Qki regulates myelinogenesis through Srebp2-dependent cholesterol biosynthesis
- Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, United States
- Cancer Research Institute of Jilin University, The First Hospital of Jilin University, China
- Cancer Biology Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, United States
- Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, and Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Liver Cancer Institute, Zhongshan Hospital, Fudan University, China
- Department of Neuroscience, Baylor College of Medicine, United States
- Department of Neurosurgery, Beijing Tongren Hospital, Capital Medical University, China
- Graduate School of Biomedical Sciences, Tufts University, United States
- Clinical Science Division, H. Lee Moffitt Cancer Center & Research Institute, United States
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, United States
- Neuroscience Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, United States
Figures
Figure 1 with 1 supplement
Deletion of Qk in mouse neural stem cells leads to hypomyelination in the central nervous system.
(A) Schema of the generation of Qk-Nestin-iCKO mice. (B) Representative images of severe hind limb paralysis in Qk-Nestin-iCKO mice 2 weeks after tamoxifen injection. Ctrl: control. (C) Latency of …
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Figure 1—source data 1
Exact p-values for statistical analysis.
- https://cdn.elifesciences.org/articles/60467/elife-60467-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Deletion of Qk in mouse neural stem cells leads to hypomyelination in the central nervous system.
(A) Kaplan–Meier curves of and log-rank test results for quaking phenotype-free survival of QkL/L mice (n = 23), Nestin-CreERT2;WT mice (n = 8), Nestin-CreERT2;QkL/+ mice (n = 122), and Nestin-CreERT…
Figure 2 with 2 supplements
Qki loss in neural stem cells does not impair formation of oligodendrocyte precursor cells or Aspa+Gstpi+myelinating oligodendrocytes.
(A–C) Representative images of and quantification of immunofluorescent staining of Pdgfrα-Qki (A), Aspa-Qki (B), and Gstpi-Qki (C) in the corpus callosum tissues in Qk-Nestin-iCKO mice (n = 3) and …
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Figure 2—source data 1
Exact p-values for statistical analysis.
- https://cdn.elifesciences.org/articles/60467/elife-60467-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Qki loss in neural stem cells impairs the differentiation of Olig2+AspaGstpi- oligodendroglial lineage cells.
(A, B) Representative images of and quantification of immunofluorescent staining of CC-1–Qki-5 (A) and Olig2-Qki (B) in the corpus callosum tissues in Qk-Nestin-iCKO mice (n = 3) and control mice (n …
Figure 2—figure supplement 2
Deletion of Qk in neural stem cells has no effects on neuronal or astrocytic populations.
(A) Representative images of and quantification of immunofluorescent staining of NeuN in the cortex tissues in Qk-Nestin-iCKO mice and control mice 2 weeks after tamoxifen injection (n = 4 …
Figure 3
Qki loss leads to defective myelin membrane assembly.
(A) Representative images of immunofluorescent staining of MBP in the corpus callosum tissues in Qk-Nestin-iCKO mice and control mice 2 weeks after tamoxifen injection. (B, C) Representative images …
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Figure 3—source data 1
Exact p-values for statistical analysis.
- https://cdn.elifesciences.org/articles/60467/elife-60467-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
Qk deletion in oligodendrocyte precursor cells leads to defective myelinogenesis without impairing differentiation of Aspa+ myelinating oligodendrocytes.
(A) Schema of the generation of Qk-Plp-iCKO mice. (B) Representative images of severe hind limb paresis in Qk-Plp-iCKO mice 2 weeks after tamoxifen injection. (C) Latency of mice falling off the …
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Figure 4—source data 1
Exact p-values for statistical analysis.
- https://cdn.elifesciences.org/articles/60467/elife-60467-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Deletion of Qk in mouse oligodendrocyte precursor cells results in hypomyelination in the central nervous system.
(A) Kaplan–Meier curves of and log-rank test results for quaking phenotype-free survival of QkL/L mice (n = 8), Plp-CreERT2;WT mice (n = 8), Plp-CreERT2;QkL/+ mice (n = 43), and Plp-CreERT2;QkL/L …
Figure 4—figure supplement 2
Deletion of Qk does not alter proliferation of oligodendrocyte precursor cells and oligodendroglial lineage cells.
(A, B) Representative images of and quantification of immunofluorescent staining of Pdgfrα and Ki67 (A) and Olig2 and Ki67 (B) in the brain region within the red dotted box in Qk-Plp-iCKO;mTmG mice …
Figure 5 with 1 supplement
Qki regulates transcription of the genes involved in myelin cholesterol biosynthesis.
