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Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of facioscapulohumeral muscular dystrophy

  1. Sujatha Jagannathan  Is a corresponding author
  2. Yuko Ogata
  3. Philip R Gafken
  4. Stephen J Tapscott  Is a corresponding author
  5. Robert K Bradley  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States
Tools and Resources
Cite this article as: eLife 2019;8:e41740 doi: 10.7554/eLife.41740
7 figures, 1 table, 3 data sets and 2 additional files

Figures

Figure 1 with 1 supplement
Quantitative mass spectrometry of DUX4-expressing cells.

(A) Schematic of the experimental set up and the subsequent data analysis steps used to measure protein fold change in cells expressing vDUX4 or iDUX4. (B) Histogram of normalized, log2-transformed DUX4/Control ratios for all peptides mapping to ZSCAN4, a DUX4 target gene (top panels), and for RPL15, a housekeeping gene (bottom panels), following 24 and 36 hr of vDUX4 expression. (C) Histogram of normalized, log2-transformed DUX4/Control ratios for all peptides mapping to ZSCAN4, a DUX4 target gene (top panels), and for RPL15, a housekeeping gene (bottom panels), in the two label-swap replicates of iDUX4 expression. (D) Box plot of normalized, log2-transformed DUX4/Control ratio for peptides corresponding to the FSHD biomarker transcripts identified by Yao et al. (2014) in the vDUX4 dataset. Each dot refers to an individual peptide that was quantified and the color represents the time point of vDUX4 expression (24 hours in blue and 36 hours in purple). (E) Box plot of normalized, log2-transformed DUX4/Control ratio for peptides corresponding to the FSHD biomarker transcripts identified by Yao et al. (2014) in the vDUX4 dataset. Each dot refers to an individual peptide that was quantified and the color denotes replicate 1 (red) or replicate 2 (purple) of iDUX4 expression.

Figure 1—figure supplement 1
Demonstration of peptide to protein summarization for two candidate genes.

(A) The mass spectra for a representative peptide from a heavy-labeled sample. (B) Histogram of log2-ransformed heavy to light ratio of peptides from cells labeled with heavy amino acids for 1 week. (C) Histogram of log2-transformed heavy to light ratio of peptides from an equal mixture of unlabeled cells with cells labeled with heavy amino acids for 1 week. (D) Histogram shows the DUX4/Control ratios (log2) for all peptides in the iDUX4 dataset (top panel), for peptides mapping to RPL15 (middle panel) or for peptides mapping to ZSCAN4 (bottom panel). (E)Box and whisker plot showing the median log2 DUX4/control ratios for RPL15 and ZSCAN4.

Concordant changes in the RNA and protein abundance of several DUX4 transcriptional targets.

(A) Table showing number of genes with fold changes in their RNA and protein levels in the iDUX4 datasets. (B) Scatter plot of log2 fold change in RNA levels (DUX4/control) versus log2 fold change in protein levels (DUX4/control) for all genes. (C) Gene Ontology analysis of genes that are up- or downregulated > 4-fold at the RNA or protein level. Multiple testing correction was performed using a Benjamini–Hochberg procedure. (D) Fluorescence micrographs of cells with and without DUX4 induction for 14 hr, stained with DAPI (blue), anti-GM130 (Golgi apparatus; in green) and phalloidin-555 (actin; in red). (E) Quantification of the percentage of cells with DUX4-positive nuclei and fragmented Golgi apparatus at 12 hr and 24 hr post doxycycline induction of DUX4 expression. Error bars represent standard deviations.

Extensive post-transcriptional buffering of stress-response genes in DUX4-expressing cells.

