2338 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Cell line (H. sapiens)2453 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Cell line (H. sapiens)54–2 myoblasts(Krom et al., 2012) (DOI:10.1016/j.ajpath.2012.07.007)See Supplementary file 3
Cell line (H. sapiens)eMHF2 iPS cellsUniversity of Washington Institute for Stem Cell and Regenerative Medicine Tom and Sue Ellison Stem Cell Core (http://depts.washington.edu/iscrm/ellison)
Cell line (H. sapiens)HFF3 fibroblastsATCCATCC:SCRC-1043; RRID:CVCL_DB29
Cell line (H. sapiens)MB073 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Cell line (Homo sapiens)MB135 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Cell line (H. sapiens)MB200 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Cell line (H. sapiens)MB2401 myoblastsFields Center for FSHD and Neuromuscular Research at the University of Rochester Medical Center (https://www.urmc.rochester.edu/neurology/fields-center.aspx)See Supplementary file 3
Antibodyalpha-TubulinSigma-AldrichSigma-Aldrich:T9026; RRID:AB_477593
AntibodyAcetyl-Histone H4EMD MilliporeEMD Millipore:06866; RRID:AB_310270
AntibodyCHD4Bethyl LaboratoriesBethyl Laboratories:A301081A; RRID:AB_873001
AntibodyDUX4 (14–3)(Geng et al., 2011)
AntibodyDUX4 (E5-5)(Geng et al., 2011)
AntibodyFITC anti-mouseJackson ImmunoResearchJackson ImmunoResearch:715095150; RRID:AB_2340792
AntibodyFLAG M2Sigma-AldrichSigma-Aldrich:F1804 or F3165; RRID:AB_262044 or RRID:AB_259529
AntibodyHDAC2AbcamAbcam:ab7029; RRID:AB_305706
AntibodyHRP anti-mouseJackson ImmunoResearchJackson ImmunoResearch:115035146; RRID:AB_2307392
AntibodyMBD2Bethyl LaboratoriesBethyl Laboratories:A301632A; RRID:AB_1211478
AntibodyMTA2AbcamAbcam:ab8106; RRID:AB_306276
AntibodyTRITC anti-rabbitJackson ImmunoResearchJackson ImmunoResearch:711025152; RRID:AB_2340588
Recombinant DNA reagentpCW57.1-MBD3L2This paperAddgene plasmid #106332Lentiviral vector expressing doxycycline-inducible MBD3L2 (generated using pCW57.1 [Addgene plasmid #41393])
Recombinant DNA reagentpGIPZ-shControlFred Hutchinson Cancer Research Center Genomics Shared Resource (http://monod.fhcrc.org/rnai/)
Recombinant DNA reagentpGIPZ-shMBD3L-1Fred Hutchinson Cancer Research Center Genomics Shared Resource (http://monod.fhcrc.org/rnai/)
Recombinant DNA reagentpGIPZ-shMBD3L-2Fred Hutchinson Cancer Research Center Genomics Shared Resource (http://monod.fhcrc.org/rnai/)
Recombinant DNA reagentpZLCv2-3xFLAG-dCas9-HA-2xNLSThis paperAddgene plasmid #106357Lentiviral vector expressing FLAG-tagged, nuclease-deficient Cas9 (generated using lentiCRISPRv2 [Addgene plasmid #52961] and pHR-SFFV-KRAB-dCas9-P2A-mCherry [Addgene plasmid #60954])
Recombinant DNA reagentpZLCv2-gD4Z4-1-3xFLAG-dCas9-HA-2xNLSThis paperAddgene plasmid #106352Lentiviral vector expressing FLAG-dCas9 and a guide RNA targeting the D4Z4 unit
Recombinant DNA reagentpZLCv2-gD4Z4-2-3xFLAG-dCas9-HA-2xNLSThis paperAddgene plasmid #106353Lentiviral vector expressing FLAG-dCas9 and a gRNA targeting the D4Z4 unit
Recombinant DNA reagentpZLCv2-gD4Z4-3-3xFLAG-dCas9-HA-2xNLSThis paperAddgene plasmid #106354Lentiviral vector expressing FLAG-dCas9 and a gRNA targeting the D4Z4 unit
Recombinant DNA reagentpZLCv2-gMYOD1-3xFLAG-dCas9-HA-2xNLSThis paperAddgene plasmid #106355Lentiviral vector expressing FLAG-dCas9 and a gRNA targeting the MYOD1 distal regulatory region
Sequenced-based reagentenChIP-/ChIP-qPCR primersThis paperSee Supplementary file 4
Sequenced-based reagentgRNAsThis paperSee Supplementary file 4
Sequenced-based reagentRT-qPCR primersThis paperSee Supplementary file 4
Sequenced-based reagentshRNAsThis paperSee Supplementary file 4
Sequenced-based reagentsiRNAsThis paperSee Supplementary file 4
Peptide, recombinant protein3X FLAG peptideSigma-AldrichSigma-Aldrich:F4799
Commercial assay or kitQIAshredderQiagenQiagen:79656
Commercial assay or kitRNeasy Mini KitQiagenQiagen:74106
Commercial assay or kitSuperScript III First-Strand Synthesis SystemInvitrogen/Thermo FisherInvitrogen/Thermo Fisher:18080051
Chemical compound, drug2-MercaptoethanolSigma-AldrichSigma-Aldrich:M3148
Chemical compound, drugDexamethasoneSigma-AldrichSigma-Aldrich:D4902
Chemical compound, drugDMEM:Nutrient Mixture F-12Gibco/Thermo FisherGibco/Thermo Fisher:11320082
Chemical compound, drugDNase IThermo FisherThermo Fisher:18068015
Chemical compound, drugDoxycyline hyclateSigma-AldrichSigma-Aldrich:D9891
Chemical compound, drugDulbecco's Modified Eagle Medium (DMEM)Gibco/Thermo FisherGibco/Thermo Fisher:11965092
Chemical compound, drugDynabeads-Protein GThermo FisherThermo Fisher:10003D
Chemical compound, drugHam's F-10 Nutrient MixGibco/Thermo FisherGibco/Thermo Fisher:11550043
Chemical compound, drugHorse serumGibco/Thermo FisherGibco/Thermo Fisher:26050070
Chemical compound, drugHyClone Fetal Bovine SerumGE Healthcare Life SciencesGE Healthcare Life Sciences:SH30071.03
Chemical compound, drugInsulinSigma-AldrichSigma-Aldrich:I1882
Chemical compound, drugKnockOut Serum ReplacementGibco/Thermo FisherGibco/Thermo Fisher:10828028
Chemical compound, drugLipofectamine RNAiMAXInvitrogen/Thermo FisherInvitrogen/Thermo Fisher:13778150
Chemical compound, drugMatrigelCorning Life ScienceCorning Life Science:354277
Chemical compound, drugMEM non-essential amino acidsGibco/Thermo FisherGibco/Thermo Fisher:11140050
Chemical compound, drugmTeSR1 mediumSTEMCELL TechnologiesSTEMCELL Technologies:85850
Chemical compound, drugOpti-MEM reducedserum mediumThermo FisherThermo Fisher:31985070
Chemical compound, drugPenicillin/streptomycinGibco/Thermo FisherGibco/Thermo Fisher:15140122
Chemical compound, drugPolybreneSigma-AldrichSigma-Aldrich:107689
Chemical compound, drugPuromycinSigma-AldrichSigma-Aldrich:P8833
Chemical compound, drugRecominant human basic fibroblast growth factorPromega CorporationPromega Corporation:G5071
Chemical compound, drugSodium pyruvateGibco/Thermo FisherGibco/Thermo Fisher:11360070
Chemical compound, drugTransferrinSigma-AldrichSigma-Aldrich:T0665
Chemical compound, drugY-27632 ROCK inhibitorMiltenyi BiotecMiltenyi Biotec:130106538
Software, algorithmCode used for proteomics data analysisThis paper (Jagannathan, 2017)The R code used for the proteomics data analysis can be accessed via github at https://github.com/sjaganna/2017-campbell_et_al
Software, algorithmGraphPad PrismGraphPad Prism (https://graphpad.com)RRID:SCR_015807Version 6
Software, algorithmImageJImageJ (http://imagej.nih.gov/ij/)RRID:SCR_003070
Software, algorithmProteome DiscovererThermo FisherRRID:SCR_014477Version 1.4

