Human DUX4 and mouse Dux interact with STAT1 and broadly inhibit interferon-stimulated gene induction

  1. Amy E Spens
  2. Nicholas A Sutliff
  3. Sean R Bennett
  4. Amy E Campbell
  5. Stephen J Tapscott  Is a corresponding author
  1. Human Biology Division, Fred Hutchinson Cancer Research Center, United States
  2. Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, United States
  3. Clinical Research Division, Fred Hutchinson Cancer Research Center, United States
  4. Department of Neurology, University of Washington, United States
7 figures, 1 table and 3 additional files

Figures

Figure 1 with 2 supplements
DUX4 suppresses interferon-stimulated gene (ISG) induction.

(A) MB135 cells expressing doxycycline-inducible DUX4 (MB135-iDUX4), parental MB135 cells, or MB135 cells expressing doxycycline-inducible DUXB (MB135-iDUXB) were untreated, treated with IFNγ, or treated with doxycycline and IFNγ. RT-qPCR was used to evaluate expression of a DUX4 target gene, ZSCAN4, and ISGs IFIH1, ISG20, CXCL9, and CD74. Ct values were normalized to the housekeeping gene RPL27. Data represent the mean ± SD of three biological replicates with three technical replicates each. See Figure 1—figure supplement 2 for biological replicates in independent cell lines. (B) MB135-iDUX4 cells were untreated, treated with either IFNβ (type 1 IFN pathway), poly(I:C) (IFIH1/MDA5 pathway), 5’ppp-dsRNA (DDX58/RIG-I pathway), or cGAMP (cGAS/STING pathway), or treated with doxycycline and the same immune reagent. RT-qPCR was used to evaluate expression of IFIH1, ISG20, CXCL9, and CD74. Ct values were normalized to the housekeeping gene RPL27. Data represent the mean ± SD of three biological replicates with three technical replicates each (unpaired t-test; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns p>0.05).

Figure 1—figure supplement 1
Schematics of constructs cloned for use in this study.

(A) Schematic depiction of each transgene used in this study highlighting the N-terminal homeodomains (light gray in DUX4, no fill in DUXB, light green in mDux), DNA-binding HMG box (dark blue in CIC and CIC-DUX4), conserved C-terminal domain (medium gray in DUX4 and CIC-DUX4, medium green in mouse Dux), (L)LxxL(L) (black in DUX4 and CIC-DUX4, dark green in mouse Dux), mutations (* and black bar F67A, * replacement of LDELL with AAEAA), and 3xFLAG-NLS cassette regions (no fill). ‘3XFLAG’ refers to a triplicated FLAG tag sequence. ‘NLS’ refers to two independent NLS sequences, derived from SV40 and the eight amino acid NLS from SMCHD1 (PPKRMRRE), which have been mapped functionally (Hiramuki and Tapscott, 2018). (B) MUSCLE alignment of the terminal ~50 aa of the human DUX4, mutated human DUX4 (mL1, dL2, mL1dL2), and mouse Dux constructs used in this study.

Figure 1—figure supplement 2
Biological replicates in independent cell lines for each DUX4 construct.

Additional subcloned MB135 cell lines of the iDUX4 (A), iDUX4-F67A (B), iDUX4-CTD (C), iDUX4mL1dL2 (D), iDUX4-CTDmL1dL2 (E), iDUX4aa339-324 (F), and iDux (G) treated with IFNγ ± doxycycline. RT-qPCR shows interferon-stimulated gene (ISG) expression graphed as a % of IFNγ-only. Data represent the mean ± SD of two or three biological replicates (see individual construct data points) with three technical replicates each (unpaired t-test, ****p<0.0001, ***p<0.005, **p<0.01, *p<0.05, ns = nonsignificant). Immunofluorescence panels show protein expression and nuclear localization using an antibody against the N-terminal (anti-DUX4 [E14-3]) or C-terminal (anti-DUX4 [E5-5]) residues of DUX4 as appropriate for the construct.

DUX4 transcriptional activity is not necessary for interferon-stimulated gene (ISG) suppression, whereas the C-terminal domain (CTD) is both necessary and sufficient.

(A) MB135 cell lines with the indicated doxycycline-inducible transgene ± doxycycline were evaluated for ZSCAN4 expression by RT-qPCR as a measure of the ability of the construct to activate a DUX4-target gene. Ct values were normalized to the housekeeping gene RPL27. Data represent the mean ± SD of three biological replicates with three technical replicates each. (B–D) MB135 cell lines with the indicated doxycycline-inducible transgene were treated with IFNγ ± doxycycline. Light gray, N-terminal boxes, homeodomains; medium gray, C-terminal box, conserved region of CTD; black, C-terminal boxes, (L)LxxL(L) motifs; * indicates sites of mutation for F67A in HD1 and mutation of first LLDELL to AADEAA. See Figure 1—figure supplement 1 for additional description of 3XFLAG and NLS cassette. RT-qPCR was used to evaluate expression of IFIH1, ISG20, CXCL9, and CD74 and Ct values were normalized to the housekeeping gene RPL27, then normalized to the IFNγ-only treatment to set the induced level to 100%. Data represent the mean ± SD of three biological replicates with three technical replicates each (unpaired t-test; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns p>0.05). See Figure 1—figure supplement 2 for additional cell lines.

The DUX4 protein interacts with STAT1 and additional immune response regulators.

Left panel, representative candidate interactors identified by mass spectrometry of proteins that co-immunoprecipitated with the DUX4-CTD and their relative ranking in the candidate list (see Supplementary file 2 for full list). Right panel, validation western blot of proteins that co-immunoprecipitate with the DUX4-CTD in cell lysates from MB135 cells expressing doxycycline-inducible 3xFLAG-DUXB or 3xFLAG-DUX4-CTD, ± IFNγ treatment. Data represent biological duplicates. See Figure 3—source data 1 for uncropped/raw images.

Figure 3—source data 1

Validation co-IP from inducible MB135 cells lines, anti-FLAG.

