PPARγ regulates expression of carbohydrate sulfotransferase 11 (CHST11/C4ST1), a regulator of LPL cell surface binding.

The transcription factor PPARγ is the key regulator of adipocyte differentiation, function and maintenance, and the cellular target of the insulin-sensitizing thiazolidinediones. Identification and functional characterization of genes regulated by PPARγ will therefore lead to a better understanding of adipocyte biology and may also contribute to the development of new anti-diabetic drugs. Here, we report carbohydrate sulfotransferase 11 (Chst11/C4st1) as a novel PPARγ target gene. Chst11 can sulphate chondroitin, a major glycosaminoglycan involved in development and disease. The Chst11 gene contains two functional intronic PPARγ binding sites, and is up-regulated at the mRNA and protein level during 3T3-L1 adipogenesis. Chst11 knockdown reduced intracellular lipid accumulation in mature adipocytes, which is due to a lowered activity of lipoprotein lipase, which may associate with the adipocyte cell surface through Chst11-mediated sulfation of chondroitin, rather than impaired adipogenesis. Besides directly inducing Lpl expression, PPARγ may therefore control lipid accumulation by elevating the levels of Chst11-mediated proteoglycan sulfation and thereby increasing the binding capacity for Lpl on the adipocyte cell surface

Chst11 is a novel PPARy target gene.

<p>(A) Chip-Seq data of PPARγ and RXRα occupancy on the Chst11 gene according to Nielssen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064284#pone.0064284-Nielsen1" target="_blank">[3]</a>. UCSC Genome Browser tracks at day 0, 1, 2 4 and 6 of differentiation are shown. Please note differences in y-axes. Two intronic PPARy-RXRα binding sites were designated site 1 and 2. (B) ChIP-PCR on 3T3-L1 preadipocytes and adipocytes. Chromatin was prepared on day 0 and day 6 of differentiation and subjected to immunoprecipitation using antibodies against PPARγ. Enriched DNA was analysed using quantitative PCR with primers located at site 1 and 2 in the Chst11 gene (dark gray and blacks bars, respectively). As a positive control, primers located at the Fabp4 PPREs (∼5500 bp from transcription start site; light gray bars) were used, primers located in the globin locus were used as a negative control (white bars). Results are shown relative to normalized ChIP recovery data of day 0 and results are representative of at least 3 independent experiments. (C and D) U2OS cells were cotransfected with a reporter construct (TKpGL3) containing Chst11 site 1 or site 2 sequences, or the parental reporter construct, together with empty (pCDNA) or PPARγ encoding expression vectors. Activation of the luciferase reporter in the absence or presence of 1 µM rosiglitazone is expressed as fold induction over that with empty reporter cotransfected with pCDNA in the absence of rosiglitazone after normalisation for Renilla luciferase activity. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (E) Chst11 mRNA expression in 3T3-L1 adipocytes that had been treated with control or PPARy siRNA from the start of differentiation and analyzed at day 5. Relative mRNA expression levels were related to control siRNA treated cells and normalized for the TFIIb reference gene. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (F) Chst11 mRNA expression in 3T3-L1 adipocytes and the effect of rosiglitazone treatment (1 µM, 24 h). Relative mRNA expression levels were normalized for the TFIIb reference gene. (G) As in panel F, but after treatment with the PPARγ antagonist GW9662 (10 µM, 24 h).</p

Knockdown of Chst11 leads to decreased lipid accumulation but not adipogenesis.

<p>(A) 3T3-L1 cells were treated with siRNA oligonucleotides targeting Chst11 or control (scrambled) oligonucleotides and Chst11 mRNA and protein expression levels were determined at day 5 after differentiation by qPCR (upper panel) and Western blotting (lower panel), respectively. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3 (B) 3T3-L1 cells were treated with siRNA oligonucleotides targeting PPARy, Chst11, Lpl, or control (scrambled) oligonucleotides, fixed and stained for lipid accumulation using Oil-red-O. Pictures are representative of 3 independent experiments. (C) 3T3-L1 cells were treated with siRNA oligonucleotides targeting Chst11, PPARy or control (scrambled) oligonucleotides and mRNA expression levels of PPARγ, C/EBPα, Lpl, adiponectin (adipoq) and Fabp4 were determined after 5 days of differentiation. Relative mRNA expression levels were related to control siRNA treated cells and normalized for the TFIIb reference gene. Samples were also generated for a western blot in which PPARγ expression was shown upon Chst11 and PPARγ knockdown. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3.</p

Knockdown of Chst11 leads to decreased Lpl activity.

<p>3T3-L1 cells were treated with siRNA oligonucleotides targeting Chst11, PPARy or control (scrambled) oligonucleotides and Lpl activity was analysed. The error bars display SEM and significance is shown by the asterisks (p<0.05), n = 3.</p

Chst11 is expressed in mature 3T3-L1 adipocytes.

<p>(A) and (B): mRNA expression profiles of Chst11 and PPARy in adipogenesis of 3T3-L1 cells during different days. Data are represented as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064284#pone-0064284-g001" target="_blank">Figure 1E</a>. (C) Representative confocal microscopy images of 3T3-L1 (pre)adipocytes. Endogenous Chst11 (red) was visualized utilizing specific antibodies, differentiated cells were identified with Nile Red (green), Hoechst was used to visualize the nuclei (blue).</p

Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60

Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesi