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. 2013 Dec 5;504(7478):163-7.
doi: 10.1038/nature12652. Epub 2013 Nov 6.

EHMT1 controls brown adipose cell fate and thermogenesis through the PRDM16 complex

Haruya Ohno  1 , Kosaku Shinoda, Kana Ohyama, Louis Z Sharp, Shingo Kajimura
Affiliations

EHMT1 controls brown adipose cell fate and thermogenesis through the PRDM16 complex

Haruya Ohno et al. Nature. .

Abstract

Brown adipose tissue (BAT) dissipates chemical energy in the form of heat as a defence against hypothermia and obesity. Current evidence indicates that brown adipocytes arise from Myf5(+) dermotomal precursors through the action of PR domain containing protein 16 (PRDM16) transcriptional complex. However, the enzymatic component of the molecular switch that determines lineage specification of brown adipocytes remains unknown. Here we show that euchromatic histone-lysine N-methyltransferase 1 (EHMT1) is an essential BAT-enriched lysine methyltransferase in the PRDM16 transcriptional complex and controls brown adipose cell fate. Loss of EHMT1 in brown adipocytes causes a severe loss of brown fat characteristics and induces muscle differentiation in vivo through demethylation of histone 3 lysine 9 (H3K9me2 and 3) of the muscle-selective gene promoters. Conversely, EHMT1 expression positively regulates the BAT-selective thermogenic program by stabilizing the PRDM16 protein. Notably, adipose-specific deletion of EHMT1 leads to a marked reduction of BAT-mediated adaptive thermogenesis, obesity and systemic insulin resistance. These data indicate that EHMT1 is an essential enzymatic switch that controls brown adipose cell fate and energy homeostasis.

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Figures

Figure 1
Figure 1. Identification of EHMT1 in the PRDM16 transcriptional complex
a, Up: schematic illustration of PRDM16. Bottom: PRDM16 complex purified from brown adipocytes were subjected to in vitro histone methylation assay. b, Immunoprecipitation of EHMT1 protein followed by Western blotting to detect PRDM16. Input was shown in lower panels. c, In vitro binding assay of 35S-labeled EHMT1 or CtBP1 and purified PRDM16 fragments. d, Histone methylation assay of PRDM16 complex from brown adipocytes expressing indicated constructs. n=3–4. e, Western blotting for indicated proteins in adipose tissues. f, Transcriptional activities of PRDM16 using a PPAR-γ responsive luciferase reporter. n=3. Error bars are s.e.m.; ** P <0.01, *** P <0.001.
Figure 2
Figure 2. EHMT1 is required for BAT-versus-muscle lineage specification
a, Morphology of BAT from wild-type and Ehmt1 myf5 KO embryos at P1. Scale bar, 2.5 mm. b, H&E staining of wild-type and Ehmt1 myf5 KO BAT. Scale bar, 600 µm. Bottom: high magnification images. Scale bar, 30 µm. c, BAT weight from wild-type (n=14) and KO embryos (n=8). d, GO analyses of RNA-sequencing data. The Log2-fold changes in the expression of skeletal muscle (group M) and BAT (group B) genes are shown. e, Immunocytochemistry for MHC in C2C12 cells expressing indicated constructs under pro-myogenic culture conditions. Scale bar, 200 µm. f, Myogenin mRNA expression in e. n=3. g, ChIP assays using indicated antibodies. n=3. Error bars are s.e.m.; * P < 0.05, ** P <0.01, *** P <0.001.
Figure 3
Figure 3. EHMT1 controls BAT thermogenesis through stabilizing PRDM16 protein
a, Cellular respiration in brown adipocytes expressing indicated constructs. n=6. b, BAT-selective gene expression in brown adipocytes expressing indicated constructs. n=3. c, Oil-Red-O staining of MEFs expressing indicated constructs under pro-adipogenic culture conditions. d, BAT-selective gene expression in c. n=3. e, Effects of EHMT1 mutants on PRDM16 transcriptional activites. n=3. f, PRDM16 protein levels in COS7 cells expressing indicated constructs. g, Regression analysis of the PRDM16 protein stability. n=3. h, Changes in rectal temperature during a cold challenge. n=4–5. Error bars are s.e.m.; * P < 0.05, ** P <0.01, *** P <0.001.
Figure 4
Figure 4. EHMT1 deficiency in BAT causes obesity and insulin resistance
a, VO2 of wild-type and Ehmt1 adipo KO mice treated with CL316,243 (0.5 mg/kg) at thermoneutrality. n=6. b, Serum FFA levels in mice treated with saline or CL316,243. c, FA oxidation in BAT. n=6–10. d, Body weight change under a high-fat diet at thermoneutrality. n=16. e, Adipose tissue weight after 4-week high-fat diet. n=16. f, H&E staining of adipose tissues. Scale bar, 100 µm. g, GTT in 9-week high-fat diet-fed mice. n=9. h, ITT in 10-week high-fat diet-fed mice. n=9. i, Serum insulin levels at the fasted and glucose-stimulated states. n=9. j, H&E staining of liver in d. Scale bar, 50 µm. k, Liver triglyceride levels in j. n=9. l, Hepatic insulin signaling as assessed by phosphorylated (S473) and total Akt levels. Error bars are s.e.m.; * P < 0.05, ** P <0.01, *** P <0.001.
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