Western blot showing anti-FLAG signal. ‘*’ marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Bottom blot (boxed in green) is relevant for this figure and was probed with anti-FLAG. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data1-v3.zip
Figure 3—source data 2

Validation co-IP from inducible MB135 cell lines, anti-DDX3X.

Western blot showing anti-DDX3X signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Top right blot (boxed in green) is relevant for this figure and was probed with anti-DDX3X. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data2-v3.zip
Figure 3—source data 3

Validation co-IP from inducible MB135 cell lines, anti-STAT1.

Western blot showing anti-STAT1 signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Middle blot (boxed in green) is relevant for this figure and was probed with anti-STAT1. The multiple bands represent the alpha (upper) and beta (lower) isoforms of STAT1. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data3-v3.zip
Figure 3—source data 4

Validation co-IP from inducible MB135 cell lines, anti-PRKDC.

Western blot showing anti-PRKDC signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Top blot (boxed in green) is relevant for this figure and was probed with anti-PRKDC. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data4-v3.zip
Figure 3—source data 5

Validation co-IP from inducible MB135 cell lines, anti-YBX1.

Western blot showing anti-YBX1 signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Bottom blot (boxed in green) is relevant for this figure and was probed with anti-YBX1. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data5-v3.zip
Figure 3—source data 6

Validation co-IP from inducible MB135 cell lines, anti-hnRNPM.

Western blot showing anti-hnRNPM signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Middle blot (boxed in green) is relevant for this figure and was probed with anti-hnRNPM. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data6-v3.zip
Figure 3—source data 7

Validation co-IP from inducible MB135 cell lines, anti-hnRNPK.

Western blot showing anti-hnRNPK signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Top blot (boxed in green) is relevant for this figure and was probed with anti-hnRNPK. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data7-v3.zip
Figure 3—source data 8

Validation co-IP from inducible MB135 cell lines, anti-PPP2R1A.

Western blot showing anti-PPP2R1A signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Bottom right blot (boxed in green) is relevant for this figure and was probed with PPP2R1A. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data8-v3.zip
Figure 3—source data 9

Validation co-IP from inducible MB135 cell lines, anti-PABPC1.

Western blot showing anti-PABPC1 signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Top blot (boxed in green) is relevant for this figure and was probed with anti-PABPC1. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data9-v3.zip
Figure 3—source data 10

Validation co-IP from inducible MB135 cell lines, anti-NCL.

Western blot showing anti-NCL signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Blot was probed with anti-NCL. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data10-v3.zip
Figure 3—source data 11

Validation co-IP from inducible MB135 cell lines, anti-CDK4.

Western blot showing anti-CDK4 signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Middle right blot (boxed in green) is relevant for this figure and was probed with anti-CDK4. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data11-v3.zip
Figure 3—source data 12

Validation co-IP from inducible MB135 cell lines, anti-hnRNPU.

Western blot showing anti-hnRNPU signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Bottom blot (boxed in green) is relevant for this figure and was probed with anti-hnRNPU. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data12-v3.zip
Figure 3—source data 13

Validation co-IP from inducible MB135 cell lines, anti-TRIM28.

Western blot showing anti-TRIM28 signal. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Top blot (boxed in green) is relevant for this figure and was probed with anti-TRIM28. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig3-data13-v3.zip
Figure 4 with 2 supplements
The DUX4-CTD preferentially interacts with pSTAT1-Y701.

(A) Western blot showing input and immunoprecipitated proteins from either 3xFLAG-iDUXB (DUXB) or a truncation series of the 3x-FLAG-iDUX4-CTD cells (iDUX4) precipitated with anti-FLAG and probed with the indicated antibodies. Serial deletions of the iDUX4-CTD were assayed as indicated. All samples were treated with IFNγ.An asterisk indicates the correct band for each FLAG-tagged construct. See Figure 4—source data 1 for uncropped/raw Western blots. (B) Input and anti-FLAG immunoprecipitation from 3xFLAG-iDUXB or 3x-FLAG-iDUX4-CTD cells co-expressing doxycycline-inducible 3xMYC-iSTAT1, -iSTAT1-Y701A, or -iSTAT1-S727A with or without IFNγ treatment and probed with the indicated antibodies. An ‘x’ indicates the endogenous (non-MYC tagged) STAT1 band. See Figure 4—source data 1 for uncropped/raw Western blots. (C) Proximity ligation assay (PLA) showing co-localization of endogenous STAT1 and pSTAT1 701 with the iDUX4-CTD compared to the interaction with the DUXB-CTD, in the nuclear compartment of IFNγ- and doxycycline-treated MB135 cells. Mean fluorescent intensity (MFI) of the nuclei in the PLA channel was measured for 10 images per cell line and treatment and plotted (unpaired t-test; ****p<0.0001).

Figure 4—source data 1

Co-IP from inducible MB135 cell lines, anti-STAT1.

Western blot showing anti-STAT1 signal for Figure 4A. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Top left blot (boxed in green) is relevant for this figure and was probed with anti-STAT1. The multiple bands represent the alpha (upper) and beta (lower) isoforms of STAT1.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data1-v3.zip
Figure 4—source data 2

Co-IP from inducible MB135 cell lines, anti-pSTAT1(Y701).

Western blot showing anti-pSTAT1(Y701) signal for Figure 4A. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple unrelated blots were imaged in this exposure/file. Top blot (boxed in green) is relevant for this figure and was probed with anti-pSTAT1(Y701). Protein ladder appears in white light channel. Signal from ECL only appears in the chemiluminescence channel. The multiple bands represent the alpha (upper) and beta (lower) isoforms of STAT1.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data2-v3.zip
Figure 4—source data 3

Co-IP from inducible MB135 cell lines, anti-pSTAT1(S727).

Western blot showing anti-pSTAT1(S727) signal for Figure 4A. * marks correct size band. This blot is probed with anti-pSTAT1(S727). Protein ladder appears in white light channel. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data3-v3.zip
Figure 4—source data 4

Co-IP from inducible MB135 cell lines, anti-FLAG.

