A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency
- Department of Human Genetics, University of Utah School of Medicine, United States
- Department of Internal Medicine, Division of Nephrology and Hypertension, and Molecular Medicine Program, University of Utah, United States
- Department of Computational and Systems Biology, University of Pittsburgh, United States
- Genetic Resource Science, The Jackson Laboratory, United States
- Medical Service, Veterans Affairs Salt Lake City Health Care System, United States
Figures
Figure 1 with 1 supplement
Lethality phenotype of NGLY1 knockdown is highly modifiable by strain background.
(A) Drosophila cross for NGLY1 knockdown in each Drosophila genetic reference panel (DGRP) strain. (B) Proportion of NGLY1 knockdown flies surviving for each cross was calculated based on the number …
Figure 1—figure supplement 1
Ncc69 expression is not correlated with survival.
Survival upon knockdown of NGLY1 was plotted versus baseline expression levels of Ncc69 in the DGRP (r = −0.022, N = 154, p=0.78). DGRP gene expression data was taken from Huang et al., 2014.
Figure 2 with 1 supplement
Gene set enrichment analysis.
Top significant ontological categories identified by GSEA. p-values are indicated by red-to-blue gradient, with red the lowest p-values and blue the highest p-values. Gene number identified in each …
Figure 2—figure supplement 1
NGLY1 knockdown causes circadian rhythm defect.
Activity monitor was used to analyze 2- to 5-day-old flies for 1 week in complete darkness after 3 days of entrainment in a 12 hr light, dark cycle. At least 15 flies were analyzed for each …
Figure 3
NGLY1 and Ncc69 interact genetically in Drosophila.
(A) Proportion of flies surviving to eclosion in ubiquitous knockdowns. NGLY1 knockdown (KD) are UAS-PnglRNAi/+; Tubulin-GAL4/+. Ncc69 KD are UAS-Ncc69RNAi/+; Tubulin-GAL4/+. NGLY1 Ncc69 double …
Figure 4 with 1 supplement
Endogenous NKCC1 is altered in NGLY1-deficient MEFs.
(A) Control (+/+) and NGLY1 null (-/-) MEFs were grown to confluency and then lysed to isolate the membrane and cytoplasmic fractions. Three separate membrane lysates for both genotypes were …
Figure 4—figure supplement 1
Full western blot of NKCC1 in NGLY1 +/+ and -/- MEFs.
Tris-acetate gel was run at 150V for 2.5 hr to sufficiently distinguish between the different molecular weights of NKCC1, and therefore the 75 kDa band of the protein marker was the bottom-most …
Figure 5 with 1 supplement
NGLY1 -/- MEFs show decreased NKCC1-specific ion flux.
(A) Bumetanide-sensitive 86Rb flux was measured in NGLY1 +/+ and NGLY1 -/- MEFs to measure NKCC1 activity. Flux was examined in three bath conditions, isotonic (iso), hypotonic (hypo), and …
Figure 5—figure supplement 1
86Rb uptake in MEFs occurs through bumetanide-sensitive and ouabain-sensitive pathways.
Either NGLY1 +/+ (A) or NGLY1 -/- (B) MEFs were incubated with DMSO as a vehicle control or with the NKCC1 inhibitor, bumetanide, or the Na+/K+-ATPase inhibitor, ouabain. 86Rb flux was measured in …
Author response image 1
Tables
Table 1
Candidate modifier genes identified from GWA.
Rank order of candidate genes was established based on the most significant associated SNP in the respective gene.
