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. 2016 May 5;62(3):443-452.
doi: 10.1016/j.molcel.2016.03.011. Epub 2016 Apr 14.

S6K1 Phosphorylation of H2B Mediates EZH2 Trimethylation of H3: A Determinant of Early Adipogenesis

Sang Ah Yi  1 , Sung Hee Um  2 , Jaecheol Lee  3 , Ji Hee Yoo  1 , So Young Bang  1 , Eun Kyung Park  1 , Min Gyu Lee  1 , Ki Hong Nam  1 , Ye Ji Jeon  1 , Jong Woo Park  1 , Jueng Soo You  4 , Sang-Jin Lee  5 , Gyu-Un Bae  5 , Jong Won Rhie  6 , Sara C Kozma  7 , George Thomas  8 , Jeung-Whan Han  9
Affiliations

S6K1 Phosphorylation of H2B Mediates EZH2 Trimethylation of H3: A Determinant of Early Adipogenesis

Sang Ah Yi et al. Mol Cell. .

Abstract

S6K1 has been implicated in a number of key metabolic responses, which contribute to obesity. Critical among these is the control of a transcriptional program required for the commitment of mesenchymal stem cells to the adipocytic lineage. However, in contrast to its role in the cytosol, the functions and targets of nuclear S6K1 are unknown. Here, we show that adipogenic stimuli trigger nuclear translocation of S6K1, leading to H2BS36 phosphorylation and recruitment of EZH2 to H3, which mediates H3K27 trimethylation. This blocks Wnt gene expression, inducing the upregulation of PPARγ and Cebpa and driving increased adipogenesis. Consistent with this finding, white adipose tissue from S6K1-deficient mice exhibits no detectable H2BS36 phosphorylation or H3K27 trimethylation, whereas both responses are highly elevated in obese humans or in mice fed a high-fat diet. These findings define an S6K1-dependent mechanism in early adipogenesis, contributing to the promotion of obesity.

