This study used pre-existing and de-identified human tissue samples from tissue repositories and commercial sources and was approved by the Fred Hutchinson Cancer Research Center and the University of Washingtion Institutional Review Boards. Animal studies were approved by the University of Washington Institutional Animal Care and Use Committee and followed the Assessment and Accreditation of Laboratory Animal Care guidelines.
Muscle biopsies, cultures, and human RNA and protein
Muscle biopsy samples were collected from the vastus lateralis muscle of clinically affected and control individuals using standardized needle muscle biopsy protocol and cell cultures were derived from biopsies as described on the Fields Center website: http://www.urmc.rochester.edu/fields-center/protocols/documents/PreparingPrimaryMyoblastCultures.pdf
. The sex, age, and severity score for the FSHD muscle biopsies were: F1998 (M, 43, 2); F0519 (M, 43, 4); F0515 (F, 48, 2); F0509 (M, 47, 2); F0531 (F, 47, 2); F2306 (F, 46, ND); F2331 (F, 56, 4); F2316 (F, 34, 5); F2319 (M, 52, ND); F2315 (F, 40, 3). Pathologic grading scale is 0–12 (from normal to severe) based on a score of 0–3 for each of four parameters: muscle fiber size/shape; degree of central nucleation; presence of necrotic/regenerating fibers or inflammation; and degree of fibrosis. Controls were selected in the same age range and sex representation. Muscle cell culture MB216 and muscle biopsy F2316 are from the same individual, otherwise the muscle cultures were derived from other individuals. RNA and protein lysates from human tissues were purchased from BioChain (Hayward, CA) and Origene (Rockville, MD).
RT-PCR for DUX4-fl, DUX4-s, and DUX4-fl3′
Total RNA was isolated from muscle biopsies and cultured cells using Trizol (Invitrogen) and then treated with DNase I for 15 minutes using conditions recommended by Invitrogen with the addition of RNaseOUT (Invitrogen) to the reaction. DNase reaction components were removed using the RNeasy (Qiagen) system and RNA eluted by two sequential applications of 30 µl of RNase-free water. Volume was reduced by speed vac and 1.5–2 µg of RNA used for first strand cDNA synthesis. RNA from adult human tissues was purchased from Biochain and had been DNase-treated by the supplier. First strand synthesis was performed using Invitrogen SuperScript III reverse transcriptase and Oligo dT primers according to manufacturer's instructions at 55° for 1 hour followed by digestion with RNase H for 20 minutes at 37°. Finally, the reactions were cleaned using the Qiaquick (Qiagen) pcr purification system and eluted with 50 µl of water. Primary pcr reactions were performed with 10% Invitrogen PCRx enhancer solution and Platinum Taq polymerase using 10–20% of the first strand reaction as template in a total reaction volume of 20 µl in thin wall MicroAmp (Applied Biosystems) reaction tubes. Nested pcr reactions used 1 µl of the primary reaction as template. Primers for Dux4-fl and -s detection in biopsy and cultured cell samples were 14A forward and 174 reverse, nested with15A (or 16A) forward and 175 reverse. Primers for 3′ detection were 182 forward and 183 reverse nested with1A forward and 184 reverse. All primer sequences are listed in .
Dux4-fl and -s in adult human tissues were detected using 14A forward and 183 reverse, then nested with 15A forward and 184 reverse primers. Pcr cycling conditions were as follows for both primary and nested pcr: 94° 5 minutes denaturation, 35 cycles of 94° for 30″, 62° for 30″ and 68° for 2.5 minutes or 1 minute depending on expected length of product. A single final extension of 7 minutes at 68° was included. Pcr products were examined on 2% NuSieve GTG (Lonza) agarose gels in TBE.
Pooled PCR for DUX4
To assess for stochastic expression of DUX4 in affected muscle cells, FSHD primary myoblasts were trypsinized and collected at confluence or after differentiation for 96 hr. Cells were counted and split into pools of 100-cell, 600-cell, or 10,000-cell aliquots. RNA was extracted from individual aliquots using Dynabeads mRNA DIRECT Kit (Invitrogen) following manufacturer's instructions. Bound polyadenylated mRNA was used directly for reverse transcription reaction with SuperScript III using on-bead oligo dT as primer. Synthesis was carried out at 52°C for 1 hr, terminated at 70°C for 15 min, followed by 15 min of RNase H treatment. 2 uL of cDNA product was used for nested DUX4-fl3′ PCR as described above.
