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PRDM16 represses the type I interferon response in adipocytes to promote mitochondrial and thermogenic programing

Megan Kissig, Jeff Ishibashi, Matthew J Harms, Hee‐Woong Lim, Rachel R Stine, Kyoung‐Jae Won, Patrick Seale
DOI 10.15252/embj.201695588 | Published online 13.04.2017
The EMBO Journal (2017) e201695588

Author Affiliations

  1. Megan Kissig1,2,
  2. Jeff Ishibashi1,2,
  3. Matthew J Harms1,2,
  4. Hee‐Woong Lim1,3,
  5. Rachel R Stine1,2,
  6. Kyoung‐Jae Won1,3 and
  7. Patrick Seale (sealep{at}upenn.edu)*,1,2
  1. 1Institute for Diabetes, Obesity & Metabolism, Smilow Center for Translational Research, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
  2. 2Department of Cell and Developmental Biology, Smilow Center for Translational Research, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
  3. 3Genetics Department, Smilow Center for Translational Research, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
  1. ↵*Corresponding author. Tel: +1 215 573 8856; Fax: +1 215 898 5408; E‐mail: sealep{at}upenn.edu
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Article Figures & Data

Figures

  • Figure 1.
    Figure 1. PRDM16 is required to repress type I IFN‐stimulated genes (ISGs) in adipocytes

    1. Relative mRNA levels of Prdm16, pan‐adipogenic genes (AdipoQ, Pparg2), and brown fat‐selective genes (Cidea, Ucp1) in R26CreER; Prdm16 fl/fl inguinal adipocytes treated with ethanol (EtOH) or 1 μM 4‐hydroxytamoxifen (4OHT) to induce knockdown of Prdm16, then differentiated +/− 1 μM rosiglitazone (rosi).

    2. Heat map depicting global gene expression levels in control (EtOH) and Prdm16 KO (4OHT) cells under control (Ctl) or rosi treatment.

    3. Gene ontology (GO) analysis of upregulated genes (blue cluster, B).

    4. Volcano plot depicting log‐fold change of gene expression in Prdm16 fl/fl (WT) and Myf5Cre; Prdm16 fl/fl (KO) adult mice. Red dots identify type I IFN‐stimulated genes (ISGs) found in the blue cluster of the heat map in (B).

    5. Immunofluorescence analysis of PRDM16 expression (red) and nuclei (DAPI, blue) in WT and R26CreER; Prdm16 fl/fl (R26Cre+) primary inguinal preadipocytes treated with 4OHT. Scale bar = 100 μm.

    6. Relative mRNA levels of Prdm16 and ISGs in WT and R26Cre+ primary inguinal preadipocytes.

    Data information: In (A, F), data represent (n = 3) mean ± standard deviation and are consistent with results from triplicate independent experiments. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

  • Figure EV1.
    Figure EV1. PRDM16 is required for repression of interferon‐stimulated genes

    1. Gene ontology (GO) of downregulated genes in Prdm16 KO cells (green cluster Fig 1B).

    2. Relative mRNA levels of Prdm16 and ISGs in Prdm16 fl/fl (WT) and R26CreER; Prdm16 fl/fl (R26Cre+) inguinal adipocytes treated with 1 μM 4‐hydroxytamoxifen (4OHT).

    3. Volcano plot comparing gene expression between young Prdm16 fl/fl (WT) and Prdm16 KO BAT. Red dots indicate type I ISGs found in the blue cluster of Fig 1B heat map.

    4. Relative mRNA levels of Prdm16 and ISGs in inguinal adipose from wild‐type mice incubated in TN (n = 5) or cold (n = 5).

    5. Relative mRNA levels of Prdm16 and ISGs in WT (n = 7) and R26Cre+ (n = 13) brown preadipose cells treated with 4OHT.

    6. Relative mRNA of Prdm16 and ISGs in brown adipocyte precursor cells transduced with CRISPR lentiviral vectors expressing Cas9 and guide RNA sequences for Rosa26 (gR26) or Prdm16 (gPrdm16a, gPrdm16b) (n = 3).

    Data information: Data are presented as mean ± standard deviation (B, E, F) and mean ± SEM (D). *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

  • Figure 2.
    Figure 2. PRDM16 blocks type I IFN signaling downstream of IFNAR receptor

    1. Relative mRNA levels of IFN‐stimulated genes (ISGs) in Prdm16 KO brown adipocyte precursors infected with control (Ctl) or PRDM16 retrovirus. Data represent (n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

    2. Western blot analysis of FLAG, phosphorylated STAT1 (pSTAT1), STAT1, phosphorylated STAT2 (pSTAT2), STAT3, and tubulin (loading control) protein in Prdm16 KO precursors infected with control (Ctl) or FLAG‐PRDM16 retrovirus.

