Research Article

Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis

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Science  11 May 2018:
Vol. 360, Issue 6389, eaan5780
DOI: 10.1126/science.aan5780
  • Compartmentalized NAD+ biosynthesis by NMNATs regulates adipogenesis through PARP-1.

    NMNATs synthesize NAD+ from nicotinamide mononucleotide (NMN) and adenosine triphosphate. Nuclear NMNAT-1 provides NAD+ for nuclear ADP-ribosylation and gene regulation by PARP-1, whereas cytoplasmic NMNAT-2 provides NAD+ for cytosolic ADP-ribosylation and cellular metabolism. Competition between NMNAT-1 and NMNAT-2 for their common substrate, NMN, promotes compartmentalized regulation of NAD+ levels, allowing for discrete nuclear and cytoplasmic events. The fluorescent images of NAD+ in the bottom panel were generated using a NAD+ sensor localized to the nucleus (left) or the cytoplasm (right).

  • Fig. 1 NMNAT-1 regulates PARP-1 activity and adipocyte differentiation.

    (A) Schematic representation of NAD+ biosynthesis by NMNATs and their subcellular localization. (B) Western blot showing the levels of PAR upon shRNA-mediated knockdown (KD) of Nmnat1 during the early phase of adipogenesis in 3T3-L1 cells. PAR levels (primarily automodification of PARP-1) represent the enzymatic activity of PARP-1. Blots of NMNAT-1, PARP-1, and SIRT1 are shown for comparison (β-tubulin was used as a control). M.W., molecular weight. (C and D) Accumulation of lipid droplets at 4 days (C) and 8 days (D) of differentiation after knockdown of Nmnat1 or Parp1 in 3T3-L1 cells. Lipids were stained using BODIPY 493/503 [green, (C)] or Oil Red O [red, (D)], and nuclei were stained using TO-PRO-3 [blue, (C)]. Scale bars in (C), 150 μm.

  • Fig. 2 NMNAT-1 and PARP-1 regulate the adipogenic transcriptional program through C/EBPβ.

    (A) Expression of adipocyte marker genes in 3T3-L1 cells at 4 days of differentiation, as determined by RT-qPCR. Each bar represents the mean + SEM (n = 3). Asterisks indicate significant differences from the corresponding control (Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001). (B) RNA-seq assay of genes regulated upon Nmnat1 or Parp1 knockdown in 3T3-L1 cells compared with control knockdown after 2 days of differentiation. The overlapping region of the Venn diagram indicates co-regulated genes. (C) Percent of promoters of NMNAT-1 and PARP-1–co-regulated genes [from (B)] that interact with binding sites for adipogenic transcription factors (TFs). The interaction between the promoter regions of the co-regulated genes and the transcription factor binding sites (TFBSs) were determined by integrating published PCHi-C data [National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) data set GSE95533] and ChIP-seq data (NCBI GEO data set GSE27826). Bars show means + SEM. (D and E) Levels of Cebpb mRNA assessed by RT-qPCR (D) and C/EBPβ protein assessed by Western blotting (E) in 3T3-L1 cells after knockdown of Nmnat1. Each bar represents the mean + SEM (n = 3). ns, not significant (Student’s t test; P > 0.05). (F) C/EBPβ binding at the Cebpa and Pparg gene promoters in 3T3-L1 cells after 4 hours of differentiation, as determined by ChIP-qPCR assays. Each bar represents the mean + SEM (n = 3). Bars marked with asterisks are significantly different from the control (Student’s t test; *P < 0.05). (G) Expression of genes whose promoters interact with C/EBPβ binding sites upon knockdown of Nmnat1 or Parp1. Significant C/EBPβ ChIP-seq peaks at 4 hours postdifferentiation were compared with PCHi-C–determined looping events to define the interactions. The expression level of those genes after 2 days of differentiation was compared with the expression level in control knockdown cells at day 0 to determine the fold change. Up-regulated genes (fold change > 1) were used in the analysis. Bars marked with different letters are significantly different from each other (Wilcoxon rank sum test; P < 0.0001).

  • Fig. 3 Nuclear NAD+ levels are regulated through compartmentalized biosynthesis.

    (A) The levels of total intracellular NAD+ (enzyme-linked NAD+ assay), PAR (Western blot), and Nmnat2 mRNA (RT-qPCR) were determined at the indicated differentiation time points in 3T3-L1 cells (means ± SEM). (B) Detection of nuclear (Nuc) and cytoplasmic (Cyto) NAD+ levels in 3T3-L1 cells by using a cpVenus-based NAD+ biosensor. Representative images of NAD+ sensor fluorescence during the early phase of differentiation are shown. (C) Changes in subcellular NAD+ levels during the early phase of differentiation of 3T3-L1 cells. NAD+ levels were calculated from sensor(488/405 nm)/control(488/405 nm) fluorescence ratios determined by flow cytometry using a standard curve. Each bar represents the mean + SEM (n = 7). Bars marked with asterisks are significantly different from the undifferentiated (0 hours) control [analysis of variance (ANOVA); **P < 0.01]. (D) Representative images of nuclear NAD+ sensor fluorescence (488/405 nm) during differentiation upon Nmnat2 knockdown. (E) Effect of Nmnat2 knockdown on nuclear NAD+ levels in 3T3-L1 cells. Relative nuclear NAD+ levels were determined from the sensor(488/405 nm)/control(488/405 nm) fluorescence ratio using flow cytometry. Each bar represents the mean + SEM (n = 3). Asterisks indicate significant differences from the control knockdown at the 0-hour time point (ANOVA; *P < 0.05). (F) Effect of Nmnat2 knockdown on the differentiation of 3T3-L1 cells. Differentiation was assessed by the expression of adipocyte marker genes. Each bar represents the mean + SEM (n = 3). Asterisks indicate significant differences from the control (Student's t test; **P < 0.01, ***P < 0.001; ****P < 0.0001). Scale bars, 10 μm in (B) and 20 μm in (D).