Data availability

The following data sets were generated
  1. 1

Additional files

Supplementary file 1

Proteins identified by enChIP-MS.

The table lists the gene name, corresponding number of peptides recovered (pseudoquant), and percent coverage for each protein identified by enChIP-MS of nine independent FLAG-dCas9 immunoprecipitations from various gD4Z4- or gMYOD1-expressing myoblast cell lines.

https://doi.org/10.7554/eLife.31023.039
Supplementary file 2

Gene ontology analysis of D4Z4-associated proteins.

The table shows the gene ontology (GO) biological process categories enriched among the D4Z4-associated proteins identified by enChIP-MS, along with the number of observed versus expected proteins in each category and the associated fold enrichment score and p-value.

https://doi.org/10.7554/eLife.31023.040
Supplementary file 3

Characteristics of myoblast cell lines used in this study.

The table summarizes details of the muscle cell lines used for this study.

https://doi.org/10.7554/eLife.31023.041
Supplementary file 4

Oligonucleotide sequences.

The table lists all oligonucleotides used in this study, including gRNAs, siRNAs, shRNAs and primers used for enChIP-qPCR, ChIP-qPCR and RT-qPCR.

https://doi.org/10.7554/eLife.31023.042
Transparent reporting form
https://doi.org/10.7554/eLife.31023.043

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