    3. Relative mRNA levels of ISGs in control (Ctl) and PRDM16‐expressing preadipocytes +/− recombinant mouse IFNα. Data represent (n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

    4. Relative mRNA levels of ISGs in WT and R26Cre+ inguinal preadipocytes treated with 4OHT and vehicle or anti‐IFNAR1 neutralizing antibody (αIFNAR1) for 4 days. Data represent (n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. *P ≤ 0.05, **P ≤ 0.01 (paired two‐way ANOVA).

    5. Western blot analysis of PRDM16, STAT1, and actin (loading control) protein in brown adipocytes expressing gR26 (control) and gPrdm16 CRISPR/Cas9 constructs and treated +/− αIFNAR1 throughout differentiation.

    6. Relative mRNA levels of brown‐selective (Ucp1, Cidea, Pgc1a) and mitochondrial (mt‐Co1, mt‐CytB, mt‐Nd1) genes in brown adipocytes expressing gR26 and gPrdm16 CRISPR/Cas9 constructs +/− αIFNAR1 throughout differentiation. Data represent (n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. *P ≤ 0.05, **P ≤ 0.01 (paired two‐way ANOVA).

    Source data are available online for this figure.

    Source Data for Figure 2 [embj201695588-sup-0002-SDataFig2.pdf]

  • Figure EV2.
    Figure EV2. PRDM16 blocks type I IFN signaling downstream of IFNAR receptor

    1. Relative mRNA levels of Prdm16, Irf7, Ifi44, and Stat1 in WT and R26Cre+ inguinal precursors treated with increasing doses of recombinant mouse IFNα.

    2. Relative mRNA levels of ISGs in brown preadipocytes treated with vehicle, anti‐IFNAR (αIFNAR) neutralizing antibody, mouse IFNα, or a combination of αIFNAR and IFNα.

    Data information: Data represent (n = 3) mean ± standard deviation. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

  • Figure 3.
    Figure 3. Type I IFN disrupts mitochondrial structure and function in adipocytes

    Brown adipocytes were treated with 1,000 U/ml mouse IFNα or vehicle (Ctl) throughout differentiation.

    1. Relative expression levels of ISGs. Data represent (n = 3) mean ± standard deviation. **P ≤ 0.01 (Student's t‐test).

    2. Oil Red O staining of lipid droplets and relative mRNA levels of pan‐adipogenic genes (Fabp4, Pparg2). Data represent (n = 3) mean ± standard deviation.

    3. Western blot analysis of STAT1, UCP1, and actin (loading control) protein levels.

    4. Relative mRNA levels of brown fat‐selective (Ucp1, Cidea, Pgc1a) and mitochondrial (Cox7a1, mt‐Co1, mt‐Cytb, mt‐Nd1) genes. Data represent (n = 3) mean ± standard deviation and are consistent with duplicate independent experiments. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

    5. Western blot analysis of mitochondrial complex proteins and actin (loading control).

    6. Relative ratio of mitochondrial DNA (mt‐Co1) to nuclear DNA (Ndufv1) (n = 6). Data are presented as mean ± standard deviation.

    7. Transmission electron micrograph of representative brown adipocytes showing mitochondria (M), lipid droplets (L), and nuclei (N). Scale bar = 500 nm.

    8. Relative oxygen consumption rates of adipocytes (n = 6). Data are presented as mean ± standard deviation. *P ≤ 0.05 (Student's t‐test).

    9. Relative mRNA levels of brown‐selective genes (Ucp1, Cidea, Pgc1a) and mitochondrial genes (mt‐Co1, mt‐CytB, mt‐Nd1) in brown adipocytes infected with control (Ctl) or PRDM16 retrovirus +/− mouse IFNα. Data represent (n = 3) mean ± standard deviation. *P ≤ 0.05, **P ≤ 0.01 (paired two‐way ANOVA).

    Source data are available online for this figure.

    Source Data for Figure 3 [embj201695588-sup-0003-SDataFig3.pdf]

  • Figure EV3.
    Figure EV3. Type I IFN disrupts mitochondrial structure and function in adipocytes

    • A, B Western blot analysis of PRDM16 and actin protein (A) and relative mRNA levels of pan‐adipogenic genes (Fabp4, Pparg2) and brown‐selective genes (Ucp1, Cidea) (B) in brown adipocytes treated with vehicle, anti‐IFNAR (αIFNAR) neutralizing antibody, mouse IFNα, or αIFNAR + IFNα.

