Supplementary Information Supplementary Figure 1 | β-Adrenergic dependent genomic localization of JMJD1A. (a) ChIP-seq tag distribution of JMJD1A surrounding TSS of JMJD1A targets. TSS, transcription start site; ISO, isoproterenol. (b) Genome browser shots showing the ISO induced JMJD1A recruitments

Histone 3 lysine 9 (H3K9) demethylase JMJD1A regulates β-adrenergic-induced systemic metabolism and body weight control. Here we show that JMJD1A is phosphorylated at S265 by protein kinase A (PKA), and this is pivotal to activate the β1-adrenergic receptor gene (Adrb1) and downstream targets including Ucp1 in brown adipocytes (BATs). Phosphorylation of JMJD1A by PKA increases its interaction with the SWI/SNF nucleosome remodelling complex and DNA-bound PPARγ. This complex confers β-adrenergic-induced rapid JMJD1A recruitment to target sites and facilitates long-range chromatin interactions and target gene activation. This rapid gene induction is dependent on S265 phosphorylation but not on demethylation activity. Our results show that JMJD1A has two important roles in regulating hormone-stimulated chromatin dynamics that modulate thermogenesis in BATs. In one role, JMJD1A is recruited to target sites and functions as a cAMP-responsive scaffold that facilitates long-range chromatin interactions, and in the second role, JMJD1A demethylates H3K9 di-methylation.

The gray shadows highlight the regions containing E1 and E2 enhancer elements and anchor points. Crosslinked chromatin samples prepared from differentiated iBATs (day 8) were treated with or without ISO (1 µM) for 1 hr, the same was used in Figure 6a. Relative interacting frequencies between the indicated regions and the anchor point were measured by qPCR. All PCR signals were normalized to digested/re-ligated bacterial artificial chromosome (BAC) templates. Error bars represent ± SEM of three independent experiments. Student's t-test was performed and *P < 0.05 and **P < 0.01 were considered statistically significant.  (a) Core rectal temperature of Jmjd1a+/+ and Jmjd1a-/-mice at indicated time points during exposure to 4°C were measured as described 8 (6-11 weeks of age, n = 5). Mice were acclimated to thermoneutrality for 24 hr prior to experiments. Student's t-test was performed and *P < 0.05 and ***P < 0.001 were considered statistically significant. (b) Oxygen consumption (VO 2 ), CO 2 production (VCO 2 ) and respiratory quotient (RQ) measured by indirect calorimetry in individually placed Jmjd1a+/+ and Jmjd1a-/-mice.
Luciferase reporter assay iBATs (6 x10 5 cells/24-well) were transfected with the indicated pGL3 reporter (150 ng) and Renilla luciferase plasmids (pRL-CMV) (9 ng) using GeneJuice (Novagen) according to the manufacturer's instructions. Two days after transfection, the cells were harvested and the firefly and Renilla luciferase activities were measured using Dual-Glo Luciferase assay system (Promega). Firefly luciferase signal was normalized to Renilla luciferase signal. All luciferase assay data represent ± SEM of triplicate samples.

Animal experiments
Mice were fed standard chow (CE-2, CLEA Japan Inc.) ad libitum in a temperature controlled environment with 12 hr light/dark cycles. Jmjd1a+/+ and Jmjd1a-/-mice previously described 5 or C57BL/6J mice were evaluated for cold-or ISO-induced changes in chromatin conformation and histone modification by 3C. For 3C assay, brown adipose tissues were taken, minced in cold PBS and cross linked as described under "Chromosome conformation capture (3C) assay". For immunoblot analysis, tissues were flash frozen in liquid nitrogen and stored at -80°C. For the cold-induced changes, mice were placed at 28°C for 4 hr and shifted to 4°C environment for 4 hr. At the end of the experiments, tissues were harvested and snap frozen. Energy expenditure was measured using indirect calorimetry (MK-5000RQ; Muromachi) as described previously 6,7 . Core body temperature was monitored using a rectal thermometer. 6-11 weeks mice were acclimated to thermoneutrality (28°C) for 24 hr prior to experiments as described 8 . All mice experiments were approved by the Animal Care and Use Committee of the University of Tokyo.

Cyclic AMP assay
Cyclic AMP assay was performed using a Lance ® Ultra cAMP kit

Flux analysis
Oxygen consumption was measured using a Seahorse XF24 extracellular flux analyzer as described previously 9 . iBATs were induced for differentiation as described above and on day 7, adipocytes were detached with EDTA (0.53 mM, pH 7.4) and re-seeded at 4.0 x10 4 cells/well into XF24 V7 cell culture microplates (Seahorse Bioscience). On the next day, the medium was replaced with pre-warmed XF24 assay medium (DMEM, 2 mM glutamine, 1 mM pyruvate and 25 mM glucose) in the presence or absence of 100 μM dobutamine (Sigma) for 1 hr. Following the measurement of dobutamine induced respiration, ATP turnover was evaluated in response to 40 μg mL -1 oligomycin (Sigma).
Mitochondrial respiration was blocked by adding both 1 μM rotenone (Sigma) and 1 μM antimycin A (Wako) and the residual OCR was considered as non-mitochondrial respiration. Proton leak was calculated by subtracting the ATP turnover and the non-mitochondrial respiration components of total basal and dobutamine induced respiration.

Immunofluorescence analysis
iBATs plated on slide glasses were fixed with 4% paraformaldehyde in PBS for 10 min at RT and permeabilized with 0.2% NP-40 in PBS for 20 min. After an incubation step with 10% goat serum (Vector Labs) for 30 min, the cells were stained with 10 μg mL -1 of anti-V5 antibody (Invitrogen) at 4°C overnight. Slides were incubated with 6 μg mL -1 of Alexa Fluor 488 conjugated AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch) and covered with VECTASHIELD mounting medium containing DAPI (Vector Labs). Immunofluorescence was captured with LEICA DMI 6000B microscope (Leica microsystems).

Baculovirus production and protein purification from Sf9 cells
For purifying human JMJD1A protein, baculovirus expressing eXact TM -tagged JMJD1A was produced.