Western blot showing anti-FLAG signal for Figure 4A. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple unrelated blots were imaged in this exposure/file. Bottom left blot (boxed in green) is probed with anti-FLAG. Protein ladder appears in white light channel. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data4-v3.zip
Figure 4—source data 5

Co-IP from dual-inducible MB135 cell lines, anti-MYC.

Western blot showing anti-MYC signal for Figure 4B. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple separate blots were imaged in this exposure/file. Top blot (boxed in green) is relevant for this figure and was probed with anti-MYC to detect the INDUCIBLE MYC-tagged STAT1 or STAT1-mutant transgene. Signal from ECL only appears in the chemiluminescence channel. Protein ladder appears in white light channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data5-v3.zip
Figure 4—source data 6

Co-IP from dual-inducible MB135 cell lines, anti-STAT1.

Western blot showing anti-STAT1 signal for Figure 4B. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple unrelated blots were imaged in this exposure/file. Middle blot (boxed in green) is probed with anti-STAT1 to detect the INDUCIBLE MYC-tagged STAT1 transgene. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data6-v3.zip
Figure 4—source data 7

Co-IP from dual-inducible MB135 cell lines, anti-pSTAT1(Y701).

Western blot showing anti-pSTAT1(Y701) signal for Figure 4B. * marks correct size band. Blot is probed with anti-pSTAT1(Y701) to detect the phosphorylated INDUCIBLE MYC-tagged STAT1 or MYC-tagged STAT1-mutant transgene. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data7-v3.zip
Figure 4—source data 8

Co-IP from dual-inducible MB135 cell lines, anti-pSTAT1(S727).

Western blot showing anti-pSTAT1(S727) signal for Figure 4B. * marks correct size band for the INDUCIBLE MYC-tagged STAT1 or mutated-STAT1. Lower band represents endogenous STAT1. Blot is probed with anti-pSTAT1(S727). Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data8-v3.zip
Figure 4—source data 9

Co-IP from dual-inducible MB135 cell lines, anti-FLAG.

Western blot showing anti-FLAG signal for Figure 4B. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple unrelated blots were imaged in this exposure/file. Lower blot (boxed in green) is probed with anti-FLAG to detect the INDUCIBLE FLAG-tagged DUXB or DUX4-CTD transgene. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig4-data9-v3.zip
Figure 4—figure supplement 1
Expression of the DUX4-CTD does not prevent translocation of STAT1 to the nucleus.

Immortalized MB135 myoblasts with doxycycline-inducible iDUX4-CTD (A) and iDUXB (B) transgenes were treated ± doxycycline and ± IFNγ, then fixed and stained for total STAT1 and either transgene. Both cell lines show good induction and nuclear translocation of STAT1 restricted to the +IFNγ condition, and good doxycycline-dependent induction of the transgene. (C) Phosphorylation of STAT1 and nuclear translocation of pSTAT1-Y701 is equally restricted to the +IFNγ condition in MB135iDUX4CTD and MB135iDUXBCTD cell lines. Immunofluorescence staining for pSTAT1-Y701 shows no phosphorylation or nuclear translocation of STAT1 in untreated cells, and strong localization of pSTAT1-Y701 to the nucleus only with IFNγ treatment. Mean fluorescent intensity (MFI) of nuclear pSTAT1-Y701 signal was measured from two fields per treatment per cell line and plotted (right). There was significant nuclear enrichment of pSTAT1-Y701 with IFNγ treatment compared to untreated cells in both immortalized transgenic cells lines (unpaired t-test, **** p<00001, ns = nonsignificant; n = 26 DUX4CTD untreated, n = 36 DUX4CTD + IFNγ, n = 43 DUXBCTD untreated, and n = 53 DUXBCTD + IFNγ). (D) Primary human foreskin fibroblasts (HFF 1°), primary MB135 myoblasts (MB135 1°), and immortalized MB135 myoblasts (MB135 immortalized) were treated ± IFNγ, then fixed and stained for total STAT1. Background staining noise was similar across cell lines in the untreated condition, and all cell lines showed good induction and nuclear localization of STAT1 in the + IFNγ condition.

Figure 4—figure supplement 2
Primary human foreskin fibroblasts (HFFs) expressing transgenic DUX4CTD show increased interaction with STAT1 and reduced MHC I activation with IFNγ treatment.

Primary HFFs (HFF 1°) expressing no transgene (noTG), constitutive 3XFLAG-DUXBCTD, or constitutive 3XFLAG-DUX4CTD were treated with IFNγ, and then fixed and used in a proximity ligation assay (PLA) to determine interaction between the FLAG-tagged transgenes and total STAT1. Post-PLA the cells were re-stained with an aFLAG antibody to determine transgene-positive (TG+) nuclei based on fluorescence. Mean fluorescent intensity (MFI) in the PLA channel of TG+ nuclei from five images per cell line was calculated and plotted (right; ‘n’ = total number of TG+ nuclei analyzed). TG+ DUX4 CTD nuclei had significantly more interaction with total STAT1 compared to TG+ nuclei of the DUXBCTD cell line (one-way ANOVA with multiple comparisons, ****p<0.0001, **p<0.01, ns = nonsignificant). The PLA signal, which relies on presence and proximity of both the aFLAG and aSTAT1 antibodies, was higher in TG+ nuclei of both cell lines as expected.

The DUX4-CTD decreases STAT1 occupancy at interferon-stimulated gene (ISG) promoters and blocks Pol-II recruitment.