| Rank order | Gene | FBgn | Human ortholog | Periphery/membrane | Proteostasis |
|---|---|---|---|---|---|
| 1 | exp | FBgn0033668 | --- | no | no |
| 2 | Ncc69 | FBgn0036279 | NKCC1/2 | yes | no |
| 3 | CG5888 | FBgn0028523 | --- | yes | no |
| 4 | CG16898 | FBgn0034480 | --- | no | no |
| 5 | bru3 | FBgn0264001 | CELF2/3/4/5/6 | no | no |
| 6 | CG31690 | FBgn0051690 | TMTC2 | no | yes |
| 7 | CG7227 | FBgn0031970 | SCARB1 | no | no |
| 8 | CR44997 | FBgn0266348 | --- | no | no |
| 9 | rgn | FBgn0261258 | Many | no | no |
| 10 | M6 | FBgn0037092 | GPM6A | yes | no |
| 11 | Rab26 | FBgn0086913 | RAB26 | yes | yes |
| 12 | Obp56i | FBgn0043532 | --- | no | no |
| 13 | 5-HT1A | FBgn0004168 | HTR1A | yes | no |
| 14 | CG33012 | FBgn0053012 | ERMP1 | no | yes |
| 15 | rst | FBgn0003285 | --- | yes | no |
| 16 | CR43926 | FBgn0264547 | --- | no | no |
| 17 | CG7337 | FBgn0031374 | WDR62 | no | no |
| 18 | hiw | FBgn0030600 | MYCBP2 | yes | yes |
| 19 | fid | FBgn0259146 | TRMT9B | no | no |
| 20 | nmo | FBgn0011817 | NLK | no | no |
| 21 | Sirup | FBgn0031971 | SDHAF4 | no | no |
| 22 | tst | FBgn0039117 | SKIV2L | no | no |
| 23 | Mdr50 | FBgn0010241 | many | yes | no |
| 24 | Cpr49Aa | FBgn0050045 | --- | no | no |
| 25 | COX7C | FBgn0040773 | COX7C | no | no |
| 26 | Eip63E | FBgn0005640 | CDK14/15 | yes | no |
| 27 | CG30048 | FBgn0050048 | PKD1 | no | no |
| 28 | CG15040 | FBgn0030940 | --- | no | no |
| 29 | SP2353 | FBgn0034070 | EGFLAM | no | no |
| 30 | Mf | FBgn0038294 | --- | no | no |
| 31 | ome | FBgn0259175 | many | no | no |
| 32 | esn | FBgn0263934 | PRICKLE1-3 | no | no |
| 33 | haf | FBgn0261509 | many | no | no |
| 34 | dally | FBgn0263930 | GPC3/5 | yes | no |
| 35 | robo2 | FBgn0002543 | ROBO1/2/3/4 | no | no |
| 36 | Gyc32E | FBgn0010197 | NPR1/2 | yes | no |
| 37 | CG8170 | FBgn0033365 | many | no | no |
| 38 | CG8405 | FBgn0034071 | TMEM259 | no | yes |
| 39 | scaf | FBgn0033033 | --- | yes | no |
| 40 | borr | FBgn0032105 | CDCA8 | yes | no |
| 41 | Syx7 | FBgn0267849 | STX7/12 | yes | no |
| 42 | DIP-delta | FBgn0085420 | many | yes | no |
| 43 | cv-c | FBgn0285955 | DLC1 | yes | no |
| 44 | Snmp2 | FBgn0035815 | CD36/SCARB1 | no | no |
| 45 | Mer | FBgn0086384 | NF2 | yes | no |
| 46 | sba | FBgn0016754 | --- | no | no |
| 47 | Hsromega | FBgn0001234 | --- | no | yes |
| 48 | CCAP-R | FBgn0039396 | NPSR1 | yes | no |
| 49 | Hrd3 | FBgn0028475 | SEL1L | no | yes |
| 50 | blue | FBgn0283709 | NEURL4 | no | yes |
| 51 | CG6262 | FBgn0034121 | TREH | no | no |
| 52 | CG45186 | FBgn0266696 | SVIL | no | no |
| 53 | Spn | FBgn0010905 | PPP1R9A | yes | no |
| 54 | dnc | FBgn0000479 | PDE4A/B/C/D | no | no |
| 55 | CG4374 | FBgn0039078 | many | no | no |
| 56 | sff | FBgn0036544 | many | no | yes |
| 57 | CG42383 | FBgn0259729 | NSFL1C | no | yes |
| 58 | Dyb | FBgn0033739 | DTNB | yes | no |
| 59 | CG34371 | FBgn0085400 | --- | no | no |
| 60 | CG4341 | FBgn0028481 | TMTC2 | no | yes |
| 61 | CG30043 | FBgn0050043 | ERMP1 | no | yes |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | Pngl | GenBank | ID:35527 Dmel_CG7865 | |
| Gene (Drosophila melanogaster) | Ncc69 | GenBank | ID: 39410 Dmel_CG4357 | |
| Gene (Mus musculus) | NGLY1 | GenBank | ID: 59007 | |
| Gene (Mus musculus) | NKCC1 | GenBank | ID: 20496 | Slc12a2 |
| Genetic reagent (Drosophila melanogaster) | Pngl-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_54853 | y1 v1; P{y+t7.7 v+t1.8=TRiP.HMJ21590}attP40 |