RT-PCR for transcripts from chromosomes 10 and 4
Pcr reactions were performed on RT reactions generated as described above and using nested primer sets to sequences in exons 1 and 2 that are common to alleles on chromosomes 4 as well as 10. Transcripts were detected using primers 1A and 187 followed by nesting with 138S and 188 (). Diagnostic polymorphisms (underlined) in the 5′ end of exon 2 were used to assign allele origins of transcripts:
Quantitative RT-PCR for DUX4-fl3′
For quantitative PCR, 1 ug of DNase'd RNA was used for first strand cDNA synthesis. Reverse transcription was performed as above, except at 52°C for the synthesis reaction followed by 15 minutes of RNase H treatment and the Qiaquick purification eluted in 30 µl of water. One round of PCR reactions were performed using the same reagents as above and 2 uL of purified cDNA template. Primers for full length detection were 92 forward and 116 reverse (). PCR cycling conditions were as follows: 95°C 5 min denaturation, 36 cycles of 95°C for 30″, 62°C for 30″ and 68°C for 1 min, and final extension of 5 min at 68°C. Sequence of the product matched DUX4. A standard curve for DUX4 template copies was generated from PCR reactions using the same primers and cycling conditions but with known dilutions of a plasmid containing full length DUX4 cDNA in water. Test sample PCR reactions and standard PCR reactions were run in triplicate and examined on the same 1% agarose/TBE gels stained with SYBR Gold (Invitrogen) for 40 min per manufacturer instructions. Fluorescence was detected with Typhoon Trio Multi-mode Imager (GE Healthcare): excitation laser 488 nm; emission filter 520DP 40, PMT 500 V, 100 µm resolution. Histogram analysis was performed to ensure no signals were saturated. Gel band intensities were quantified with ImageQuant TL v2005 (GE Healthcare) software. Estimates for the copies of DUX4 full length template in the test samples were interpolated from the line of best fit of the dilutional standards, with the lowest visible dilutional signal setting the detection limit. The interpolated number was doubled to adjust for the single-stranded cDNA input in contrast to the double-stranded plasmid standard input. This resulted in an estimated copy number of DUX4 full-length per ug of total RNA. Final copy number estimates per cell were calculated based on assumptions of 100% efficient reverse transcription and 3.3 pg of total RNA per cell.
Open reading frame PCR for DUX4-fl
To assess for the full coding region of DUX4, three rounds of PCR were performed on cDNA, totaling 36 cycles. Conditions for each round were as follows: 95°C for 5′, 3 cycles of 95°C for 30″ and 68°C for 1′33″, 3 cycles of 95°C for 30″ and 65°C for 30″ and 68°C for 1′33″, 6 cycles of 95°C for 30″ and 62°C for 30″ and 68°C for 1′33″. 3 uL of primary PCR was used in the secondary PCR, and 3 uL of secondary PCR were used in the tertiary PCR. Primers for successive rounds of pcr (133, 134, 135, 136, 137, and 138G) are listed in .
3′ RACE for DUX4 in human testes
3′ RACE was performed on total RNA using Invitrogen Gene Racer kit essentially as described. Prior to pcr with gene specific primers and the GeneRacer 3′ primers the RT reaction was cleaned using Qiaquick (Qiagen) spin columns as described above. Gene specific forward primers were 182 and 1A (nesting). Pcr products were gel purified, cloned into TOPO 4.0 (Invitrogen) and sequenced.
Generation of induced pluripotent stem (iPS) cells
iPS cells were generated by forced expression of human OCT4, SOX2, and KLF4 using the retroviral vectors essentially as previously described (1). MLV vectors (pMXs-hOCT4, pMXs-hSOX2, and pMXs-hKLF4) were purchased from Addgene (www.addgene.com
, Cambridge, MA) and vector preparations were generated by transient transfection of Phoenix-GP cells (2) with pCI-VSV-G and vector plasmids (1
1 ratio), replacing the culture medium 16 and 48 hours later, harvesting and filtering (0.45 um pore size) conditioned medium after a 16 hour exposure to cells, and concentrating 50 to 100-fold by centrifugation (3). Transduction with MLV vectors was performed with polybrene (4ug/ml concentration) (Sigma-Aldrich Corp., St. Louis, MO) added to the medium. iPS cell colonies were identified by their characteristic morphology, cloned by microdissection, and expanded on irradiated mouse embryo fibroblasts (6000 rads) for further characterization. Typically, 5×104
fibroblasts cultured in DMEM plus 10% FBS were seeded to a 9.4 cm2
well on day minus 1, the medium was replaced with medium containing vectors and polybrene on day 0, and changed again to medium with DMEM plus 10% FBS on day 1. Cells were detached with trypsin and seeded to five 55 cm2
dishes on day 2 and medium changed on day 4. On day 6 cells are again detached with trypsin and 5×105
cells seeded to 55 cm2
dishes containing 7×105
irradiated mouse embryo fibroblasts (6000 rads) in human ES cell culture medium (see below). Medium is replaced every other day and colonies with typical morphology of iPS cells appear between day 20 and day 30 post infection. Colonies are mechanically dissected using drawn Pasteur pipettes and seeded to mouse embryo fibroblast feeder layers for culture and passaged every 2–3 days using 2 u/ml dispase.