(A) Bar graph showing the five canonical pathways most affected by Qki on the basis of differentially expressed genes in Qk-Plp-iCKO mice and control mice (n = 2 mice/group). Blue and red indicate …
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Figure 5—source data 1
Exact p-values for statistical analysis.
- https://cdn.elifesciences.org/articles/60467/elife-60467-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Qki regulates transcription of the genes involved in myelin cholesterol biosynthesis.
(A) Graph of the five enriched molecular and cellular functions most affected by Qki on the basis of differentially expressed genes in Rosa26-CreERT2;QkL/L mice (n = 3 mice) and control mice (n = 5 …
Figure 6 with 1 supplement
Qki-5 interacts with Srebp2 to regulate transcription of the genes involved in cholesterol biosynthesis.
(A) Representative images of and quantification of immunofluorescent staining of Srebp2 in Aspa+Qki- oligodendrocytes in Qk-Nestin-iCKO mice (n = 3) and Aspa+Qki+ oligodendrocytes in control mice (n …
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Figure 6—source data 1
Exact p-values for statistical analysis.
- https://cdn.elifesciences.org/articles/60467/elife-60467-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Qki-5 cooperates with Srebp2 to regulate transcription of the genes involved in cholesterol biosynthesis.
(A) Global distribution of the ChIP-seq events of Qki-5, Srebp2, and Pol II in differentiated oligodendrocytes. UTR: untranslated region. (B) Venn diagram of the overlap of Qki-5-bound genes in …
Figure 7
Qki transcriptionally enhances Srebp2-mediated cholesterol biosynthesis.
(A) Venn diagram of the overlap of Qki-5-bound genes in Qki-5 ChIP-seq from freshly isolated mouse oligodendrocytes and the genes with markedly lower expression in Qk-Plp-iCKO mice than in control …
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Figure 7—source data 1
Exact p-values for statistical analysis.
- https://cdn.elifesciences.org/articles/60467/elife-60467-fig7-data1-v2.xlsx
Figure 8
Model of Qki’s roles in regulating cholesterol biosynthesis and fatty acid metabolism during central nervous system myelination and myelin maintenance.
Qki regulates cholesterol biosynthesis in a Srebp2-dependent manner during de novo myelinogenesis but not during myelin maintenance. In contrast, Qki regulates fatty acid metabolism during both de …
Author response image 1
Qki is moderately expressed in GFAP+ astrocytes.
Representative images and quantification of immunofluorescent staining of Qki and GFAP in the cortex/corpus callosum/hippocampus tissues in WT mice at P21. Arrow: Qki+GFAP+ cells. Arrow head: …
Author response image 2
GFP is expressed in a small subpopulation of GFAP+ astrocytes, and Qki loss does not alter GFAP expression.
(A) Representative images of immunofluorescent staining of GFP and GFAP in the corpus callosum tissues in Qk- Nestin-iCKO and control mice two weeks after tamoxifen injection. Arrowhead: …
Videos
Video 1
Defect in motor coordination displaying tremors and ataxia in Qk-Nestin-iCKO mice.
Video 2
Defect in motor coordination displaying tremors and ataxia in Qk-Plp-iCKO mice.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Mouse monoclonal anti-MBP | BioLegend | Cat# SMI-94R | |
| Antibody | Mouse monoclonal anti–β-Amyloid | BioLegend | Cat# SIG-39220 | |
| Antibody | Rabbit polyclonal anti-PLP | Abcam | Cat# ab105784 | |
| Antibody | Goat polyclonal anti-Iba1 | Abcam | Cat# ab107159 | |
| Antibody | Rabbit polyclonal anti-Hmgcs1 | Abcam | Cat# ab155787 | |
| Antibody | Rabbit polyclonal anti-Fdps | Abcam | Cat# ab153805 | |
| Antibody | Rabbit polyclonal anti-Lss | Abcam | Cat# ab80364 | |
