DUX4 protein, human

DNA methylation was analyzed by BSS assay. BS conversion was performed on 1 μg of genomic DNA using the EpiTect Bisulfite Kit (Qiagen) as per the manufacturer’s instructions, and 200 ng of converted genomic DNA was used per PCR. For the 4qA BSS analysis, converted DNA was amplified by nested PCR using oligonucleotide primers and thermocycling conditions that amplify 4qA but not 4qB; the initial PCR was performed with oligonucleotide primers BSS1438F (5’-GTTTTGTTGGAGGAGTTTTAGGA) and BSS3742R (5’-AACATTCAACCAAAATTTCACRAAA) and then followed by nested PCR with oligonucleotide primers BSS1438F and BSS3626R (5’-AACAAAAATATACTTTTAACCRCCAAAAA) using 10% of the first PCR product as template. Polymorphic nucleotide changes that preferentially amplify the 4A subtelomeric region are underlined. The BSS3742R sequence does not exist in 4B or 10B and utilizes a polymorphic change at bp 7946 in FJ439133 to eliminate 10A166, and BSS3626R utilizes polymorphic changes at bp 7827 in FJ439133 to eliminate 10A, 4B, and 10B [15 (link)]. All PCRs were performed using GoTaq Hot Start Polymerase (Promega) as follows: 94°C for 2 min, 25 cycles of 94°C for 15 sec, 58°C for 20 sec, and 72°C for 50 sec, followed by a final extension at 72°C for 10 min. The 593-bp PCR product spans the end of full-length DUX4 exon 1 to the beginning of DUX4 exon 3, therefore allowing specific analysis of the methylation status of the most distal 4qA D4Z4 repeat, which contains 57 CpGs (Additional file 1: Figure S1A). For the 4qA-L BSS analysis, converted DNA was similarly amplified by nested PCR. The initial PCR was performed with oligonucleotide primers BSS4qALF (5’-TTATTTATGAAGGGGTGGAGTTTGTT) and BSS3742R, and then followed by nested PCR with oligonucleotide primers 4qALF and BSS3626R using 10% of the first PCR product as template. All PCRs were performed using GoTaq Hot Start Polymerase (Promega) as follows: 94°C for 2 min, 25 cycles of 94°C for 15 sec, 58°C for 20 sec, and 72°C for 30 sec followed by a final extension at 72°C for 10 min. The 354-bp PCR product spans the 3’ end of the extended 4qA-L D4Z4 repeat to the beginning of DUX4 exon 3, therefore allowing specific analysis of the methylation status of the most distal 4qA D4Z4 repeat sequence, which contains 30 CpGs (Additional file 1: Figure S1D). When no PCR product was obtained with either the 4qA- or 4qA-L-specific BS PCRs, DNA methylation status of same distal D4Z4 region was analyzed using primer BSS3702R (5’-AAAACCAACRAACTCCCTTACAC) instead of BSS3626R. BSS3702R amplifies distal D4Z4 from both 10A and 4A. For the DUX4 5’ region, BS-converted DNA was amplified by nested PCR as described above. The initial PCR was performed with oligonucleotide primers BSS167F (5’-TTTTGGGTTGGGTGGAGATTTT) and BSS1036R (5’-AACACCRTACCRAACTTACACCCTT), and then followed by nested PCR with oligonucleotide primers BSS475F (5’-TTAGGAGGGAGGGAGGGAGGTAG) and BSS1036R. A polymorphic nucleotide change at bp 6748 in FJ439133 (underlined) was used to preferentially amplify the 4A subtelomeric region. This 578-bp PCR product contains 61 CpGs to preferentially analyze the methylation status of the DUX4 5’ region of chromosome 4-type D4Z4 repeats (Additional file 1: Figure S1E).
All BS PCR products were cloned into the pGEM-T Easy Vector system I (Promega) for sequencing analysis. At least 10 clones were sequenced for each subject and their methylation status was analyzed using web-based analysis software BISMA (http://biochem.jacobs-university.de/BDPC/BISMA/) [36 (link)] with the default parameters. Default parameters have a lower threshold of 90% identity to the reference sequence, a lower threshold of BS conversion rate of 95%, and remove identical sequences derived from the same genomic template based on conversion artifacts. To remove PCR amplification bias, 1 CpG in BSS3626R primer and 2 CpGs in BSS1036R primer were removed from the analysis; therefore, a total of 56 CpGs, 30 CpGs, and 59 CpGs were analyzed for the 4qA, 4qA-L, and DUX4 5’ region, respectively. The “R” designation in primer sequences represents a purine (A or G).