| Genetic reagent (Drosophila melanogaster) | Tubulin-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_5138 | y1 w*; P{w+mC = tubP-GAL4}LL7/TM3, Sb1 Ser1 |
| Genetic reagent (Drosophila melanogaster) | Tubulin-GAL80 | Bloomington Drosophila Stock Center | RRID:BDSC_5190 | y1 w[*]; P{w[+mC]=tubP-GAL80}LL9 P{w[+mW.hs]=FRT(w[hs])}2A/TM3, Sb1 |
| Genetic reagent (Drosophila melanogaster) | Drosophila Genetics Reference Panel | Bloomington Drosophila Stock Center | Set of 194 strains, example strain: BDSC:55014, RRID:BDSC_55014 | |
| Genetic reagent (Drosophila melanogaster) | Pdf-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_6899 | P{w[+mC]=Pdf-GAL4.P2.4}X, y1 w[*] |
| Genetic reagent (Drosophila melanogaster) | UAS-Pngl-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_42592 | y1 sc* v1 sev21; P{y+t7.7 v+t1.8=TRiP.HMS02424}attP40 |
| Genetic reagent (Drosophila melanogaster) | UAS-Ncc69-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_28682 | y1 v1; P{y+t7.7 v+t1.8=TRiP.JF03097}attP2 |
| Cell line (Mus musculus) | Mouse embryonic fibroblasts (MEF) | Jackson Labs | Primary line from mouse #027060,https://www.jax.org/strain/027060 | |
| Antibody | Anti-NKCC1 (Rabbit polyclonal) | Cell Signaling | Cat#14581, RRID:AB_2798524 | IB: 1:1000 |
| Antibody | Anti-TCF11/NRF1 (Rabbit monoclonal) | Cell Signaling | Cat#8052, RRID:AB_11178947 | IB: 1:1000 |
| Antibody | IRDye 800CW Goat-anti-rabbit | Abcam | Cat#216773 | IB: 1:10,000 |
| Commercial assay or kit | Cell Fractionation Kit | Cell Signaling | Cat#9038 | |
| Chemical compound, drug | Bortezomib | EMD Millipore | Cat# 179324-69-7 | |
| Software, algorithm | Genome Wide Association | Chow et al., 2016 | ||
| Software, algorithm | Gene Set Enrichment Analysis | Subramanian et al., 2005 | ||
| Software, algorithm | Evolution Rate Covariation | Clark et al., 2012 | ||
| Software, algorithm | R | https://www.r-project.org/ |
Additional files
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Supplementary file 1
NGLY1 DGRP cross progeny counts.
The number of eclosed flies were scored for each resulting genotype. The ‘no marker’ column represents flies expressing the NGLY1 RNAi. The largest balanced genotype was used as ‘expected’ for percent survival.
- https://cdn.elifesciences.org/articles/57831/elife-57831-supp1-v3.xlsx
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Supplementary file 2
GWA analysis for survival in NGLY1 DGRP screen.
Single-nucleotide polymorphisms (SNPs) are listed by chromosome position and rs ID.
- https://cdn.elifesciences.org/articles/57831/elife-57831-supp2-v3.zip
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Supplementary file 3
Top associated SNPs.
The top 125 variants. SNPs are listed in rank order of significance.
- https://cdn.elifesciences.org/articles/57831/elife-57831-supp3-v3.xlsx
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Supplementary file 4
Gene set enrichment analysis (GSEA).
Gene Ontology (GO) terms are listed by rank significance. Individual genes within each category are listed with the FBgn#.
- https://cdn.elifesciences.org/articles/57831/elife-57831-supp4-v3.xlsx
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Supplementary file 5
Evolutionary rate covariance (ERC).
Co-evolving genes are listed by rank significance (sumnlogpvbest). Genes that are known to cause a Congenital Disorder of Glycosylation (CDG) are highlighted in red.
- https://cdn.elifesciences.org/articles/57831/elife-57831-supp5-v3.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57831/elife-57831-transrepform-v3.docx
