Histone H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis

Abstract

PPARγ promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, we find that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARγ locus. H3K9me2 and G9a levels decrease during adipogenesis, which correlates inversely with induction of PPARγ. Removal of H3K9me2 by G9a deletion enhances chromatin opening and binding of the early adipogenic transcription factor C/EBPβ to PPARγ promoter, which promotes PPARγ expression. Interestingly, G9a represses PPARγ expression in an HMT activity-dependent manner but facilitates Wnt10a expression independent of its enzymatic activity. Consistently, deletion of G9a or inhibiting G9a HMT activity promotes adipogenesis. Finally, deletion of G9a in mouse adipose tissues increases adipogenic gene expression and tissue weight. Thus, by inhibiting PPARγ expression and facilitating Wnt10a expression, G9a represses adipogenesis.

Figure 1

H3K9me2 is enriched on the entire PPARγ locus in preadipocytes. ChIP-Seq analyses of histone methylations were done in 3T3-L1 preadipocytes with the sequence read length of 50 bp. (A) Positional distribution profiles of global H3K4me3, H3K9me2 and H3K27me3 around gene body±5 kb regions in 3T3-L1 preadipocytes. TSS, transcription start site; TES, transcription end site. The numbers between TSS and TES indicate the sites (percentage) of gene body. (B, C) Inverse correlation of global H3K9me2 levels with H3K4me3 and H3K27me3 levels in promoter and gene body regions. The horizontal line indicates the mean level of H3K9me2 enrichment and the vertical bar indicates the spread variations (root mean square (RMS)). RPKM, reads per kb per million read sequenced. (D) Inverse correlation of H3K9me2 level with gene expression. The horizontal line indicates the mean level of H3K9me2 enrichment and the vertical bar indicates the spread variations (RMS). The gene expression levels in 3T3-L1 preadipocytes were analysed by RNA-Seq. (E, F) Profiles of H3K4me3 (green), H3K9me2 (blue) and H3K27me3 (red) on PPARγ and Wnt6-Wnt10a loci are visualized using the UCSC genome browser. (G) ChIP analyses of histone methylations on promoters of positive and negative regulators of adipogenesis in 3T3-L1 preadipocytes. Profiles of H3K4me3, H3K9me2 and H3K27me3 on the entire gene loci of these adipogenesis regulators are shown in Figure 2. The locations of quantitative PCR (qPCR) primers for ChIP are PPARγ1 (+0.5 kb), PPARγ2 (+0.5 kb), C/EBPα (+0.3 kb), C/EBPβ (+0.3 kb), C/EBPδ (+0.2 kb), Krox20 (+0.5 kb), KLF4 (+0.3 kb), CREB (+0.2 kb), Wnt6 (+0.5 kb), Wnt10a (+0.8 kb) and GAPDH (+0.8 kb) (also see Supplementary Tables S2 and S3). Data are representative of three independent experiments. qPCR data in all figures are presented as mean values±s.d.

Figure 2

Histone methylation profiles on adipogenesis regulator genes in preadipocytes. ChIP-Seq profiles of H3K4me3, H3K9me2 and H3K27me3 on gene loci encoding positive (A–G) and negative (H–N) regulators of adipogenesis in 3T3-L1 preadipocytes (day 0) are visualized using the UCSC genome browser. Panels (A) and (H) are shown as controls.

Figure 3

H3K9me2 and G9a levels decrease during adipogenesis. (A) ChIP analyses of H3K4me3, H3K9me2 and H3K27me3 on PPARγ1 and PPARγ2 promoters before (day 0) and after (day 6) 3T3-L1 adipogenesis. The schematic of PPARγ1 and PPARγ2 promoters and the locations of Taqman probes for PCR quantitation of ChIP are shown at the top. Wnt10a promoter serves as a control. (B) ChIP-Seq profiles of H3K9me2 on PPARγ locus (highlighted) before (day 0) and after (day 7) 3T3-L1 adipogenesis. (C) qRT–PCR analysis of PPARγ and G9a expression before and after 3T3-L1 adipogenesis. (D) Western blot analysis of G9a, PPARγ and H3K9me2 levels during 3T3-L1 adipogenesis. The 54.5-kDa PPARγ1 and 57.5-kDa PPARγ2 bands as well as the 30-kDa and 42-kDa C/EBPα isoforms are indicated. The p85α subunit of phosphoinositol-3-phosphate kinase is used as a loading control. (E) Western blot analysis of protein levels in preadipocytes and adipocytes isolated from mouse inguinal white adipose tissue. Source data for this figure is available on the online supplementary information page.