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Figures

Figure 1
Figure 1. S6K1 Is Required for H3K27 Trimethylation during Adipogenic Commitment
(A) Schematic representation of 10T1/2 cell differentiation into adipocyte. (B) Immunoblot analysis of cytoplasmic and nuclear extracts from 10T1/2 cells treated with BMP4 for the indicated times, with quantification graphs of S6K1 indicated in the left-hand panel. (C) Immunoblot analysis of cytoplasmic and nuclear extracts from 10T1/2 cells treated with BMP4 in either the absence or presence of rapamycin (100 nM), which was added for 1 hr prior to the addition of BMP4. (D) Immunoblot analysis of 10T1/2 cells treated with BMP4 for 4 days in the presence or absence of PF-4708671 (20 μM). (E) Immunoblot analysis of 10T1/2 cells expressing S6K1 siRNA (#03) in the presence of BMP4 (2 days). (F) Immunoblot analysis of 10T1/2 cells treated with BMP4 for 1 hr in the presence or absence of PF-4708671 (20 μM). (G) Immunoblot analysis of HeLa cells stimulated with serum for 15 min after pretreatment with rapamycin (100 nM) or PF-4708671 (20 μM) for 1 hr. See also Figure S1.
Figure 2
Figure 2. S6K1 Phosphorylates H2BS36 and S6K1-Mediated H2B Phosphorylation Induces Trimethylation of H3K27
(A) In vitro phosphorylation of recombinant H2A, H2B, H3, or H4 with active S6K1 and 32P-ATP. (B) In vitro phosphorylation of H2B with active S6K1 and 32P-ATP after preincubation of S6K1 with the indicated concentration of PF-4708671 for 30 min. (C) In vitro phosphorylation of H2B 7–26 and 27–46 peptides with active S6K1 and 32P-ATP. (D) Peptide sequences containing S6K1 target motifs in H2B or single site mutation (top panel) and in vitro phosphorylation of the indicated peptides with active S6K1 and 32P-ATP (bottom panel). (E) Immunoblot analysis of 10T1/2 cells expressing S6K1 siRNA in the presence of BMP4 (2 days). (F) Immunoblot analysis of S6K1 immunoprecipitates (IP) and whole-cell lysates (WCLs) from 10T1/2 cells. (G) Immunoblot analysis of immunoprecipitates with S6K1 antibody from 10T1/2 cells in the presence or absence of BMP4. (H) Immunoblot analysis of 10T1/2 cells treated with BMP4 for the indicated time. (I) Immunoblot analysis of 10T1/2 cells treated with BMP4 in the presence or absence of PF-4708671 (20 μM) for the indicated time. (J) Immunoblot analysis of 10T1/2 cells transfected with or without the GFP-H2BS36D vector in the presence of BMP4 and PF-4708671 (20 μM) for 24 hr. (K) Immunoblot analysis of immunoprecipitates with GFP antibody from 10T1/2 cells transfected with GFP-H2B36D or GFP-H2BS36A vectors in the presence of BMP4. See also Figure S2.
Figure 3
Figure 3. S6K1-Mediated H2BS36 Phosphorylation Suppresses the Expression of Wnt Genes through the Recruitment of EZH2 during Adipogenic Commitment
(A) The mRNA levels of Wnt genes in 10T1/2 cells expressing S6K1 siRNA during commitment. (B) The mRNA levels of Wnt genes in 10T1/2 cells expressing WT, constitutively active, and dominant-negative S6K1 vectors during commitment. (C and D) 10T1/2 cells treated with BMP4 or co-treated with BMP4 and rapamycin (100 nM) for 24 hr, followed by ChIP-qPCR analyses for S6K1 (C) and H2BS36p (D) antibodies in the promoter regions of the Wnt genes. (E) The mRNA levels of Wnt genes in 10T1/2 cells expressing GFP-WT H2B, GFP-H2BS36D, or GFP-H2BS36A vectors during commitment. (F) The mRNA levels of Wnt genes in 10T1/2 cells expressing the GFP-H2BS36D vector in the presence or absence of PF-4708671 (20 μM) during commitment. (G and H) 10T1/2 cells treated with BMP4 or co-treated with BMP4 and rapamycin (100 nM) for 24 hr, followed by ChIP-qPCR analyses for EZH2 (G) and H3K27me3 (H) antibodies in the promoter regions of the Wnt genes. (I and J) 10T1/2 cells transfected with GFP-WT H2B, GFP-H2BS36D, or GFP-H2BS36A vectors and incubated with BMP4 for 24 hr, followed by ChIP-qPCR analyses for EZH2 (I) and H3K27me3 (J) antibodies in the promoter region of the Wnt genes. In (A)–(J), data are presented as the mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001. See also Figures S3 and S4.
Figure 4
Figure 4. S6K1-Mediated H2BS36 Phosphorylation Is Required for Adipogenic Commitment and Correlates with BMI
(A) The mRNA levels of PPARγ and Cebpa genes in 10T1/2 cells expressing S6K1 siRNA during commitment. (B) The mRNA levels of PPARγ and Cebpa genes in 10T1/2 cells expressing WT, constitutively active, and dominant-negative S6K1 vectors during commitment. (C) The mRNA levels of PPARγ and Cebpa genes in 10T1/2 cells expressing GFP-WT H2B, GFP-H2BS36D, or GFP-H2BS36A vectors during commitment. (D) The mRNA levels of PPARγ and Cebpa genes in 10T1/2 cells expressing GFP-H2BS36D vector in the presence or absence of PF-4708671 (20 μM) during commitment. (E) The mRNA levels of adipocytic marker genes in fully differentiated adipocytes from 10T1/2 cells expressing GFP-WT H2B, GFP-H2BS36D, or GFP-H2BS36A vectors during commitment. (F) The mRNA levels of adipocytic marker genes in fully differentiated adipocytes from 10T1/2 cells expressing GFP-H2BS36D vector in the presence or absence of PF-4708671 (20 μM) during commitment. (G) Epididymal white adipose tissues (eWAT) from WT and S6K1-knockout (S6K1−/−) mice. (H) The mRNA levels of Wnt genes in eWAT from WT and S6K1−/− mice. (I) The mRNA levels of adipogenic marker genes in eWAT from WT and S6K1−/− mice. (J) Immunoblot analysis of eWAT from WT and S6K1−/− mice. (K) Immunoblot analysis of human abdominal subcutaneous WAT from normal or overweight specimens. (L) Semiquantitative analysis of pS6K1, H3K27me3, and H2BS36p level in individual WAT samples. (M) Immunoblot analysis of eWAT from WT mice fed normal chow diet (NCD) or high-fat diet (HFD) for 10 weeks. (N) Mechanisms of metabolic regulation by S6K1 in the nucleus (1) and the cytoplasm (2). In (A)–(F), (H), and (I), data are presented as the mean ± SEM (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001.
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