PWWP2B Fine‐Tunes Adipose Thermogenesis by Stabilizing HDACs in a NuRD Subcomplex

Abstract Histone deacetylases (HDACs) are widely involved in many biological processes, as well as in control of brown and beige adipose physiology, but the precise molecular mechanisms by which HDACs are assembled into transcriptional machinery to fine‐tune thermogenic program remain ill‐defined. PWWP domain containing 2b (PWWP2B), which is identified as a component of the nucleosome remodeling and deacetylation complex (NuRD), interacts and stabilizes HDAC1/2 at the thermogenic gene promoters to suppress their expression. Ablation of Pwwp2b promotes adipocyte thermogenesis and ameliorates diet‐induced obesity in vivo. Intriguingly, Pwwp2b is not only a brown fat‐enriched gene but also dramatically induced by cold and sympathetic stimulation, which may serve as a physiological brake to avoid over‐activation of thermogenesis in brown and beige fat cells.

. Pwwp2b knockout mice are more resistance to HFD-induced obesity.
a) Food intake of high-fat-diet. n=7. b) Fat and liver weights of mice after a HFD-feeding for 11 weeks. n=10-11, male mice.
The expression of adipogenic genes was analyzed by QPCR. n=3.
b) Representative images of cells in (a) during differentiation taken by a phase-contrast microscope. Scale bar in day -2, 0 and 3 is 100μm; scale bar in day 6 is 50μm.
c) Pwwp2b knockdown cells generated as in (a) were stained by Oil red O on differentiation day 6.
d) Gene expression levels in Pwwp2b knockdown cells generated as in (a) detected by QPCR. n=3.
e) The PWWP2B level in lentiviral Pwwp2b-transduced adipocytes. The lentivirus bearing Pwwp2b infected brown preadipocytes. Cells were induced to mature adipocytes and PWWP2B protein level was analyzed on day 6.
f) Gene expression analysis in adipocytes in (e) was performed on day 6. n=3.
Rbbp4 or Rbbp7 plasmids were co-transfected with Hdac1 plasmids to HEK293T cells.
Western blot analysis was performed 48 h after transfection.   Figure S8. ZFP516 recruits PWWP2B to regulate thermogenic program.. a) Overexpressed ZFP516 interacts with endogenous PWWP2B in brown fat cells. ZFP516 was overexpressed by lentiviral transduction to preadipocytes. Immunoprecipitation assay was performed in mature adipocytes (day 6). b) PWWP2B competes LSD1 to associate with ZFP516. Plasmids were transfected to HEK293T cells following the immunoprecipitation assay. c) PRDM16 promotes PWWP2B interacting with ZFP516. Plasmids were transfected to HEK293T cells following the immunoprecipitation assay.
d) The interaction between PWWP2B and PRDM16 can not be detected. The plasmid bearing Prdm16 was co-transfected with a Pwwp2b or Pparα plasmid to HEK293T cells. The interaction between PPARα and PRDM16 was used as a positive control. The red arrows point to PWWP2B in western blot. e) Knocking down Zfp516 abolished PWWP2B association with gene promoters. Lentivirus bearing HA-Pwwp2b infected brown preadipocyte following standard induction to mature adipocytes. Lentiviral Zfp516 shRNA was used to transduce adipocytes on differentiation day 2 and 4. ChIP was performed on day 6 with HA antibody or control IgG. ChIP was repeated for 3 times and representative data were shown. n=3.
f) The Zfp516 knockdown efficiency in cells of (e). n=3. g) PWWP2B enhances the interaction between ZFP516 and HDAC1. Plasmids were transfected to HEK293T cells. Immunoprecipitation was performed 48 h after transfection. *P<0.05, **P<0.01 by unpaired two-tailed Student's t-test. The data shown are mean±SEM.