Figure 4

Characterization of immortalized G9a^flox/flox brown preadipocytes. SV40T-immortalized G9a^flox/flox brown preadipocyte cell line #1 was infected with retroviruses MSCVhygro expressing Cre (MSCVhygro-Cre) or vector (Vec) alone. After selection with 150 μg/ml hygromycin for 2 weeks, cells were maintained at subconfluence. (A, B) Confirmation of G9a deletion by qRT–PCR (A) and western blot (B). (C) Cell morphology under the microscope. (D) To analyse the short-term cell growth rates, 1 × 10⁵ cells were plated at day 0 and the cumulative cell numbers were determined every day for 5 days. (E) Western blot analysis of histone methylation and acetylation in nuclear extracts. me1, me2 and me3 refer to mono-, di- and trimethylation, respectively. ac, acetylation. Source data for this figure is available on the online supplementary information page.

Figure 5

G9a represses adipogenesis. (A–C) G9a represses adipogenesis of brown preadipocytes. G9a^flox/flox brown preadipocytes described in Figure 4 were infected with MSCVhygro-Cre. Adipogenesis was induced at standard condition at day 0. Whole cell extracts and RNA samples were prepared at indicated time points for western blot and qRT–PCR, respectively. (A) Morphological differentiation at day 6. Cells were stained with Oil Red O. Top panels, stained dishes; lower panels, representative fields under the microscope. (B) Western blot analysis of expression of adipogenesis makers and G9a during adipogenesis. (C) qRT–PCR analysis of expression of adipogenic makers and G9a during adipogenesis. (D–G) Knockdown of G9a promotes adipogenesis of 3T3-L1 white preadipocytes. 3T3-L1 cells were infected with lentiviral shRNA targeting G9a or control (Con), followed by puromycin selection for 7 days. (D) Western blot analysis of undifferentiated, subconfluent cells using antibodies indicated on the right. (E) qRT–PCR analysis of G9a and PPARγ expression in undifferentiated, subconfluent 3T3-L1 cells. (F, G) Adipogenesis was induced at suboptimal condition using 1/3 of adipogenic cocktail. After differentiation, cells were stained with Oil Red O (F) or collected for qRT–PCR analysis of adipocyte marker expression (G). (H, I) Overexpression of G9a inhibits 3T3-L1 adipogenesis. Cells were infected with MSCVhygro expressing FLAG-tagged G9a (F-G9a). Adipogenesis was induced at standard condition. (H) Oil Red O staining after differentiation. (I) qRT–PCR analysis of adipocyte marker expression before and after adipogenesis. Source data for this figure is available on the online supplementary information page.

Figure 6

G9a directly represses PPARγ expression but facilitates Wnt10a expression. G9a^flox/flox brown preadipocytes described in Figure 4 were infected with MSCVhygro-Cre. (A, B) G9a directly represses PPARγ expression in preadipocytes. Cells maintained at subconfluence were collected for (A) qRT–PCR analysis of expression of adipogenic transcription factors and (B) ChIP assays of G9a, H3K9me2, H3K9ac and RNA polymerase II (Pol II) levels on PPARγ1/γ2 promoters (+0.5 kb). (C) G9a enzymatic activity is required for repressing PPARγ expression in preadipocytes. G9a^flox/flox brown preadipocytes were infected with MSCVhygro-F-G9a expressing FLAG-tagged G9a (F-G9a) or with MSCVhygro-F-G9aΔSET expressing G9a lacking the SET domain. After hygromycin selection, cells were infected with WZLneo expressing Cre (WZLneo-Cre), followed by selection with G418. Gene expression in subconfluent preadipocytes was analysed by qRT–PCR. (D) FAIRE analysis of chromatin opening on PPARγ1 promoter. (E–G) Deletion of G9a promotes chromatin opening of, and C/EBPβ binding to, PPARγ2 promoter in the early phase of adipogenesis. Adipogenesis of Vec- and Cre-infected G9a^flox/flox brown preadipocytes was induced under standard condition. Samples were collected at indicated time points for qRT–PCR of PPARγ2 and C/EBPβ expression (E), ChIP of C/EBPβ and H3K9me2 around −0.3 kb C/EBPβ-binding site on PPARγ2 promoter (F), and FAIRE analysis of chromatin opening on PPARγ2 promoter (G). (H) Wnt10a expression in preadipocytes requires G9a. Cells maintained at subconfluence were collected for qRT–PCR. (I) Western blot analysis of β-catenin levels in the cytosolic and membrane fractions. β-Actin serves as the loading control. (J) G9a facilitates Wnt10a expression in preadipocytes independent of its enzymatic activity. The experiment was done as in (C). (K) ChIP of G9a and Pol II levels on Wnt10a promoter in subconfluent preadipocytes. qPCR data are presented as means±s.d. and are representative of 3–4 independent experiments. Source data for this figure is available on the online supplementary information page.