Stem cell culture
iPS cells and Human ES cells were grown in a solution of DMEM
1) with 3.151 g/L glucose, supplemented with L-Glutamine (Invitrogen), non-essential amino acids (10 mM (100×) liquid, Invitrogen, # 11140-076), sodium pyruvate (100 mM (100×), liquid, # 11360-070), 20% knockout serum replacer (# 10828010) (Invitrogen, Carlsbad, CA), 1mM beta-mercapto-ethanol (Sigma, St. Louis MO), and 5 ng/ml basic fibroblast growth factor (Peprotech, #AF-100-18B ). Cells were generally cultured in 0.1% gelatin coated dishes containing irradiated mouse embryo fibroblasts at a density of 1.3×104
. When cells were used as a source of RNA, DNA, or protein, they were cultured on matrigel (1
60 dilution, BD biosciences, #356234) coated dishes in medium conditioned by exposure to confluent layers of mouse embryo fibroblasts over a 3 day period. Cells were passaged a minimum of 4 times under these conditions before DNA, RNA, or protein was harvested.
Detection of embryonic antigens in iPS cells
iPS cells were evaluated for the presence of tissue non-specific alkaline phosphatase activity by fixing colonies in phosphate buffered saline solution containing 0.5% gluteraldehyde, and washing ×3 in PBS. A staining buffer containing 100 mM Tris pH 8.5, 100 mM NaCL, 50 mM MgCl2, 0.1 mg/ml 5-Bromo-4-chloro-3-indolyl phosphate (xphos) and 1 mg/ml p-Nitro-Blue tetrazolium chloride (NBT) (Sigma-Aldrich, St. Louis, MO, USA) was used to detect tissue non-specific alkaline phosphatase activity. Stage Specific embryonic antigen 4 (SSEA4) was detected using mouse monoclonal MC-813-70 and goat anti-mouse FITC conjugated secondary. TRA-1-60 was detected using mouse monoclonal TRA-1-60 (Millipore, Billerica, MA), and goat anti-mouse FITC conjugated secondary (Millipore, Billerica, MA). Human NANOG was detected with a goat polyclonal flurophore (Northern Lights) conjugated antibody (NL493, R & D systems, Minneapolis, MN). Human OCT4 was detected with a rabbit polyclonal (Abcam, Cambridge, MA) and goat anti rabbit secondary conjugated with the Alexa 488 flurophore (Invitrogen, Carlsbad, CA). Cell karyotypes were determined by the University of Washington Cytogenetics laboratory.
Teratoma formation and staining
Induced pluripotent stem cells were detached from culture dishes with dispase (2 units/ml working concentration), 2×106
cells resuspended in F12
1 mixture) medium without supplements, and injected into the femoral muscle of SCID-Beige mice (CB17.B6-Prkd
/Crl Charles River, Stock # 250). Mice were maintained under biosafety containment level 2 conditions and palpable tumor masses developed approximately 6 weeks later. When a tumor mass was palpable the mice were sacrificed and tumor tissue fixed for several days in phosphate buffered saline solution containing 4% formaldehyde, and imbedded in paraffin. Sections of the tumor (5 micron thickness) were placed on slides and stained with hematoxylin and eosin using standard protocols.
Embryoid body formation
Human iPS were prepared for embryoid body formation by expanding cell numbers on mouse irradiated feeder layers, detaching colonies with dispase, triturating with a Pasteur pipette, and seeding colony fragments to dense layers of mouse embryo fibroblast feeders (5×104
) prior to EB formation. Four days later densely grown colonies from a 55 cm2
dish were treated with dispase and gently detached by pipetting or scraping. Colony fragments were washed several times and seeded (1
1) to Ultra Low Attachment 55 cm2
culture dishes (Corning, Corning, NY) in DMEM supplemented with 20% Fetal Bovine Serum. Every three days, EB's were allowed to gravity settle and the medium was gently removed and replaced. RNA and chromatin was harvested three weeks later for analysis.
Analysis of gene expression in iPS cells
iPS cells were grown without MEF feeders for preparation of RNA to be used in gene expression analysis. Cells were seeded to matrigel coated dishes and filtered conditioned medium from mouse embryo fibroblasts was used for culture. RNA was purified from cells using standard techniques and treated with DNAse to remove residual genomic DNA from the cells. cDNA synthesis was primed with oligo dT and reverse transcriptase. In all cases a tube was processed in parallel without the addition of reverse transcriptase to serve as a control for possible DNA contamination. The presence of RNA transcripts were detected using 28 thermal cycles with the primer pairs for OCT4, SOX2, hTERT, NANOG, KLF4, cMYC, and GAPDH indicated in . RNA was replaced with water as a negative control for the reaction.
The Chromatin Immunoprecipitation (ChIP) analysis of repressive histone modifications at the 5′-region of DUX4
was performed on primary fibroblasts, induced pluripotent stem (iPS) cells and corresponding embryoid bodies (EB) derived from unaffected individuals and FSHD patients, following a previously described protocol 
. Briefly, cells were cross-linked with formaldehyde at 1.42% final concentration for 15 min at room temperature, quenched, and sonicated to generate 500–100 bp DNA fragments. 25 µg aliquots (representing approximately 500,000 cells) of chromatin were used for each immunoprecipitation with anti-Histone H3K9me3 antibodies (Abcam) and nonimmune IgG fraction used as a mock control. After reverse cross-linking and DNA purification, the IP products were analyzed by real time PCR. The 5′-region of the DUX4 gene was analyzed using the 4q-specific D4Z4 primers, 4qHox or Q-PCR, that detect internal D4Z4 units including the last repeat unit 
. The real-time PCR signals obtained for IP antibodies were normalized to mock control IgG and to input to account for the number of D4Z4 repeats. Data are presented as mean ± stdev and represent the results of at least three independent immunoprecipitations followed by real-time PCR analysis done in triplicates.
Generation of antibodies to DUX4
We generated monoclonal antibodies to the amino- and carboxy-terminus of DUX4 for this study. The full characterization of these antibodies will be published separately 
. Briefly, the N-terminal 159 amino acids and the C-terminal 76 amino acids of DUX4 were fused to glutathione-s-transferase tags, respectively, and injected into the animals as immunogens. Mouse monoclonals were produced at the Antibody Development core facility at the Fred Hutchinson Cancer Research Center and will be commercially available. Rabbit monoclonals were produced in collaboration with and will be available through Epitomics (Burlingame, CA). Hybridoma clones were screened for specificity by ELISA, western blot and immunofluorescence in C2C12 myoblasts transfected with DUX4. The C-terminal antibodies P4H2, P2B1 and E5-5 are specific to DUX4 and do not recognize DUX4c, whereas the N-terminal antibodies P2G4 and E14-3 recognize both DUX4 and DUX4c.
For western blotting, protein lysates were prepared by resuspension in standard Laemmli buffer and sonicated briefly. Equivalent amounts of test samples were loaded onto 4–12% gradient gel and transferred to nitrocellulose membrane, which were then blocked with 5% non-fat dry milk in PBS 0.1% Tween-20. Custom monoclonal antibodies (Epitomics, Burlington, CA) raised against DUX4 were used to probe the blots and detected by ECL reagent (Pierce, Rockford, IL). Membranes were stripped and reprobed with anti-α-tubulin antibody (Sigma-Aldrich, St Louis, MO) for loading control. Immunoprecipitation was performed on samples resuspended in PBS with protease inhibitor cocktail (Roche) by incubating overnight at 4°C with pooled anti-DUX4 rabbit monoclonal antibodies bound to protein A- and G-coupled Dynabeads (Invitrogen, Carlsbad, CA). Samples were eluted directly into Laemmli buffer and analyzed on western blot as described. For immunofluorescence, cells were fixed in 2% paraformaldehyde for 7 min and permeabilized in 1% Triton X-100 in PBS for 10 min at room temperature. Cells were probed with pairs of rabbit and mouse primary antibodies raised against N- or C-terminus of DUX4 diluted in PBS overnight at 4°C. Double labeling was detected with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen) at 1
500 in PBS for 1 hr and counterstained with DAPI.
DUX4 IHC on frozen tissue
Immunohistochemistry was performed by the FHCRC Experimental Histopathology Shared Resource. Six-micron sections of OCT embedded frozen de-identified human testes tissue were sectioned and fixed for 10 minutes in 10% neutral buffer formalin. The slides were rehydrated in TBS-T wash buffer, permeablized with 0.1% triton X-100 for 10 minutes, and then endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide (Dako, Carpinteria, CA) for 8 minutes. Five minute incubation in 50% acetone and 50% methanol was used for antigen retrieval on a subset of slides. Protein block containing 0.25% casein and 0.1% Tween 20 was applied for 10 minutes. Slides were incubated over night at 4 degrees C with a 1
5 dilution of either clone E5-5 or P2B1 in a 0.3 M NaCl antibody diluent containing 1% BSA. Staining was developed using Mach2 HRP-labeled polymers (Biocare Medical, Concord, CA). The staining was visualized with 3,3′-diaminobenzidine (DAB, Dako, Cupertino, CA) for 8 minutes, and the sections were counter-stained with hematoxylin (Dako) for 2 minutes. Concentration matched isotype control slides were run for each tissue sample (Jackson ImmunoResearch).