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doi: 10.1038/nature07777

Full Methods

Generation of Jhdm2a-/-

Mice. A targeting vector was constructed using a bacterial

artificial chromosome (BAC) clone. A loxP site and a β-Geo cassette flanked by two loxP

sites were introduced into the mouse Jhdm2a locus (Fig. S1A). E14 embryonic stem (ES)

cells (derived from 129Sv strain) having undergone homologous recombination thus

having 3loxP sites were isolated using standard procedures. Chimeric mice were mated

with wild-type C57BL/6J mice to generate F1 with a 3loxP allele. The F1 mice were

crossed with EIIa-Cre mice in C57BL/6J background to obtain offspring with 1loxP allele

(Jhdm2a+/-

mice). Jhdm2a-/-

mice were obtained by mating Jhdm2a+/-

mice. These mice

were backcrossed to the C57BL/6J strain for five generations. Correct genotypes were

confirmed by PCR amplification of genomic DNA.

Animal experiments. All animal experiments were performed according to procedures

approved by the Institutional Animal Care and Use Committee (IACUC). Mice were

maintained on a diet of standard rodent chow or a high-fat diet containing 60% fat-

derived calories (58Y1, TestDiet, Richmond, IN) with 12 hr light and dark cycles. Body

weight was measured weekly for a period of 8 months. For diet-induced obesity, 4 week-

old mice were fed a high-fat diet for 2 months and body weight was measured weekly.

The food intake of mice was measured using singly housed mice. Before measurement of

food intake, the mice were acclimated to the housing environment for at least a week, and

the intake data was collected during a 2-week period.

For cold exposure, 12-week old mice were individually housed in plastic cages at 4 for

5 hr. Core body temperature was intra-rectally monitored. Brown adipose tissue was

dissected 5 hr after cold exposure and subjected to quantitative RT-PCR (qRT-PCR).

Total RNA was purified using an RNeasy or RNeasy lipid tissue kit (Qiagen). After

DNase treatment, first-strand cDNA was synthesized using the Improm II Reverse

Transcription System (Promega). qPCR was performed using SYBR GreenER

(Invitrogen).


doi: 10.1038/nature07777 SUPPLEMENTARY INFORMATION

Body composition was evaluated by EchoMRI-100 (Echo Medical Systems) and

metabolic studies were performed using TSE LabMaster Calorimetry Module in the

Clinical Nutrition Research Unit at the University of North Carolina.

Analysis of blood samples. Leptin and adiponectin in serum were assayed using the

mouse leptin ELISA kit (CRYSTAL CHEM) and Circulex mouse adiponectin ELISA kit

(MBL), respectively, according to the manufacture’s instructions. Other hormones were

measured at the Vanderbilt Diabetes Research and Training Center. The blood level of

triglyceride and total cholesterol were analyzed in the Animal clinical chemistry and gene

expression core facility at the University of North Carolina. Serum free fatty acid was

measured using the HR series NEFA-HR kit (Wako Diagnostics).

Affymetrix microarray hybridization and data anlaysis: 7 µg of total RNA was used to

synthesize cDNA. A custom cDNA kit from Life Technologies was used with a T7-(dT)24

primer for this reaction. Biotinylated cRNA was then generated from the cDNA reaction

using the BioArray High Yield RNA Transcript Kit. The cDNA was then fragmented in

fragmentation buffer (5X fragmentation buffer: 200mM Tris-acetate, pH8.1, 500mM

KOAc, 150mM MgOAc) at 94oC for 35 minutes before the chip hybridization. 15 µg of

fragmented cDNA was then added to a hybridization cocktail (0.05 µg/µl fragmented

cDNA, 50 pM control oligonucleotide B2, BioB, BioC, BioD, and cre hybridization

controls, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 100mM MES, 1M

[Na+], 20mM EDTA, 0.01% Tween 20). 10 µg of cDNA was used for hybridization.

Arrays were hybridized for 16 hours at 45oC in the GeneChip Hybridization Oven 640.

The arrays were washed and stained with R-phycoerythrin streptavidin in the GeneChip

Fluidics Station 400. After this, the arrays were scanned with the Hewlett Packard

GeneArray Scanner. Affymetrix GeneChip Microarray Suite 5.0 software was used for

washing, scanning, and basic analysis. Sample quality was assessed by examination of 3’

to 5’ intensity ratios of certain genes. Data analysis was carried out in Genespring GX

10.0 software (Agilent Technologies) using the RMA algorithm. Probes exhibiting at least

a 2 fold reduction in mRNA level in Jhdm2a KO samples versus WT samples were

subjected to pathway analysis and gene ontology enrichment analysis with significance


SUPPLEMENTARY INFORMATIONdoi: 10.1038/nature07777

threshold set to p<0.05. Data mapping onto the PPAR pathway was carried out using

GenMAPP 2.0 (Gladstone Institutes).