| Antibody | Mouse monoclonal anti-RNA polymerase II CTD repeat YSPTSPS | Abcam | Cat# ab817 | |
| Antibody | Rabbit monoclonal anti-MAG | Cell Signal Technology | Cat# 9043 | |
| Antibody | Rabbit polyclonal anti-GFP | Cell Signal Technology | Cat# 2555 | |
| Antibody | Rabbit monoclonal anti-Pdgfrα | Cell Signal Technology | Cat# 3174 | |
| Antibody | Mouse monoclonal anti-Ki67 | Cell Signal Technology | Cat# 9449 | |
| Antibody | Rabbit polyclonal anti-Aspa | Millipore | Cat# ABN1698 | |
| Antibody | Rabbit polyclonal anti-Olig2 | Millipore | Cat# AB9610 | |
| Antibody | Mouse monoclonal anti-NeuN | Millipore | Cat# MAB377 | |
| Antibody | Mouse monoclonal anti-Apc (CC-1) | Millipore | Cat# OP80 | |
| Antibody | Rabbit polyclonal anti-Gstpi | MBL International | Cat# 311 | |
| Antibody | Mouse monoclonal anti-Qki (Pan) | Sigma-Aldrich | Cat# SAB5201536 | |
| Antibody | Rabbit polyclonal anti-Hmgcs2 | Sigma-Aldrich | Cat# SAB2107997 | |
| Antibody | Rabbit polyclonal anti-Srebp2 | Sigma-Aldrich | Cat# HPA031962 | |
| Antibody | Mouse monoclonal anti–β-actin | Sigma-Aldrich | Cat# A5441 | |
| Antibody | Mouse monoclonal anti-Flag | Sigma-Aldrich | Cat# F1804 | |
| Antibody | Rabbit polyclonal anti-Hmgcr | Thermo Fisher Scientific | Cat# PA5-37367 | |
| Antibody | Rabbit polyclonal anti-Srebp2 | Cayman Chemical | Cat# 10007663 | |
| Antibody | Mouse monoclonal anti-Gfap | BD Biosciences | Cat# 556330 | |
| Antibody | Normal rabbit IgG | Santa Cruz Technology | Cat# sc-2027 | |
| Antibody | Rabbit polyclonal anti–Qki-5 | This paper | immunized with a short synthetic peptide (CGAVATKVRRHDMRVHPYQRIVTADRAATGN) | |
| Antibody | Mouse monoclonal anti-AnkG | Sigma-Aldrich | MABN466 | |
| Antibody | Rabbit polyclonal anti–Sox9 | Sigma-Aldrich | AB5535 | |
| Antibody | Rabbit polyclonal anti-Caspr | Gift from Dr. Rasband lab | ||
| Antibody | Mouse monoclonal anti-PanNav | Gift from Dr. Rasband lab | (K58/35) | |
| Chemical compound | Tamoxifen | Sigma-Aldrich | Cat# T5648 | |
| Chemical compound | 4-hydroxytamoxifen | Sigma-Aldrich | Cat# H7904 | |
| Chemical compound | Poly-L-ornithine | Sigma-Aldrich | Cat# P3655 | |
| Chemical compound | 3,3',5-Triiodo-L-thyronine | Sigma-Aldrich | Cat# T2877 | |
| Chemical compound | Anti-Flag M2 Magnetic Beads | Sigma-Aldrich | Cat# M8823 | |
| Chemical compound | Citrate buffer | Poly Scientific R&D Corp | Cat# s2506 | |
| Chemical compound | FluoroMyelin Red | Thermo Fisher Scientific | Cat# F34652 | |
| Chemical compound | Penicillin-Streptomycin | Thermo Fisher Scientific | Cat# 15140122 | |
| Recombinant protein | Laminin Mouse Protein, Natural | Thermo Fisher Scientific | Cat# 23017015 | |
| Chemical compound | Neurobasal Medium | Thermo Fisher Scientific | Cat# 21103049 | |
| Chemical compound | B-27 | Thermo Fisher Scientific | Cat# 17504044 | |
| Chemical compound | GlutaMAX Supplement | Thermo Fisher Scientific | Cat# 35050061 | |
| Chemical compound | Puromycin Dihydrochloride | Thermo Fisher Scientific | Cat# A1113802 | |
| Recombinant protein | Dynabeads Protein G | Thermo Fisher Scientific | Cat# 10004D | |
| Chemical compound | HardSet Antifade Mounting Medium with DAPI | Vector Laboratories | Cat# H1500 | |
| Chemical compound | NeuroCult Basal Medium | Stemcell Technologies | Cat# 05700 | |
| Chemical compound | NeuroCult Proliferation Supplement | Stemcell Technologies | Cat# 05701 | |
| Recombinant protein | Recombinant Human EGF | ProteinTech | Cat# AF-100-15 | |
| Recombinant protein | Recombinant Human FGF-basic (146 a.a.) | ProteinTech | Cat# 100-18C | |
| Chemical compound | DNase (RNase-free) | Qiagen | Cat# 79254 | |
| Chemical compound | cOmplete, Mini Protease Inhibitor Cocktail | Roche Diagnostics | Cat# 11836153001 | |