    • C. Relative mRNA levels of general adipocyte markers (Fabp4, Pparg2), mitochondrial genes (Cox7a1, mt‐Cytb), and brown fat‐selective genes (Ucp1, Cidea) in primary inguinal adipocytes treated with IFNα or vehicle (Ctl) +/− 1 μM rosiglitazone (Rosi).

    • D. Relative mRNA levels of general adipocyte markers (Fabp4, Pparg2), brown fat‐selective genes (Ucp1, Cidea), and mitochondrial genes (mt‐Cytb, mt‐Co1) in brown adipocytes treated with vehicle (Ctl) or mouse IFNα for varying periods during differentiation.

    Data information: Data represent (n = 3) mean ± standard deviation. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

  • Figure 4.
    Figure 4. PRDM16 opposes type I IFN signaling in vivo

    • A–D Prdm16 fl/fl (WT) and Myf5Cre; Prdm16 fl/fl (KO) mice treated with IFNα or phosphate‐buffered saline (PBS) for 2 weeks prior to analysis of brown adipose tissue (BAT). Experimental groups: WT+PBS (n = 4), KO+PBS (n = 3), WT+IFN (n = 6), KO+IFN (n = 4). (A) Western blot analysis of PRDM16, STAT1, and GAPDH (loading control) protein levels. (B) qPCR analysis of Ifi44 and Stat1 mRNA levels. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01 (paired two‐way ANOVA). (C) Hematoxylin and eosin (H&E) and anti‐UCP1 immunohistochemical staining. Scale bar = 50 μm. (D) Relative mRNA levels of brown fat‐specific genes (Ucp1, Cidea) and mitochondrial genes (mt‐Co1, mt‐CytB). Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01 (paired two‐way ANOVA).

    • E. Volume of O2 (VO2) consumed before and after norepinephrine injection. Experimental groups: WT+PBS (n = 9), KO+PBS (n = 6), WT+IFN (n = 6), KO+IFN (n = 7). Data are presented as mean ± SEM. **P ≤ 0.01 (paired two‐way ANOVA).

    Source data are available online for this figure.

    Source Data for Figure 4 [embj201695588-sup-0004-SDataFig4.pdf]

  • Figure EV4.
    Figure EV4. PRDM16 opposes type I IFN signaling in vivo

    • A, B Relative mRNA levels of ISGs (A) and mitochondrial genes (B) in brown adipose of Prdm16 fl/fl (WT) and Myf5Cre; Prdm16 fl/fl (KO) mice treated with IFNα or phosphate‐buffered saline (PBS) for 2 weeks.

    • C, D Relative mRNA levels of ISGs (C), as well as brown fat‐selective genes (Ucp1, Cidea) and mitochondrial genes (mt‐Co1, mt‐CytB) (D) in inguinal tissue from the same experimental mice in (A, B).

    Data information: Experimental groups: WT+PBS (n = 4), KO+PBS (n = 3), WT+IFN (n = 6), KO+IFN (n = 4). Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01 (paired two‐way ANOVA).

  • Figure 5.
    Figure 5. PRDM16 represses ISGs through direct binding at gene promoters

    1. ChIP‐seq stack‐height profiles in reads per million (RPM) for PRDM16 and H3K27 acetylation (Ac) at Ifi44 and Oas3 in Prdm16 KO brown adipocyte precursors that express PRDM16 or a control (Ctl) retrovirus.

    2. ChIP‐qPCR analysis of PRDM16 binding at ISG promoters/enhancers in control (Ctl) or PRDM16‐expressing brown preadipose cells. Ins1 and 18S were used as non‐specific binding site controls. Data represent (n = 3) mean ± standard deviation and are consistent with results from duplicate independent experiments. **P ≤ 0.01 (Student's t‐test).

    3. Western blot analysis of STAT1 and PRDM16 protein levels in Prdm16 KO brown preadipose cells transduced with retroviral vectors that express different forms of PRDM16: wild‐type (WT), CtBP‐binding mutant (CtBP1/2), PR‐domain deletion mutant (∆PR), DNA‐binding mutant (R998Q), or empty vector (Ctl). Loading control, actin.

    4. Relative mRNA levels of ISGs in cells from (C). Data represent (n = 3) mean ± standard deviation.

    Source data are available online for this figure.

    Source Data for Figure 5 [embj201695588-sup-0005-SDataFig5.pdf]

  • Figure EV5.
    Figure EV5. PRDM16 represses ISGs through direct binding at gene promoters

    • A, B Relative mRNA levels of Prdm16 and ISGs (A) and relative mRNA levels of adipogenic (Fabp4) and brown fat‐selective genes (Pgc1a, Cidea, Ucp1) in Prdm16 KO brown adipocytes cells transduced with retroviral vectors expressing wild‐type (WT) or DNA‐binding mutant (R998Q) PRDM16, or empty vector (Ctl).