(A) Scatter plot of log2 fold change in RNA levels (DUX4/control) versus log2 fold change in protein levels (DUX4/control). Genes that are upregulated at the RNA level but no significant change at the protein level fall within the blue rectangle. (B) RNA and protein fold change across the different datasets for representative genes that show post-transcriptional buffering. (C) Changes in the RNA levels of the chaperone HSPA5 over the duration of vDUX4 expression (or control expression) as measured by qRT-PCR. The region shaded in blue represents the region where the vDUX4 sample significantly deviates from the Control because of the induction of the unfolded protein response (UPR). (D) Levels of phosphorylated eIF2α in control cells versus DUX4-expressing cells over an expression time course as detected by immunoblots. (E) Percentage of [35S]-Methionine incorporation (representing translation efficiency) in DUX4-expressing cells normalized over control cells during the time course of DUX4 expression.

DUX4 induces post-transcriptional gene regulation.

(A) Scatter plot of log2 fold change in RNA levels (DUX4/control) versus log2 fold change in protein levels (DUX4/control). The post-transcriptional regulation of genes results in drastic changes in protein level without major changes in RNA levels (highlighted in gold). (B) Schematic representation of RNA- and protein-level changes for the genes involved in mRNA surveillance. Colors represent a heat map of actual fold changes in RNA levels (top) and in protein levels (bottom). Proteins with fewer than two quantified peptides are outlined by dotted lines. (C) Scatter plot of log2 fold change in RNA levels (DUX4/control) versus log2 fold change in protein levels (DUX4/control) for genes in the ubiquitin proteasome pathway. (D)Model for DUX4-induced post-transcriptional gene regulation.

Tool allowing easy access to the data generated in this study.

Screenshot of the Shiny web server showing a sample analysis of the RNA- and protein- level data for a DUX4 transcriptional target, ZSCAN4.

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Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Cell line
(Homo sapiens)
MB135PMID:28171552
Cell line
(Homo sapiens)
MB135-iDUX4PMID:28171552
AntibodyRabbit anti-
GM130 antibody
Bethyl
Laboratories Inc.
Cat # A303-402A-T(1:250)
AntibodyRabbit anti-
Dux4 antibody
AbcamCat # ab124699(1:500)
Commercial
 assay, kit
ActinRed 555
ReadyProbes Reagent
ThermoFisher
Scientific
Cat # R37112
Chemical
compound, drug
DAPISigmaCat # D9542(1:1000)
Chemical
compound, drug
L-LYSINE:2HCL
UNLABELED
Cambridge Isotope
Laboratories Inc.
Cat # ULM-8766-0.1
Chemical
compound, drug
L-ARGININE:HCL
UNLABELED
Cambridge Isotope
Laboratories Inc.
Cat # ULM-8347-0.1
Chemical
compound, drug
L-LYSINE:2HCL
(13C6, 99%; 15N2, 99%)
Cambridge Isotope
Laboratories Inc.
Cat # CNLM-291-H-0.05
Chemical
compound, drug
L-ARGININE:HCL
(13C6, 99%; 15N4, 99%)
Cambridge Isotope
Laboratories Inc.
Cat # CNLM-539-H-0.05
OtherHam's F12 for SILACPierceCat # 88424
OtherDialysed FBS for SILACPierceCat # 88212

Data availability

Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaino et al., 2013) with the dataset identifier PXD010221. To enable easy access to processed peptide-spectrum match data, we have also deposited peptide-level data to Dryad (doi:10.5061/dryad.ck06k75). The previously published RNA-seq data are available through the NCBI SRA database under accession number GSE85461.

The following data sets were generated
  1. 1
    PRIDE PRoteomics IDEntifications database
    1. S Jagannathan
    2. Y Ogata
    3. PR Gafken
    4. SJ Tapscott
    5. RK Bradley
    (2018)
    ID PXD010221. Proteomics analysis of DUX4-expressing myoblasts.
  2. 2
    Dryad Digital Repository
    1. S Jagannathan
    2. Y Ogata
    3. P Gafken
    4. S Tapscott
    5. R Bradley
    (2018)
    Data from: Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of FSHD.
    https://doi.org/10.5061/dryad.ck06k75
The following previously published data sets were used
  1. 1
    NCBI Gene Expression Omnibus
    1. S Jagannathan
    (2016)
    ID GSE85461. Model systems of DUX4 expression recapitulate the transcriptional profile of FSHD cells.

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