(A, left four panels) Chromatin immunoprecipitation using anti-STAT1 or IgG from MB135-iDUX4-CTD cells untreated, IFNγ-treated, or IFNγ and doxycycline treated. Ab1: 50:50 mix of STAT1 antibodies Abcam ab239360 and ab234400; Ab2: Abcam ab109320. ChIP-qPCR analysis relative to a standard curve constructed from purified input DNA was used to determine the quantity of DNA per IP sample, which was then graphed as a % of input. Data represent the mean ± SD of two biological replicates with three technical replicates each (unpaired t-test; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns p>0.05). (A, right panel) RT-qPCR of RNA from cells used for STAT1 ChIP showing induction of interferon-stimulated genes (ISGs) by IFNγ and suppression by DUX4-CTD. (B) CUT&Tag data showing the intensity of Pol-II signal across a 2000 bp window centered on the TSS of ISGs (left) or IFNγ-unchanged genes (right) in untreated, IFNγ-treated, or IFNγ and doxycycline-treated MB135-iDUX4-CTD cells.

Figure 6 with 1 supplement
Endogenous DUX4 suppresses interferon-stimulated gene (ISG) induction in a sarcoma cell line expressing a CIC-DUX4 fusion gene.

(A, left panel) RT-qPCR of the indicated genes in MB135 parental or Kitra-SRS that express a CIC DUX4-fusion gene containing the DUX4 CTD. Cells were transfected with control or CIC- and DUX4-targeting siRNAs. Ct values were normalized to the housekeeping gene RPL27. Data represent the mean ± SD of three biological replicates with three technical replicates each (unpaired t-test; ****p<0.0001, ***p<0.001, ** p<0.01,*p<0.05, ns p>0.05). (A, right panel) Western blot showing lysates from MB135 or Kitra-SRS cells treated with control or CIC- and DUX4-targeting siRNAs ± IFNγ and probed with the indicated antibodies. See Figure 6—source data 1 for uncropped/raw western blots. (B) RT-qPCR of the indicated genes in MB135 with an inducible CIC (MB135-iCIC) or an inducible CIC-DUX4 fusion gene (MB135-iCIC-DUX4). Cells were untreated, IFNγ-treated, or IFNγ and doxycycline-treated. Ct values were normalized to the housekeeping gene RPL27, then normalized to the IFNγ-only treatment to set the induced level to 100%. Data represent the mean ± SD of three biological replicates with three technical replicates each (unpaired t-test; **p<0.01, ns p>0.05). (C) Proximity ligation assay (PLA) of KitraSRS cells showing association of the endogenous CIC-DUX4 fusion protein with either total STAT1 or phosphorylated STAT1-Y701 exclusively when cells were treated +IFNγ. Mean fluorescent intensity (MFI) was quantified from 200 nuclei per condition and plotted for both pairs of antibodies (unpaired t-test; ****p<0.0001).

Figure 6—source data 1

Parental MB135 anti-CIC.

Western blot showing anti-CIC signal for Figure 6B. * marks correct size band. Blot was probed with anti-CIC. NOTE: gel was loaded and transferred with samples ordered as labeled here. The image has been flipped in the article, and the labels flipped appropriately to mirror the protein layout in the Kitra-SRS experiment. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig6-data1-v3.zip
Figure 6—source data 2

Parental MB135 anti-GAPDH.

Western blot showing anti-GAPDH signal for Figure 6B. * marks correct size band. Blot was probed with anti-GAPDH. Note that gel was loaded and transferred with samples ordered as labeled here. The image has been flipped in the article and the labels flipped appropriately to mirror the protein layout in the Kitra-SRS experiment. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig6-data2-v3.zip
Figure 6—source data 3

KitraSRS anti-CIC.

Western blot showing anti-CIC signal for Figure 6B. * marks correct size band. Blot was cut into two pieces, this piece was probed with anti-CIC. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig6-data3-v3.zip
Figure 6—source data 4

KitraSRS anti-GAPDH.

Western blot showing anti-GAPDH signal for Figure 6B. * marks band of correct size. Blot was cut into two pieces, this piece was probed with anti-GAPDH. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig6-data4-v3.zip
Figure 6—figure supplement 1
Knockdown of the CIC-DUX4 fusion protein in Kitra-SRS cells rescues upregulation of MHC I in response to IFNγ.

MB135 parental myoblasts (A) and Kitra-SRS sarcoma cells (B) were treated with control siRNAs or a combination of siRNAs targeting CIC and DUX4, then left untreated or treated with IFNγ. While knockdown of the endogenous CIC in MB135 cells had no effect on MHC I upregulation in IFNγ-treated cells, knockdown of the CIC-DUX4 fusion protein in Kitra-SRS cells increased MHC I-positive cells from 27.9% to 48.1%.

Figure 7 with 1 supplement
Conservation of interferon-stimulated gene (ISG) repression in facioscapulohumeral dystrophy (FSHD) myoblasts and ISG repression and STAT1 interaction by mouse Dux.

(A) FSHD MB200 myoblasts were differentiated into myotubes, which results in the expression of endogenous DUX4 in a subset of myotubes. Cultures were treated ± IFNγ, and DUX4 and IDO1 were visualized by immunofluorescence. Representative images of untreated and IFNγ-treated (two fields, F1 and F2) cells are shown, with white arrows highlighting DUX4+ myotubes that lack IDO1 signal. Mean fluorescent intensity (MFI) of the αDUX4 and αIDO1 nuclear signal was measured in the IFNγ-treated cells only. Data represent the mean ± SD of nuclear MFI from three images, total nuclei per condition listed as ‘n’ (unpaired t-test; **p<0.01). (B, left panel) RT-PCR of the indicated genes in MB135-iDux cells untreated or treated with IFNγ ± doxycycline. Ct values were normalized to the housekeeping gene RPL27, then normalized to the IFNγ-only treatment to set the induced level to 100%. Data represent the mean ± SD of three biological replicates with three technical replicates each (unpaired t-test; ****p<0.0001, **p<0.01). (B, right panel) Western blot showing input and immunoprecipitated proteins from either 3xFLAG-iDux or 3x-FLAG-iDUXB cells ± IFNγ precipitated with anti-FLAG and probed with the indicated antibodies. See Figure 7—source data 1 for uncropped/raw Western blots. (C) A model supported by the data showing how the DUX4-CTD might prevent STAT1 ISG induction. (Top) In the absence of the DUX4-CTD, pSTAT1 Y701 (black ‘P’) dimerizes, translocates to the nucleus, binds its GAS motif in the ISG promoter, acquires secondary phosphorylation at S727 (gray ‘P’), and recruits a stable transcription complex that includes Pol-II to drive transcription of ISGs. (Bottom) In the presence of the DUX4-CTD, STAT1 is phosphorylated, translocates to the nucleus, and binds its GAS motif as evidenced by the pSTAT1 S727 in complex with the CTD. However, diminished steady-state occupancy of STAT1 at the ISG promoters and absence of Pol-II recruitment indicate that the STAT1-DUX4-CTD complex does not stably bind DNA and fails to recruit Pol-II and the pre-initiation complex. The (L)LXXL(L) motifs (black bars in DUX4-CTD) are necessary to interfere with transcription suppression and likely prevent STAT1 from interacting with a factor in the pre-initiation complex or recruit a co-repressor.