| Chemical compound | RNase A | Thermo Fisher Scientific | Cat# 12091021 | |
| Chemical compound | Proteinase K (PK) Solution | Promega | Cat# MC5005 | |
| Commercial assay | Neural Tissue Dissociation Kit | Miltenyi Biotec | Cat# 130-092-628 | |
| Commercial assay | In-Fusion HD Cloning Plus | Takara Bio | Cat# 638910 | |
| Commercial assay | RNeasy Mini Kit | Qiagen | Cat# 74104 | |
| Commercial assay | QIAquick PCR Purification Kit | Qiagen | Cat# 28104 | |
| Commercial assay | SuperScript III First-Strand Synthesis SuperMix for qRT-PCR | Thermo Fisher Scientific | Cat# 11752250 | |
| Commercial assay | SuperSignal West Pico PLUS Chemiluminescence System | Thermo Fisher Scientific | Cat# 34579 | |
| Commercial assay | iTaq Universal SYBR Green Supermix | Bio-rad Laboratories | Cat# 1725122 | |
| Commercial assay | DC Protein Assay Kit | Bio-rad Laboratories | Cat# 5000121 | |
| Commercial assay | Total Cholesterol Assay Kits | Cell Biolabs | Cat# STA-390 | |
| Commercial assay | KAPA Hyper Prep Kit | Kapa Biosystems | Cat# 004477 | |
| Commercial assay | In Situ Cell Death Detection Kit | Millipore Sigma | 11684795910 | |
| Deposited data | Gene expression profile | This paper | GEO: GSE145116 | |
| Deposited data | Gene expression profile | This paper | GEO: GSE145117 | |
| Deposited data | ChIP sequencing data | This paper | GEO: GSE144756 | |
| Deposited data | ChIP sequencing data | Zhou et al., 2020 | GEO: GSE126577 | |
| Experimental model | Mouse: B6.(Cg)-Nestin-CreERT2 | Imayoshi et al., 2008 | N/A | |
| Experimental model | Mouse: B6.(Cg)-Plp1-CreERT2 | The Jackson Laboratory | Stock No.: 005975 | |
| Experimental model | Mouse: B6.129-Rosa26-CreERT2 | The Jackson Laboratory | Stock No.: 008463 | |
| Experimental model | Mouse: B6.129(Cg)-ROSAmT/mG | The Jackson Laboratory | Stock No.: 007676 | |
| Experimental model | Mouse: B6.(Cg)-Qk-loxP | Shingu et al., 2017 | N/A | |
| Recombinant DNA reagent | pLKO-puro Flag-Srebp2 | Addgene | Cat# 32018 | |
| Recombinant DNA reagent | pLKO-puro 3X Flag-Srebp2 | This paper | N/A | |
| Software and algorithm | ImageJ | National Institutes of Health | https://imagej.nih.gov/ij/ | |
| Software and algorithm | HISAT2 | Johns Hopkins University | https://ccb.jhu.edu/software/hisat2/index.shtml | |
| Software and algorithm | StringTie | Johns Hopkins University | https://ccb.jhu.edu/software/stringtie/ | |
| Software and algorithm | DESeq2 | Bioconductor | http://bioconductor.org/packages/DESeq2/ | |
| Software and algorithm | IPA | Qiagen | https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/ | |
| Software and algorithm | Trim Galore | Babraham Institute | http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ | |
| Software and algorithm | Bowtie | Johns Hopkins University | http://bowtie-bio.sourceforge.net/tutorial.shtml | |
| Software and algorithm | SAMtools | Li et al., 2009 | https://github.com/samtools/samtools | |
| Software and algorithm | MACS2 | Feng et al., 2012 | https://github.com/taoliu/MACS/ | |
| Software and algorithm | deeptools | Ramírez et al., 2016 | https://github.com/deeptools/deepTools | |
| Software and algorithm | ngsplot | Shen et al., 2014 | https://github.com/shenlab-sinai/ngsplot | |
| Software and algorithm | Prism 8 | GraphPad Software | https://www.graphpad.com/scientific-software/prism/ | |
| Software and algorithm | HOMER | UCSD | http://homer.ucsd.edu/homer |
Additional files
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Supplementary file 1
A complete list of the sequences of the primer pairs used in this study.
- https://cdn.elifesciences.org/articles/60467/elife-60467-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60467/elife-60467-transrepform-v2.docx
