Adaptive thermogenesis is an important component of energy homeostasis and a metabolic defense against obesity. We have cloned a novel transcriptional coactivator of nuclear receptors, termed PGC-1, from a brown fat cDNA library. PGC-1 mRNA expression is dramatically elevated upon cold exposure of mice in both brown fat and skeletal muscle, key thermogenic tissues. PGC-1 greatly increases the transcriptional activity of PPARgamma and the thyroid hormone receptor on the uncoupling protein (UCP-1) promoter. Ectopic expression of PGC-1 in white adipose cells activates expression of UCP-1 and key mitochondrial enzymes of the respiratory chain, and increases the cellular content of mitochondrial DNA. These results indicate that PGC-1 plays a key role in linking nuclear receptors to the transcriptional program of adaptive thermogenesis.

Previously, we have isolated and characterized an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice. The key regulator of this enhancer is a cell type-restricted nuclear factor termed ARF6. Target sequences for ARF6 in the aP2 enhancer exhibit homology to a direct repeat of hormone response elements (HREs) spaced by one nucleotide; this motif (DR-l) has been demonstrated previously to be the preferred binding site for heterodimers of the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). We have cloned a novel member of the peroxisome proliferator-activated receptor family designated mPPART2, and we demonstrate that a heterodimeric complex of mPPAR~/2 and RXR~ constitute a functional ARF6 complex. Expression of mPPAR,/2 is induced very early during the differentiation of several cultured adipocyte cell lines and is strikingly adipose-specific in vivo. mPPAR-/2 and RXRc~ form heterodimers on ARF6-binding sites in vitro, and antiserum to RXRc~ specifically inhibits ARF6 activity in adipocyte nuclear extracts. Moreover, forced expression of mPPAR~/2 and RXR~ activates the adipocyte-specific aP2 enhancer in cultured fibroblasts, and this activation is potentiated by peroxisome proliferators, fatty acids, and 9-cis retinoic acid. These results identify mPPAR~/2 as the first adipocyte-specific transcription factor and suggest mechanisms whereby fatty acids, peroxisome proliferators, 9-cis retinoic acid, and other lipids may regulate adipocyte gene expression and differentiation.

The levels of histone mRNA are rapidly reduced after treatment of cultured cells with hydroxyurea or cytosine arabinonucleoside. The histone mRNA for the replicative histone variants is destroyed rapidly, with a half-life of [10][11][12][13][14][15] min. The levels of mRNA coding for the replacement histone variant H3.3 were unchanged after treatment with DNA synthesis inhibitors. In addition to the rapid destruction of histone mRNA, there was a reduction to 1/5th in the rate of transcription of the histone genes. Lymphoma cells (S49) arrested in GI by cyclic AMP produce and contain significant levels of histone mRNA. Hydroxyurea reduces the rate of transcription and the levels of histone mRNA in the GI-arrested cells.Histones are the basic proteins that are complexed with DNA to form the eukaryotic chromosome. It is generally thought that histones are synthesized when DNA is synthesized (1, 2). However, in one mammalian cell line, S49 mouse lymphoma cells, histones are also synthesized in G1 and incorporated into chromosomes during the subsequent S phase (3).The levels of histone mRNA are also regulated, both during the cell cycle and in response to DNA The isolation of mouse histone genes (14) allows us to directly measure both synthesis and degradation of histone mRNA. We report here that treatment of mouse cells with inhibitors of deoxynucleotide synthesis reduces the level of replication variant histone mRNAs, but not the level of a replacement variant mRNA, H3.3. This reduction is due to both a decreased rate of histone mRNA synthesis and an increased rate of degradation. In G1 lymphoma cells, which are not synthesizing DNA but are synthesizing histone mRNA, the inhibitors also reduce the rate of histone mRNA synthesis. METHODSCell Growth. Mouse myeloma 66-2 cells were grown, synchronized by isoleucine starvation (15), and treated with DNA synthesis inhibitors as described in the figure legends. When double drug treatments with cycloheximide and a DNA synthesis inhibitor were performed the cycloheximide was added 5 min prior to the addition of the DNA synthesis inhibitor. The final concentration of cycloheximide was 0.1 mM. Cells washed with fresh medium after drug treatment retained their viability. "Deathless" S49 mouse lymphoma cells were grown and synchronized as described by Lemaire and Coffino (16).Nuclear Transcription. Nuclei were isolated and incubated for RNA synthesis essentially as described by Marzluff (17). The final salt concentrations in the transcription were 70 mM KCI and 6 mM MgCl2. We used 100 uCi (1 Ci = 3. with an equal volume of DNase I (Sigma) at 25 /g/ml, 0.6 M NaCl, 50 mM Tris-HCl at pH 7.5, and 20 mM MgCl2. NaDodSO4 and EDTA were then added to final concentrations of 1% and 10 mM, respectively, followed by the addition of ammonium acetate to 0.3 M. RNA was extracted with an equal volume of water-saturated phenol at 55°C for 5 min, precipitated with ethanol, and washed three times with 75% (vol/vol) ethanol. The RNA was separated from unincorporated nucleoside triphosph...

Previously, we identified a novel transcription factor, ARF6, as a key regulator of the tissue-specific adipocyte P2 (aP2) enhancer. In order to identify the proteins which comprise the adipocyte ARF6 complex, we have purified this DNA binding activity from a cultured adipocyte cell line. We have developed a system for growth and differentiation of HIB-1B brown adipocytes in suspension culture that facilitates the production of large quantities of adipocyte nuclear extract. ARF6 was purified from HIB-1B nuclear extract by a combination of conventional and sequence-specific DNA affinity chromotography. Chemical sequencing and mass spectral analysis of tryptic peptides derived from the purified polypeptides identifies the ARF6 complex as a heterodimer of the retinoid X receptor alpha (RXR alpha) and the murine peroxisome proliferator activated receptor gamma (PPAR gamma). Of the known PPAR gamma isoforms, PPAR gamma is the predominant form expressed in adipose tissue. These results suggest that PPAR gamma 2 serves a unique function among PPAR family members as an important regulator of adipocyte-specific gene expression.

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