Figure 7—source data 1

Mouse Dux co-IP, anti-STAT1.

Western blot showing anti-STAT1 signal for Figure 7B. * marks correct size bands. Blot was probed intact for STAT1. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel. The double bands marked by the * represent the alpha (upper) and beta (lower) isoforms of endogenous STAT1.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig7-data1-v3.zip
Figure 7—source data 2

Mouse Dux co-IP, anti-pSTAT1(Y701).

Western blot showing anti-pSTAT1(Y701) signal for Figure 7B. * marks correct size bands. Blot was stripped from previous exposure and re-probed with anti-pSTAT1(Y701). Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel. The double bands marked by the * represent the alpha (upper) and beta (lower) isoforms of endogenous STAT1.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig7-data2-v3.zip
Figure 7—source data 3

Mouse Dux co-IP, anti-pSTAT1(S727).

Western blot showing anti-pSTAT1(S727) signal for Figure 7B. * marks correct size band. Blot was stripped from previous exposure and re-probed with anti-pSTAT1(S727). Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel. Only STAT1-alpha can be phosphorylated at S727, hence the lack of double band.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig7-data3-v3.zip
Figure 7—source data 4

Mouse Dux co-IP, anti-FLAG.

Western blot showing anti-FLAG signal for Figure 7B. * marks correct size band. Blot was physically cut to probe with multiple antibodies, multiple unrelated blots were imaged in this exposure/file. Lower blot (boxed in green) is probed with anti-FLAG to detect inducible FLAG-tagged transgenes. Note that the image has been flipped in the article and labeled appropriately. Protein ladder only appears in the ‘white light’ exposure. Signal from ECL only appears in the chemiluminescence channel.

https://cdn.elifesciences.org/articles/82057/elife-82057-fig7-data4-v3.zip
Figure 7—figure supplement 1
Mouse Dux contains a triplication of the (L)LxxL(L)-containing region.