  • Fig. 4 Substrate competition between NMNAT-1 and NMNAT-2 regulates nuclear NAD+ levels during differentiation.

    (A) Schematic representation of substrate competition between NMNAT-1 and NMNAT-2. (B to D) Supplementation with exogenous NMN disrupts NMNAT-1 and NMNAT-2 substrate competition. Effects on total intracellular NAD+ (B) and nuclear NAD+ levels [(C) and (D)] upon supplementation with 1 mM NMN are shown. Bar graphs [(B) and (D)] represent means + SEM (n = 7). Asterisks indicate significant differences from the undifferentiated (0 hours) control (ANOVA; *P < 0.05; **P < 0.01). Representative images (C) show changes in nuclear NAD+ sensor fluorescence ratios during differentiation. Scale bars, 20 μm. Nuclear NAD+ levels (D) were, determined by the sensor(488/405 nm)/control(488/405 nm) fluorescence ratios using flow cytometry. (E) Western blots showing the rescue of PARP-1 enzymatic activity during early differentiation upon supplementation with 5 mM NMN. PAR levels indicate PARP-1 enzymatic activity. (F and G) Supplementation with exogenous NMN (5 mM) inhibits the differentiation of control 3T3-L1 and SVF cells (F), but not NMNAT-1– and PARP-1–depleted cells (G). The expression of adipocyte marker genes determined by RT-qPCR was used to assess the extent of differentiation. Each bar represents the mean + SEM (n = 3). Asterisks indicate significant differences versus the corresponding control (Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001); ns, not significant (Student’s t test; P > 0.05).

  • Fig. 5 NMNAT-2 induction is associated with enhanced glucose metabolism during the early phase of adipogenesis.

    (A) Schematic representation of the potential role of NMNAT-1 and NMNAT-2 during adipocyte differentiation. (B) Nmnat2 knockdown does not affect the expression of genes involved in glucose metabolism after 8 hours of differentiation, as determined by RT-qPCR. None of the minor differences are significant (Student’s t test; P > 0.05). (C) Nmnat2 knockdown alters glucose flux during the differentiation of 3T3-L1 cells. Mass isotopomer analysis of citrate m+2 in cells with or without Nmnat2 knockdown. Asterisks indicate significant differences from the corresponding control (Student’s t test; *P < 0.05); ns, not significant (Student’s t test; P > 0.05). (D and E) Expression of NMNAT-2 in 3T3-L1 cells differentiated in the presence of various extracellular glucose levels (D) or the glycolysis inhibitor 2-deoxyglucose (2-DG) (E) by Western blotting. (F) Regulation of C/EBPβ binding to target gene promoters in 3T3-L1 cells by ChIP-qPCR upon inhibition of glycolysis with 2-DG. The assays were done 8 hours postdifferentiation. Asterisks indicate significant differences from the corresponding control (Student’s t test; **P < 0.01). TSS, transcription start site. Throughout, bars represents means + SEM.

  • Fig. 6 Model for the coordination of transcription and glucose metabolism during adipocyte differentiation through compartmentalized NAD+ biosynthesis.

    In undifferentiated 3T3-L1 cells, NMN is used mostly by NMNAT-1 to synthesize nuclear NAD+, which supports PARP-1 activity. Active PARP-1 ADP-ribosylates the adipogenic transcription factor C/EBPβ, which inhibits its chromatin binding and transcriptional activities, preventing differentiation in the absence of an adipogenic signal. During differentiation, the expression of genes involved in glucose metabolism increases, leading to a rapid induction of glucose flux in 3T3-L1 cells. Concurrently, NMNAT-2 is rapidly induced to support the high local NAD+ demands caused by enhanced glucose metabolism, thereby limiting NMN availability in the nucleus for NMNAT-1 to synthesize nuclear NAD+. Reduced nuclear NAD+ concentrations lead to reduced PARP-1 activity, allowing C/EBPβ to initiate the adipogenic transcription program. Competition for the NAD+ precursor, NMN, between the nuclear and cytoplasmic NMNATs results in changes in nuclear NAD+ levels, allowing cells to coordinate glucose metabolism and transcription.

Supplementary Materials

  • Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis

    Keun Woo Ryu, Tulip Nandu, Jiyeon Kim, Sridevi Challa, Ralph J. DeBerardinis, W. Lee Kraus

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