    Data information: Data represent (n = 3) mean ± standard deviation. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

  • Figure EV6.
    Figure EV6. PRDM16 blocks IRF1 function at IRF‐E elements

    1. Schematic showing the ChIP‐seq track of PRDM16 binding at Ifi44 promoter and the identified IFN‐stimulated response element (ISRE)/IRF‐binding element (IRF‐E) that was inserted into the luciferase reporter plasmid (pGL4.24‐Ifi44p).

    2. Relative mRNA levels of IRF genes in brown preadipose cells.

    3. Relative mRNA levels of Ifnar1 and Irfs in brown preadipocytes (D0) and mature brown adipocytes (D8).

    4. Western blot analysis of IRF1 and actin protein levels and relative mRNA levels of ISGs in Prdm16 KO brown adipocytes cells transduced with lentiviral short‐hairpin RNA directed against Irf1 (shIrf1) or a scrambled control (shScr) and either retroviral expression vectors expressing human IRF1 (hIRF1) or puromycin control (Ctl).

    5. Relative mRNA levels of Irf1 and ISGs and of brown fat‐selective genes (Ucp1, Cidea) and mitochondrial genes (mt‐Co1, mt‐CytB) in brown adipocytes transduced with lentiviral short‐hairpin RNA directed against Irf1 (shIrf1) or a scrambled control (shScr).

    6. Western blot analysis of IRF1 and actin protein levels and relative mRNA levels of ISGs in Prdm16 KO brown adipocytes cells transduced with CRISPR lentiviral vectors expressing Cas9 and guide RNA sequences for Rosa26 (gR26) or Irf1 (gIrf1a, gIrf1b).

    7. Western blot analysis of IRF1 and actin (loading control) protein levels in cells from Fig 5C.

    8. Relative mRNA levels of Irf1 in Prdm16 fl/fl (WT) and R26CreER; Prdm16 fl/fl (R26Cre+) inguinal adipocytes treated 1 μM 4OHT and increasing doses of IFNα.

    9. Transcriptional activity of a Gal4 UAS‐driven luciferase gene in response to expression of GAL4 DNA‐binding domain alone (Gal4), IRF1, or GAL4‐IRF1+/− PRDM16.

    10. ChIP‐qPCR showing IRF1 binding at Ifi44 transcriptional start site (Tss) in WT and Prdm16 KO cells +/− IFNα.

    Data information: Data represent (n = 3) mean ± standard deviation (B–F, H, J) and (n = 3) mean ± SEM (I). *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).

  • Figure 6.
    Figure 6. PRDM16 blocks the activation of ISGs by IRF1

    • A, B Western blot analysis of IRF1 and actin (loading control) protein levels (A) and relative mRNA levels of ISGs (B) in Prdm16 KO brown preadipose cells transduced with lentiviral short‐hairpin RNA directed against Irf1 (shIrf1a, shIrf1b) or a scrambled control (shScr).

    • C, D Western blot analysis of IRF1 and actin (loading control) protein levels (C) and relative mRNA levels of ISGs (D) in cells from in NIH3T3 cells transfected with CMV6 (Ctl) or CMV6‐IRF1.

    • E. Transcriptional activity of the Ifi44 promoter in NIH3T3 cells upon expression of IRF1 and wild‐type (WT) or DNA‐binding mutant (R998Q) forms of PRDM16.

    • F. Transcriptional activity of the Ifi44 promoter in response to IRF1 expression and increasing amounts of PRDM16 expression.

    • G. Transcriptional activity of the IFN regulatory factor‐binding element (IRF‐E)/IFN‐stimulated response element (ISRE) in Ifi44 in response to IRF1 and/or PRDM16.

    • H. ChIP‐qPCR analysis of IRF1 binding at ISGs in brown preadipose cells transduced with PRDM16 or control (Ctl) retrovirus. Ins1 and 18S were used as non‐specific binding site controls.

    • I. Proposed model for PRDM16‐action at ISG promoter regions.

    Data information: Data represent (n = 3) mean ± standard deviation (B, D) and (n = 3) mean ± SEM (E–H) and are consistent with results from duplicate independent experiments. *P ≤ 0.05, **P ≤ 0.01 (Student's t‐test).Source data are available online for this figure.

    Source Data for Figure 6 [embj201695588-sup-0006-SDataFig6.pdf]

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Volume 37, Issue 12
15 June 2018
The EMBO Journal: 37 (12)
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