(A) Mouse Dux protein sequence with homeodomains in bold and (L)LxxL(L) motifs underlined. (B) Alignment of a partial triplication of the mouse Dux protein with aa258-440 aligning with aa441-623 and aa624-650 aligning with aa258-284.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-STAT1 (phospho 701) [M135] (mouse monoclonal)AbcamCat# ab29045; RRID:AB_778096See 'Materials and methods' for dilution by application
AntibodyAnti-STAT1 [1/Stat1] (mouse monoclonal)AbcamCat# ab281999See 'Materials and methods' for dilution by application
AntibodyAnti-hnRNP M1-M4 [EPR13509(B)] (rabbit monoclonal)AbcamCat# ab177957; RRID:AB_2820246See 'Materials and methods' for dilution by application
AntibodyAnti-human DNA PKcs [Y393] (rabbit monoclonal)AbcamCat# ab32566; RRID:AB_731981See 'Materials and methods' for dilution by application
AntibodyAnti-PABPC1 (rabbit polyclonal)AbcamCat# ab21060; RRID:AB_777008See 'Materials and methods' for dilution by application
AntibodyAnti-STAT1 (phospho S727) [EPR3146] (rabbit monoclonal)AbcamCat# ab109461; RRID:AB_10863745See 'Materials and methods' for dilution by application
AntibodyAnti-STAT1 [EPR21057-141] (rabbit monoclonal)AbcamCat# ab234400See 'Materials and methods' for dilution by application
AntibodyAnti-STAT1 [EPR23049-111] (rabbit monoclonal)AbcamCat# ab239360See 'Materials and methods' for dilution by application
AntibodyAnti-STAT1 [EPR4407] (rabbit monoclonal)AbcamCat# ab109320; RRID:AB_10863383See 'Materials and methods' for dilution by application
AntibodyAnti-YBX1 [EP2708Y] (rabbit monoclonal)AbcamCat# ab76149; RRID:AB_2219276See 'Materials and methods' for dilution by application
AntibodyAnti-mouse IgG for IP HRP (rat monoclonal)AbcamCat# AB131368; RRID:AB_2895114See 'Materials and methods' for dilution by application
AntibodyIsotype control (rabbit polyclonal)BioLegendCat# CTL-4112See 'Materials and methods' for dilution by application
AntibodyAnti-DDX3X [D19B4] (mouse monoclonal)Cell Signaling TechnologyCat# 8192; RRID:AB_10860416See 'Materials and methods' for dilution by application
AntibodyAnti-hnRNP K [R332] (rabbit monoclonal)Cell Signaling TechnologyCat# 4675; RRID:AB_10622190See 'Materials and methods' for dilution by application
AntibodyAnti-IDO1 [D5J4E] (rabbit monoclonal)Cell Signaling TechnologyCat# 86630; RRID:AB_2636818See 'Materials and methods' for dilution by application
AntibodyAnti-MYC [71D10] (rabbit monoclonal)Cell Signaling TechnologyCat# 2278; RRID:AB_490778See 'Materials and methods' for dilution by application
AntibodyAnti-Nucleolin [D4C70] (rabbit monoclonal)Cell Signaling TechnologyCat# 14574; RRID:AB_2798519See 'Materials and methods' for dilution by application
AntibodyAnti-phospho Rbp1 CTD (Ser5) [D9N5I] (rabbit monoclonal)Cell Signaling TechnologyCat# 13523; RRID:AB_2798246See 'Materials and methods' for dilution by application
AntibodyAnti-PP2A A subunit [81G5] (rabbit monoclonal)Cell Signaling TechnologyCat# 2041; RRID:AB_2168121See 'Materials and methods' for dilution by application
AntibodyAnti-pSTAT1 Y701 [58D6] (rabbit monoclonal)Cell Signaling TechnologyCat #9167See 'Materials and methods' for dilution by application
AntibodyAnti-TIF1 (TRIM28) [C42G12] (rabbit monoclonal)Cell Signaling TechnologyCat# 4124; RRID:AB_2209886See 'Materials and methods' for dilution by application
AntibodyAnti-rabbit secondary antibody (goat mixed monoclonal)EpiCypherCat# 13-0047Used in CUT&Tag
AntibodyAnti-DUX4 [P2G4] (mouse monoclonal)Geng et al., 2011N/ASee 'Materials and methods' for dilution by application
AntibodyAnti-DUX4 [E14-3] (rabbit monoclonal)Geng et al., 2011N/ASee 'Materials and methods' for dilution by application
AntibodyAnti-DUX4 [E5-5] (rabbit monoclonal)Geng et al., 2011N/ASee 'Materials and methods' for dilution by application
AntibodyAnti-mouse IgG HRP (goat superclonal)InvitrogenCat# A28177See 'Materials and methods' for dilution by application
AntibodyAnti-CIC (rabbit polyclonal)InvitrogenCat# PA5-83721See 'Materials and methods' for dilution by application
AntibodyFITC-conjugated anti-rabbit (donkey monoclonal)Jackson ImmunoResearchCat# 711-095-152; RRID:AB_2315776See 'Materials and methods' for dilution by application
AntibodyTRITC-conjugated anti-mouse (donkey monoclonal)Jackson ImmunoResearchCat# 715-025-020; RRID:AB_2340764See 'Materials and methods' for dilution by application
AntibodyAnti-CDK4 (rabbit polyclonal)ProteinTechCat# 11026-1-AP; RRID:AB_2078702See 'Materials and methods' for dilution by application
AntibodyAnti-HAT1 (rabbit polyclonal)ProteinTechCat# 11432-1-AP; RRID:AB_2116435See 'Materials and methods' for dilution by application
AntibodyAnti-HNRNPU (rabbit polyclonal)ProteinTechCat# 14599-1-AP; RRID:AB_2248577See 'Materials and methods' for dilution by application
AntibodyAnti-FLAG [M2] (mouse monoclonal)Sigma-AldrichCat# F1804; RRID:AB_262044See 'Materials and methods' for dilution by application
AntibodyAnti-FLAG [M2] (mouse monoclonal)Sigma-AldrichCat# F3165; RRID:AB_259529See 'Materials and methods' for dilution by application
AntibodyAnti-rabbit IgG HRP (goat superclonal)Thermo FisherCat# A27036; RRID:AB2536099See 'Materials and methods' for dilution by application
Cell line (Homo sapiens)MB200 (male, FSHD2), immortalizedFields Center for FSHD and Neuromuscular Researchhttps://www.urmc.rochester.edu/neurology/fields-center.aspx
Cell line (H. sapiens)MB135 (female), immortalizedGeng et al., 2012N/A
Cell line (H. sapiens)MB135-iDUX4 (SSc7, female)Jagannathan et al., 2016N/A
Cell line (H. sapiens)HFF-DUX4CTDThis studyN/APrimary HFF cells transduced with the constitutive pRRLSIN-3XFLAG-NLS-DUX4CTD lentiviral expression construct
Cell line (H. sapiens)HFF-DUXB-CTDThis studyN/APrimary HFF cells transduced with the constitutive pRRLSIN-3XFLAG-NLS-DUXBCTD lentiviral expression construct
Cell line (H. sapiens)MB135-i3XFLAG-CIC (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-i3XFLAG-CIC-DUX4 (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDux-CA (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDux-CTD (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4 (ASc4, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4 (NSc2, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4-CTD (AES150-1, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4-CTD (AES150-5, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4-CTDmL1dL2 (AES150-1, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4-CTDmL1dL2 (AES150-3, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4-F67A (ASc10, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4-F67A (ASc6, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4aa154-271 (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4aa154-308 (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4aa154-372 (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4aa339-424 (NSc10, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4aa339-424 (NSc5, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4aa339-424 (NSc8, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4dL2 (NSc1, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4mL1 (NSc3, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4mL1dL2 (NSc2, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4mL1dL2 (NSc3, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUX4mL1dL2 (NSc8, female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135-iDUXB (female)This studyN/AImmortalized MB135 myoblasts transduced with the specified inducible lentiviral expression construct
Cell line (H. sapiens)MB135 (female), primaryDr. Rabi Tawil, Fields Center for FSHD Research, University of Rochester Medical CenterN/APrimary myoblast cells derived from patient muscle biopsy sample
Cell line (H. sapiens)Primary human foreskin fibroblasts (‘HFF,’ male)Dr. Dusty Miller, Fred Hutchinson Cancer CenterN/APrimary human foreskin fibroblast cells derived from patient foreskin tissue
Chemical compound, drugRIG-I ligandGift of Dr. Dan Stetson Lab, UWN/A
Chemical compound, drug2'3'-cGAMPInvivogenCat# tlrl-nacga23
Chemical compound, drugRecombinant human IFN-beta proteinR&D SystemsCat# 8499-IF-010-CF
Chemical compound, drugRecombinant human IFN-gammaR&D SystemsCat# 285IF100CF