Twenty micrograms of total protein extracts were separated by electrophoresis (4–12% PAGE-SDS) in MOPS buffer at 100 V for 3h30 and transferred to a nitrocellulose membrane at 260 mA for 1h45 in a blotting buffer (PBS, 25 mM Tris, 192 mM Glycine, 20% methanol). Protein transfer was confirmed by Ponceau red staining of the membrane. After rinsing in PBS and blocking with PBS-milk 5% for 1 h at RT, the membrane was incubated overnight with either primary antibodies: MAb 9A12 mouse anti-DUX4 (1/1000), rat anti-DUX4c 860 serum (1/1000), or rabbit anti-DUX4c serum (1/1000) diluted in PBS-2% BSA followed by rinsing in PBS and incubation 1 h at RT with secondary antibodies coupled to HRP at 1/5000 dilution. Revelation was performed with either the Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific) for endogenous protein detection or Lumi-Light Western Blotting Substrate (Roche) for overexpressed protein detection on Hyperfilm ECL (Amersham).

Putative LEUTX enhancer regions 1 and 2 were predicted from Tet-On DUX4 hESC NET-CAGE dataset.⁹ (link) Putative CRX enhancer and promoter regions were predicted from NET-CAGE data introduced in this study. The guide RNAs targeting the each of the putative enhancers or promoters were designed using the Benchling CRISPR tool (https://benchling.com), targeting them to the proximal promoters (−400 to −50 base pairs from transcription start site) or +/−200 base pairs of the putative enhancer midpoint. Guide sequences were selected according to their on- and off-target score and position. Guide RNA transcriptional units (gRNA-PCR) were prepared by PCR amplification with Phusion polymerase (Thermo Fisher), using as template U6 promoter and terminator PCR products amplified from pX335 together with a guide RNA sequence-containing oligo to bridge the gap. The oligos for guide RNA transcriptional units are as in (Balboa et al., 2015).⁶⁷ (link) PCR reaction contained 50 pmol forward and reverse primers, 2 pmol guide oligo, 5 ng U6 promoter and 5 ng terminator PCR products in a total reaction volume of 100μL. The PCR reaction program was 98°C/10 sec, 56°C/30 sec, 72°C/12 sec for 35 cycles. Amplified gRNA-PCRs were purified and transfected to HEK293 cells.
HEK 293 cells were seeded on tissue culture treated 24-well plates one day prior to transfection (5 × 104 cells/well). Cells were transfected using FuGENE HD transfection reagent (Promega) in fibroblast culture medium with 500 ng of dCas9VP192 transactivator encoding plasmid and 200 ng of guide RNA-PCR product or TdTomato guide RNA plasmid. Cells were cultured for 72 h post-transfection, after which samples were collected for qRT-PCR. Successful activation of LEUTX and CRX was confirmed by qPCR.
In order to introduce LEUTX guides to DD-dCas9 activator cell line, guide cassettes containing either four guide oligos targeting LEUTX promoter or five guide oligos targeting enhancers 1 or 2 were assembled in a GoldenGate reaction using the four different LEUTX promoter guide oligos and 5 different guide oligos targeting enhancers 1 and 2 as described in (Balboa et al., 2015).⁶⁷ (link) Guide cassettes containing both promoter and enhancer guides was further cloned together. Finally, the guide cassettes were cloned to piggyBac vector. Primer sequences for promoter and enhancer guide oligos are provided in Table S12. See Figure S5 for LEUTX enhancer validation.