MATR3 is an endogenous inhibitor of DUX4 in FSHD muscular dystrophy

. Author manuscript; available in PMC: 2023 Oct 23.

Published in final edited form as: Cell Rep. 2023 Sep 12;42(9):113120. doi: 10.1016/j.celrep.2023.113120

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER

Antibodies

Mouse monoclonal anti-FLAG M2	Sigma-Aldrich	Cat# F1804, RRID: AB_262044
Mouse monoclonal anti-HA.11 (Clone 16B12)	Covance	Cat# MMS-101R, RRID: AB_291262
Rabbit polyclonal anti MATR3	Thermo Fisher Scientific	Cat# PA5-57720, RRID: AB_2643782
Mouse monoclonal anti-6xHis	Clontech	Cat# 631212, RRID: AB_2721905
Mouse monoclonal anti-GST	Sigma-Aldrich	Cat# G1160, RRID: AB_259845
Mouse monoclonal anti-Tubulin (clone DM1A)	Sigma-Aldrich	Cat# T9026, RRID: AB_477593
Anti-mouse IgG-HRP	Jackson ImmunoResearch	Cat # 715-035-150, RRID: AB_2340770
Anti-rabbit IgG-HRP	Jackson ImmunoResearch	Cat # 711-035-152, RRID: AB_10015282
Rabbit monoclonal anti-DUX4 E5-5	Abcam	Cat# ab124699, RRID: AB_10973363
Mouse monoclonal anti-DUX4 P2B1 (for PLA)	Sigma-Aldrich	Cat# SAB5200019, RRID: AB_2612761
Mouse monoclonal anti-myosin	DSHB	Cat# MF20, RRID: AB_2147781
Rabbit monoclonal anti-WDR5 (clone D9E1I)	Cell Signaling Technology	Cat# 13105, RRID:AB_2620133
Alexa 555 goat anti-rabbit	Molecular Probes	Cat# A-27039, RRID: AB_2536100
Alexa 555 goat anti-mouse	Molecular Probes	Cat # A-21422, RRID: AB_141822
Alexa Fluor 488 goat anti-mouse	Molecular Probes	Cat# A-32723, RRID: AB_2633275

Bacterial and virus strains

Rosetta2 (DE3) pLysS competent cells	Novagen	Cat #71403

Biological samples

FSHD primary muscle cells	Richard Fields Center for FSHD Research Biobank	Table S6 of this paper
Healthy primary muscle cells	Richard Fields Center for FSHD Research Biobank	Table S6 of this paper

Chemicals, peptides, and recombinant proteins

Staurosporine	Sigma-Aldrich	19123
Doxycycline	Sigma-Aldrich	D9891
D-Biotin	Sigma-Aldrich	2031
DL-Dithiothreitol	Sigma-Aldrich	D9779
Trypsin-EDTA (0,5%)	Gibco	15400054
Dimethyl sulfoxide	Sigma-Aldrich	D2650
L-Glutamine	Thermo Fisher Scientific	25030081
Dexamethasone	Sigma-Aldrich	D4902
Paraformaldehyde	Electron Microscopy Sciences	157–8
GST recombinant protein	This paper	N/A
GST-MATR3 _2–288 recombinant protein	This paper	N/A
HIS-DUX4 dbd recombinant protein	This paper	N/A

Critical commercial assays

Caspase-Glo 3/7 luminescent assay	Promega	G8091
CellTiter-Glo luminescent assay	Promega	G7570
CASP3/7 assay GREEN reagent	Essen BioScience	4440
Duolink in situ Red kit	Sigma-Aldrich	DUO92101
QuikChange XL Site-Directed Mutagenesis Kit	Agilent Technologies	200517
Gateway LR Clonase Enzyme mix	Invitrogen	11791019
Lipofectamine 3000 Transfection Reagent	Thermo Fisher Scientific	L3000001
Lipofectamine LTX Reagent with PLUS Reagent	Thermo Fisher Scientific	15338-100
PureLink RNA Mini Kit	Thermo Fisher Scientific	12183025
DNAseI (PureLink DNase Set)	Thermo Fisher Scientific	12185010
SuperScript III First-Strand Synthesis System	Thermo Fisher Scientific	18080051
iTaq Universal SYBR Green Supermix	Biorad	1725122
Polyfect Transfection Reagent	QIAGEN	301107
Slide-A-Lyzer dialysis cassettes	Thermo Fisher Scientific	66380
Protein G Sepharose beads	GE Healthcare	71708300
Strep-Tactin Sepharose beads	IBA Lifesciences	2-1201-002
anti-HA-agarose beads	Sigma-Aldrich	A2095
Glutathione-Sepharose beads	GE Healthcare	17513201
anti-FLAG M2 Affinity Gel	Sigma-Aldrich	A2220
His-Select Nickel Affinity gel beads	Sigma-Aldrich	H0537