Chemical compound, drugPolyinosinic-polycytidylic acid sodium salt [poly(I:C)]SigmaCat# P1530
Commercial assay or kitiTaq SYBR Green SupermixBio-RadCat# 1725124
Commercial assay or kitCUTANA Non-Hot Start 2X PCR Master Mix for CUT&TagEpiCypherCat# 15-1018Used in CUT&Tag
Commercial assay or kitCUTANA pAG-Tn5 for CUT&TagEpiCypherCat# 15-1017Used in CUT&Tag
Commercial assay or kitIllumina TruSeq RNA Sample Prep v2 KitIlluminaCat# RS-122-2001
Commercial assay or kitDnase Amp gradeInvitrogenCat# 18068015
Commercial assay or kitOligo(dT) 12–18 primerInvitrogenCat# 18418012
Commercial assay or kitRNaseOUT Recombinant Ribonuclease InhibitorInvitrogenCat# 10777019
Commercial assay or kitSuperscript IVInvitrogenCat# 18091050
Commercial assay or kitLipofectamine RNAiMAXLife TechnologiesCat# 13778150
Commercial assay or kitNucleoSpin RNA kitMacherey-NagelCat# 740955
Commercial assay or kitLipofectamine 2000Thermo FisherCat# 11668019
Commercial assay or kitSuperscript IV First-Strand Synthesis SystemThermo FisherCat# 18091050
OtherAgencourt AMPure XP beadsBeckman CoulterCat# A63880Used in CUT&Tag
OtherHyclone FBSFisherCat# SH3007103Used to supplement F-10 for cell culture of myoblast lines
OtherGibco Penicillin-Streptomycin (10,000 U/ml)Fisher ScientificCat# 15-140-122Anti-fungal to supplement cell culture media
OtherDynabeads Protein G beadsInvitrogenCat# 10003DUsed in fractionated anti-FLAG immunoprecipitation
OtherProLong Glass antifade Mountant with NucblueInvitrogenCat# P36983Used to mount slides for proximity ligation assays
OtherMillicell EZ Slide 8-well glass slidesMilliporeSigmaCat# PEZGS0816Used to culture cells for proximity ligation assays
OtherProtein-A agarose beadsMilliporeSigmaCat# 16-156Used in ChIP-qPCR
OtherPierce phosphatase inhibitorsPierceCat# PIA32957Used in ChIP-qPCR, CUT&Tag
OtherPierce protease inhibitors (EDTA-free)PierceCat# PIA32955Used in ChIP-qPCR, CUT&Tag
OtherRecombinant human basic fibroblast growth factorPromegaCat# G5071Used to supplement F-10 for cell culture of myoblast lines
OtherDexamethasoneSigma-AldrichCat# D4902Used to supplement F-10 for cell culture of myoblast lines
OtherDoxycycline hyclateSigma-AldrichCat# D9891Used to induce doxycycline-inducible transgenes
OtherDuolink In Situ Detection Reagents Green kitSigma-AldrichCat# DUO92014Used in PLA
OtherDuolink In Situ PLA Probe Anti-Mouse MINUSSigma-AldrichCat# DUO92004Used in PLA
OtherDuolink In Situ PLA Probe Anti-Rabbit PLUSSigma-AldrichCat# DUO92002Used in PLA
OtherInsulinSigma-AldrichCat# I1882Used in differentiating MB200 myoblasts into myotubes
OtherPolybreneSigma-AldrichCat# 107689Used in transducing cell lines with lentivirus
OtherPuromycin dihydrochlorideSigma-AldrichCat# P833Used as a selective agent for puromycin-resistant cell lines
OtherTransferrinSigma-AldrichCat# T-0665Used in differentiating MB200 myoblasts into myotubes
OtherOptiMEM Reduced Serum MediumThermo FisherCat# 31985070Used for lipofection
Recombinant DNA reagentpMD2.GDidier Trono LabAddgene#12259; RRID:Addgene_12259VSV-G envelope expressing plasmid
Recombinant DNA reagentpsPAX2Didier Trono LabAddgene#12260; RRID:Addgene_12260Lentiviral packaging plasmid
Recombinant DNA reagentpRRLSIN.cPPT.PGK-GFP.WPREDidier Trono LabAddgene#12252Constitutive lentiviral expression vector (empty backbone)
Recombinant DNA reagentpCW57.1David Root LabAddgene#41393Doxycycline-inducible lentiviral expression vector (empty backbone)
Recombinant DNA reagentpCW57.1-3xFLAG-CICThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3xFLAG-CIC/DUX4This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DuxThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4-dL2This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4-F67AThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4-mL1This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4-mL1dL2This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4(aa339-424)This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4aa154-271This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4aa154-308This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4aa154-372This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4CTDThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUX4CTDmL1dL2This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUXBThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3XFLAG-NLS-NLS-DUXBCTDThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3xMYC-STAT1This studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3xMYC-STAT1-S727AThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpCW57.1-3xMYC-STAT1-Y701AThis studyN/ALentiviral expression plasmid for doxycycline-inducible transgene expression
Recombinant DNA reagentpRRLSIN-3XFLAG-NLS-NLS-DUX4CTDThis studyN/ALentiviral expression plasmid for constitutive transgene expression
Recombinant DNA reagentpRRLSIN-3XFLAG-NLS-NLS-DUXBCTDThis studyN/ALentiviral expression plasmid for constitutive transgene expression
Sequence-based reagentIFIH1_FGeng et al., 2012. Dev Cell. doi: 10.1016/j.devcel.2011.11.013.RT-qPCR primersCTAGCCTGTTCTGGGGAAGA
Sequence-based reagentIFIH1_RGeng et al., 2012. Dev Cell. doi: 10.1016/j.devcel.2011.11.013.RT-qPCR primersAGTCGGCACACTTCTTTTGC
Sequence-based reagentISG20_FGeng et al., 2012. Dev Cell. doi: 10.1016/j.devcel.2011.11.013.RT-qPCR primersGAGCGCCTCCTACACAAGAG
Sequence-based reagentISG20_RGeng et al., 2012. Dev Cell. doi: 10.1016/j.devcel.2011.11.013.RT-qPCR primersCGGATTCTCTGGGAGATTTG
Sequence-based reagenth16q21_FMaston et al., 2012ChIP-qPCR primers (gene desert region)AAACAAGCATCAGGGTGGAC
Sequence-based reagenth16q21_RMaston et al., 2012ChIP-qPCR primers (gene desert region)GATCCCACAAAGGAAAGGAAC
Sequence-based reagentGBP1_FOrigene Cat# HP205803RT-qPCR primersTAGCAGACTTCTGTTCCTACATCT
Sequence-based reagentGBP1_ROrigene Cat# HP205803RT-qPCR primersCCACTGCTGATGGCATTGACGT
Sequence-based reagentCXCL10_FPrimer Bank ID 323422857c1, https://pga.mgh.harvard.edu/primerbank, Wang et al., 2012. Nucleic Acids Res. doi: 10.1093/nar/gkr1013.RT-qPCR primersGTGGCATTCAAGGAGTACCTC
Sequence-based reagentCXCL10_RPrimer Bank ID 323422857c1, https://pga.mgh.harvard.edu/primerbank, Wang et al., 2012. Nucleic Acids Res. doi: 10.1093/nar/gkr1013.RT-qPCR primersTGATGGCCTTCGATTCTGGATT
Sequence-based reagentIDO1_FPrimerBank ID 323668304c1, https://pga.mgh.harvard.edu/cgi-bin/primerbank/new_search2.cgi, Wang et al., 2012. Nucleic Acids Res. doi: 10.1093/nar/gkr1013.RT-qPCR primersGCCAGCTTCGAGAAAGAGTTG
Sequence-based reagentIDO1_RPrimerBank ID 323668304c1, https://pga.mgh.harvard.edu/cgi-bin/primerbank/new_search2.cgi, Wang et al., 2012. Nucleic Acids Res. doi: 10.1093/nar/gkr1013.RT-qPCR primersATCCCAGAACTAGACGTGCAA
Sequence-based reagentCXCL10_FRosowski et al., 2014ChIP-qPCR primersAAAGGAACAGTCTGCCCTGA
Sequence-based reagentCXCL10_RRosowski et al., 2014ChIP-qPCR primersGCCCTGCTCTCCCATACTTT
Sequence-based reagentGBP1_FRosowski et al., 2014ChIP-qPCR primersTGGACAAATTCGTAGAAAGACTCA
Sequence-based reagentGBP1_RRosowski et al., 2014ChIP-qPCR primersGCACAAAAACTGTCCCCAAC
Sequence-based reagentIDO1_FRosowski et al., 2014ChIP-qPCR primersCACAGTCATTGTATTCTCTTTGCTG
Sequence-based reagentIDO1_RRosowski et al., 2014ChIP-qPCR primersGCATATGGCTTTCGTTACAGTC
Sequence-based reagentCD74_FUCSC Genome Browser, Zeisel et al., 2013. Bioinformatics. doi: 10.1093/bioinformatics/btt145.RT-qPCR primersCGCGACCTTATCTCCAACAA
Sequence-based reagentCD74_RUCSC Genome Browser, Zeisel et al., 2013. Bioinformatics. doi: 10.1093/bioinformatics/btt145.RT-qPCR primersCAGGATGGAAAAGCCTGTGT
Sequence-based reagentCXCL9_FUCSC Genome Browser, Zeisel et al., 2013. Bioinformatics. doi: 10.1093/bioinformatics/btt145.RT-qPCR primersTCTTTTCCTCTTGGGCATCA
Sequence-based reagentCXCL9_RUCSC Genome Browser, Zeisel et al., 2013. Bioinformatics. doi: 10.1093/bioinformatics/btt145.RT-qPCR primersTAGTCCCTTGGTTGGTGCTG
Transfected construct (human)Control (non-sil.) siRNAQIAGENCat# 1022076Non-targeting control siRNA
Transfected construct (human)FlexiTube siRNA Hs_CIC_6QIAGENCat# SI04275656siRNA targeting CIC
Transfected construct (human)FlexiTube siRNA Hs_CIC_8QIAGENCat# SI04368469siRNA targeting CIC
Transfected construct (human)GeneSolution siRNA Hs_DUX4_11QIAGENCat# SI04239753siRNA targeting DUX4.