Figure 7

Inhibiting G9a methyltransferase activity promotes PPARγ expression and adipogenesis. G9a^flox/flox brown preadipocytes described in Figure 4 were infected with MSCVhygro-Cre or Vec alone, followed by BIX treatment and adipogenesis assay. In all, 8 μM of BIX was used except as indicated in (A). (A–C) Inhibiting G9a methyltransferase activity promotes PPARγ expression in preadipocytes. BIX was added when cells reached confluence. Forty-eight hours later, cells were collected for western blot of H3K9me2 and H3K9ac (A), ChIP of H3K9me2 and H3K9ac levels on PPARγ1/γ2 promoters at +0.5 kb (B), and qRT–PCR of PPARγ and Wnt10a expression (C). (D, E) Inhibiting G9a methyltransferase activity promotes adipogenesis. BIX was added when cells reached confluence. Forty-eight hours later, BIX was removed and adipogenesis was induced under standard condition. Six days later, cells were stained with Oil Red O (D) or collected for qRT–PCR of adipocyte marker expression (E). qPCR data are presented as means±s.d. and are representative of three independent experiments. Source data for this figure is available on the online supplementary information page.

Figure 8

aP2-Cre-mediated deletion of G9a promotes adipogenesis ex vivo. Primary white preadipocytes were isolated from inguinal white adipose tissues of littermate control (Con, with the genotype G9a^flox/flox) and G9a knockout mice (KO, with the genotype G9a^flox/flox;aP2-Cre). (A) Adipogenesis assay. (B) qRT–PCR of gene expression during adipogenesis. (C) aP2-Cre-mediated deletion of G9a allele was determined by quantitative PCR analysis of tissue genomic DNA. qPCR was performed in triplicates for littermate group 1. Similar results were obtained for littermate groups 2 and 3 (data not shown). qPCR data are presented as means±s.e.m.

Figure 9

Deletion of G9a increases adipose tissue weight in mice. Adipose-specific G9a knockout (KO) mice carried the genotype of G9a^flox/flox;aP2-Cre. Other age-matched littermates were used as control (Con) (see Materials and methods). All data were from 16- to 22-week-old male mice. Female mice data are shown in Supplementary Figure S7B and C. (A) Representative pictures of Con and KO mice. (B) Growth curves of Con (n=10) and KO (n=7) mice fed with normal chow. (C) Increased fat mass in KO mice. Body composition was measured in non-anaesthetized mice using the Bruker Minispec NMR analyzer. (D–G) Increased adipose tissue weights and adipocyte sizes in KO mice. (D) Representative pictures of epididymal WAT (epi-WAT), inguinal WAT (ing-WAT) and interscapular BAT isolated from Con and KO mice. (E) The average tissue weights in Con (n=8) and KO (n=6) mice are presented as % of body weight. (F, G) H&E staining of paraffin sections of ing-WAT from Con and KO mice are shown in (F). The cell sizes in the sections were quantified with ImageJ (G). (H) qRT–PCR analysis of gene expression in ing-WAT of Con (n=8) and KO (n=8) mice. (I) Model on how G9a represses adipogenesis. G9a inhibits PPARγ expression by adding H3K9me2 on the entire PPARγ locus while facilitates Wnt10a expression independent of HMT activity. All values are presented as the mean±s.e.m. *P<0.05; **P<0.01; ***P<0.005. Similar results were obtained from male and female mice, but only the male mice data are shown here.