Deposited data

Raw and analyzed RNA-seq data	This paper	GEO: GSE210008 and Table S5
Raw and analyzed proteomics data	This paper	ProteomeXchange: PXD011073, https://doi.org/10.6019/PXD011073 and Table S1
DUX4 core target gene list	Table S4 (Jagannathan S et al.)³⁴	https://doi.org/10.1093/hmg/ddw271

Experimental models: Cell lines

HEK293T cells	ATCC	CRL-3216
Flp-In T-REx 293 cells	Thermo Fisher Scientific	R78007
DUX4 Flp-In T-Rex cells (HEK-iSH-DUX4)	This manuscript	N/A

Oligonucleotides

See Table S2 for a list of primers for cloning	This paper	N/A
See Table S3 for a list of siRNAs	N/A	N/A
See Table S4 for a list of primers for RT-qPCR	This paper	N/A

Recombinant DNA

pCMV_FLAG-MATR3	(Salton et al.)⁷⁸	RRID: Addgene_32880
pCMV_FLAG-SMARCC2	(Xi et al.)⁷⁹	RRID: Addgene_19142
pECE-M2-CDC27	(Banko et al.)⁸⁰	RRID: Addgene_31661
pCDNA-3xFLAG-RUVBL1	(Venteicher et al.)⁸¹	RRID: Addgene_51635
pCMV_FLAG-MATR3 _{2_288}	This paper	N/A
pCMV_FLAG-MATR3 _{2_322}	This paper	N/A
pCMV_FLAG-MATR3 _{2_798}	This paper	N/A
pCMV-FLAG-MATR3 _{289_end}	This paper	N/A
pCS2-mkgDUX4 vector	(Snider et al.)⁵²	RRID: Addgene_21156
pET-GB1-DUX4 dbd	This paper	N/A
pGEX-2tk- MATR3 _{2_288}	This paper	N/A
pCDNA5/FRT/TO STREP-HA	Dr. Superti-Furga G, CeMM Research Center for Molecular Medicine	Austrian Academy of Sciences, 1090 Vienna, Austria
pCMV-dR8.91	Broad Institute	http://www.broadinstitute.org/rnai/trc
VSV-G/pMD2G	Broad Institute	http://www.broadinstitute.org/rnai/trc
pFUGW:GFP	Dr. Shanahan CM, King’s College London, London, UK	https://doi.org/10.1091/mbc.E16-06-0346
pFUGW:GFP- MATR3-full length	This paper	N/A
pFUGW:GFP- MATR3 _{2_288}	This paper	N/A
pLBC2-BS-RFCA-BCVIII	Dr. Mullighan CG, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA	https://doi.org/10.1038/ng.3691
pLBC2-BS-RFCA-BCVIN-att-MATR3-full length	This paper	N/A
pLBC2-BS-RFCA-BCVIII-att-MATR3 _{2_288}	This paper	N/A

Software and algorithms

GraphPad PRISM 8	GraphPad Software	https://www.graphpad.com/
ImageJ	National Institutes of Health	https://imagej.nih.gov/ij/
CFX Manager Software 3.1	Biorad	https://www.bio-rad.com/it-it/sku/1845000-cfx-manager-software?ID=1845000
MaxQuant (v 1.5.2.8)	(Cox J. et al.)⁸²	https://doi.org/10.1021/pr101065j
STAR aligner (v 2.5.3a)	(Dobin A et al.)⁸³	https://doi.org/10.1093/bioinformatics/bts635
CRAN package pheatmap (v.1.0.12)	CRAN package	https://cran.r-project.org/package=pheatmap
CRAN package “venn” (v1.11).	CRAN package	https://CRAN.R-project.org/package=venn
FeatureCounts (v 1.6.4)	(Liao Y et al.)⁸⁴	https://doi.org/10.1093/bioinformatics/btt656