Additional files

Supplementary file 1

Processed RNAseq data for MB135iDUX4, MB135iDUX4-F67A, and MB135iDUX4-CTD.

Processed RNAseq data for MB135iDUX4, MB135iDUX4-F67A, and MB135iDUX4-CTD myoblasts untreated, treated with IFNγ, or treated with IFNγ following doxycycline-induction of the integrated transgene. Please see ‘Materials and methods’ for RNAseq analysis description. Raw data have been uploaded to GEO with the identifier GSE186244.

https://cdn.elifesciences.org/articles/82057/elife-82057-supp1-v3.xlsx
Supplementary file 2

Processed proteomics data for MB135iDUX4-CTD and MB135iDUX4-CTDmL1dL2.

Processed proteomics data for MB135iDUX4-CTD (‘longCTD’) and MB135iDUX4-CTdmL1dL2 (‘mL1dL2’) treated and processed as described in ‘Materials and methods’ under ‘Liquid chromatography mass spectroscopy (LC-MS).’ Raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD029215.

https://cdn.elifesciences.org/articles/82057/elife-82057-supp2-v3.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/82057/elife-82057-mdarchecklist1-v3.pdf

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  1. Amy E Spens
  2. Nicholas A Sutliff
  3. Sean R Bennett
  4. Amy E Campbell
  5. Stephen J Tapscott
(2023)
Human DUX4 and mouse Dux interact with STAT1 and broadly inhibit interferon-stimulated gene induction
eLife 12:e82057.
https://doi.org/10.7554/eLife.82057