Fig. 1

Expression of H3.3K36M in preadipocytes increases H3K27me3 to inhibit adipogenesis and adipogenic gene induction. a–c Ectopic expression of H3.3K36M in preadipocytes inhibits adipogenesis. SV40T-immortalized brown preadipocytes were infected with retroviral vector (Vec) expressing FLAG-tagged wild-type (WT) or K36M mutant histone H3.3, followed by adipogenesis assay. a Western blot analysis of histone modifications. Histone extracts of preadipocytes were subjected to Western blot analysis using antibodies indicated on the left. me2 and me3 refer to di-methylation and tri-methylation, respectively. Long exposure of histone H3 Western blot reveals the relative levels of ectopic H3.3 and endogenous H3. b Seven days after induction of differentiation, cells were stained with Oil Red O. Upper panels, stained dishes; lower panels, representative fields under microscope. Scale bars = 30 μm. c qRT-PCR of Pparg, Cebpa, and Fabp4 expression at day 0 (D0) and day 7 (D7) of adipogenesis. qRT-PCR data are presented as means ± SEM. Three technical replicates from a single experiment were used. d–f WT and K36M cells were collected at D0, D2, and D7 for RNA-Seq analysis. d Schematic of identification of K36M-sensitive and K36M-resistant up-regulated genes at D2 of adipogenesis. The threshold for up-regulation or down-regulation is twofold. e Gene ontology (GO) analysis of gene groups defined in d. f Expression levels of representative genes are shown in RPKM (reads per kilo base of transcript per million mapped reads) values. g Genome browser views of ChIP-Seq and RNA-Seq data on Pparg and Cebpa loci during adipogenesis