Fig. 10: PRDM16 interacts with DNMT1 to maintain brown adipocyte function.

A ChIP assay of DNMT1 binding to Myod1 promoter in control or Prdm16 knockdown BAT1 brown adipocytes treated with or without isoproterenol (n = 4/group). Data are expressed as mean ± SEM. Indicates statistical significance between different treatments analyzed by Kruskal–Wallis non-parametric ANOVA H test by rank followed by Pairwise Comparisons test between groups, H(3) = 9.816, p = 0.020. B Co-IP of DNMT1 and FLAG-PRDM16 in HEK293T cells. Data are representative from two independent experiments. C Co-IP of DNMT1 and various fragments of PRDM16. HA-tagged fragments of PRDM16 were expressed along with full-length DNMT1 in HEK293T cells. Cell lysates were immunoprecipitated with anti-DNMT1 antibodies followed by immunoblotting with HA or DNMT1 antibodies. Color-coded domain architecture of PRDM16 shows a PR/SET domain (PR), an N-terminal zinc-finger domain containing seven C2H2 zinc finger motifs (ZF1), a proline rich domain (PRR), a repression domain (RD), a second C-terminal zinc-finger domain containing three C2H2 zinc finger motifs (ZF2), and an acidic activation domain (AD). Data are representative from two independent experiments. D Co-IP of PRDM16 and various fragments of DNMT1. HA-tagged fragments of DNMT1 were expressed along with full-length PRDM16 in HEK293T cells. Cell lysates were immunoprecipitated with anti-HA antibodies followed by immunoblotting with HA or PRDM16 antibodies. Color-coded domain architecture of DNMT1 shows the N-terminal independently folded domain (NTD), replication foci-targeting sequence (RFTS) domain, a Zn-finger like CXXC motif, two bromo adjacent homology (BAH1 and BAH2) domains, and the catalytic domain. Data are representative from two independent experiments. E DNMT1 protein levels in Prdm16-overexpressed HEK293T cells treated with cycloheximide (CHX) (60 µg/ml) for various time. Data are representative from two independent experiments. F The interaction between PRDM16 and DNMT1 on Myod1 promoter in Prdm16 overexpressed BAT1 brown adipocytes measured by ChIP and Re-ChIP assay via sequential immunoprecipitation of PRDM16 and then DNMT1 (n = 4/group). Data are expressed as mean ± SEM. *Indicates statistical significance between two groups by Mann–Whitney’s nonparametric U test. G, H Expression of miR-133a, miR133b, miR-206 and miR-1 in iBAT of female D1KO and fl/fl mice fed with a regular chow diet (G, n = 4 for fl/fl and 6 for D1KO) and in BAT1 brown adipocytes with Myod1 overexpression (H, n = 6/group). Data are expressed as mean ± SEM. *indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in (G) and by two-tailed unpaired student’s t-test in (H). I, J Dnmt1 and miR-133a expression (I) and BAT-specific gene expression (J) in BAT1 brown adipocytes transfected with Dnmt1 siRNA, miR-133a inhibitor or both (J) (n = 3/group). Data are expressed as mean ± SEM. *Indicates statistical significance analyzed by one-way ANOVA followed by Fisher’s LSD multiple comparisons test. In (I), for Dnm1 expression, F = (3,8) = 4.62, p = 0.037; for miR-133 expression, F(3,8) = 21.370, p < 0.0001. In (J), for Ucp1 expression, F = (3,8) = 4.827, p = 0.033; for Prdm16 expression, F = (3,8) = 10.863, p = 0.003; for Pgc1β expression, F(3,8) = 11.213, p = 0.003. K Schematic illustration of the interaction between UTX-regulated PRDM16 and DNMT1 in the maintenance of brown fat identity and suppression of myogenic remodeling in mature brown adipocytes. In brief, in mature brown adipocytes, UTX maintains the persistent demethylation of the repressive mark H3K27me3 at Prdm16 promoter, leading to high expression of Prdm16; PRDM16 then recruits the DNA methyltransferase DNMT1 to Myod1 promoter, causing Myod1 promoter hypermethylation, and suppressing Myod1 expression. In addition, reduced Myod1 expression relieves the inhibition on Prdm16 by miR-133, further increasing Prdm16 expression. The interaction between PRDM16 and DNMT1 coordinately serves to maintain brown adipocyte identity while repressing myogenic remodeling in mature brown adipocytes, thus promoting their active brown adipocyte thermogenic function. Suppressing this interaction by HFD feeding induces brown adipocyte-to-myocyte remodeling, which limits brown adipocyte thermogenic capacity and compromises diet-induced thermogenesis, leading to the development of obesity.