Cell culture, viral infection, and differentiation. Primary myoblasts were isolated and

established from neonatal mice as reported1. Primary myoblasts were cultured in

DMEM with 2% horse serum for the differentiation to primary myotubes. HIB1B cells

were cultured in DMEM containing 10% FCS. To generate a Jhdm2a knockdown (KD)

HIB1B cell line, undifferentiated cells were infected with a lentiviral vector containing an

RNAi construct for Jhdm2a or control, and were selected with puromycin. The RNAi

construct for Jhdm2a was generated using the sequence: 5’-gcaggtgtcactagccttaat-3’.

Differentiation of HIB1B cells was performed as previously described 2. For the

overexpression of Jhdm2a gene in HIB1B cells and primary myocytes, cells were

infected with a retroviral vector expressing Flag-hJhdm2a or a control before the cells

were subjected to complete differentiation.

In vitro glycerol release, O2 consumption, and ββββ-oxidation assays. Soleus muscles and

intrascapular brown fat were surgically isolated. Tissue pieces were incubated in DMEM

containing 2% fatty acid-free bovine serum albumin with or without the presence of 10

µM isoproterenol (sigma) at 37°C for 2 hrs. Glycerol content was measured by using

commercial kit (zenbio). To measure O2 consumption, brown adipocytes were harvested

as previously described 3. O2 consumption of isolate cells was measured by using the

Clark-style oxygen electrode (Diamond general) at baseline, and after treatment with 10

µM isoproterenol. Measurement of in vitro β-oxidation of [1-14

C] palmitic acid was

performed using primary muscle cells. Cells were exposed to [1-14

C] palmitic acid (0.2

µCi/ml) for 60 min at 30°C and acidified with 1N HCl for an additional 30 min at 30°C.

The produced 14CO2 was trapped with a paper filter pre-soaked with NaOH. The total

level of released radiolabelled CO2 was measured by scintillation counting.

Chromatin immunoprecipitation (ChIP) analysis. Soleus muscles, primary myocytes, or

HIB1B cells with or without isoproterenol treatment were used for ChIP analysis with

histone modification-specific antibodies and anti-JHDM2A as described previously 4.



Primer sequences used for PCR analysis are listed in Supplementary Table 1.

Statistical analysis. All results are presented as the mean standard error (S.E.).

Statistical comparisons were by Student’s t tests. Statistical significance was set at p<0.05.

1. Rando, T.A. & Blau, H.M. Primary mouse myoblast purification, characterization,

and transplantation for cell-mediated gene therapy. J Cell Biol 125, 1275-1287

(1994).

2. Ross, S.R. et al. Hibernoma formation in transgenic mice and isolation of a brown

adipocyte cell line expressing the uncoupling protein gene. Proc Natl Acad Sci U

S A 89, 7561-7565 (1992).

3. Bachman, E.S. et al. betaAR signaling required for diet-induced thermogenesis

and obesity resistance. Science 297, 843-845 (2002).

4. Okada, Y., Scott, G., Ray, M.K., Mishina, Y. & Zhang, Y. Histone demethylase

JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis. Nature

450, 119-123 (2007).



Plin

Cyp27a1

Olr1

Acadl

AcadmAcaa1a

Acox1

Adipoq

Fabp4Apoa1

Apoa2

Apoc3

Aqp7

Fabp7

Cd36

Cpt1aCpt1b

Cpt2Cyp4a10

Cyp4a12bCyp4a14

Cyp7a1

Cyp8b1

Dbi

Fabp3

Fabp2

Fabp1

Acsl1

Gk2Gyk

Hmgcs2

Ilk

Fabp6Fabp5

Lpl

Me1

Pck1

Pdpk1

Pltp

Ppara

Ppard

Pparg

Rxra

RxrgScd1Scd2

Scp2

Sorbs1

Acsl6

Ubc

Ucp1

Nr1h3

Slc27a6

Acaa1b

Slc27a1

Slc27a2

Slc27a5

Slc27a4

Scd3

Acsl5Acsl4

Fads2

Angptl4

Fabp5l2

Apoa5

Ehhadh

Acsl3

Pck2

Cpt1cAcox3

Mmp1aMmp1b

Acox2

Rxra

Rxra

Cd36

Fabp4

Fabp1

Fabp3

Slc27a1

Slc27a3

Rxrb

Rxrb

Rxrb

Rxrg

Rxrg

LiverSkeletal Muscle

Skeletal MuscleAdipocyte

Adipocyte

Ketogenesis

Lipid Transport

Lipogenesis

Cholesterol Metabolism

Fatty Acid Transport

Fatty Acid Oxidation

Lipid Metabolism

AdaptiveThermogenesis

Ubiquitylation

Gluconeogenesis

Legend: KO/WT > 2 foldKO/WT > 1 foldKO/WT < -1 foldKO/WT < -2 foldNo criteria metNot found

PPAR SIGNALING PATWAY

AdipocyteDifferentiation

Cell Survival

Fig. S7

Figure S7. Jhdm2a deficiency affects PPAR signaling pathway in muscle.

Affymetrix microarray fold change data from RNA samples corresponding to Jhdm2a KO muscle vs. WT muscle was overlayed onto the PPAR signaling pathway using GenMAPP 2 software. Entities are colored by expression fold change in KO versus WT as indicated.

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