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. 2023 Mar 23;12:e83979. doi: 10.7554/eLife.83979

Figure 4. O-GlcNAc Transferase (OGT) activity is necessary for chromatin and transcriptional dynamics in response to the sugar diet environment.

(A) (left) Average CATaDa signal on a CD (light yellow) and SD (dark yellow) with OSMI centered at OGT + Pcl peaks (compare to Figure 2D); (right) Average log2 Dam::Pcl/Dam signal on a CD (light pink) and SD (dark pink) with OSMI centered at OGT + Pcl peaks (compare to Figure 2). (B) Taste responses (y-axis) to stimulation of the labellum with 30, 10, and 5% sucrose (x-axis) of age-matched male Gr5a>Pcl (pink) and transgenic controls (shades of gray) on CD supplemented with vehicle (DMSO, n = 14–23) or 10 µM OSMI (n = 20–42). Two-way repeated measure analysis of variance (ANOVA): (1) DMSO, main effect of genotype (****p < 0.0001) and genotype x concentration (*p < 0.05); Tukey multiple comparisons test for 30, 10, and 5% sucrose concentrations: Gr5a>wcs vs. Gr5a>Pcl p = 0.0165, p = 0.0056, p = 0.0025; Pcl>wcs vs. Gr5a>wcs, ns. (2) OSMI: main effect of genotype p = 0.3194 and genotype × concentration p = 0.6893. (C) Taste responses (y-axis) to stimulation of the labellum with 30, 10, and 5% sucrose (x-axis) in Gr5a>OGT;Pclc429 (purple), Gr5a>OGT (green), Pclc429/+ (pink), and transgenic controls (gray) on CD or SD, n = 20–37. Two way repeated measure ANOVA, main effect of diet: ****p < 0.0001. Tukey multiple comparisons test, ****p < 0.0001 and *p < 0.05. (D) Taste responses (y-axis) to stimulation of the labellum with 30, 10, and 5% sucrose (x-axis) in Gr5a>OGT (green) and transgenic controls (shades of gray) on a CD supplemented with vehicle or 8 µM EEDi, n = 20–22. Two-way repeated measure ANOVA, main effect of Gr5a>OGT genotype compared to each control genotype: ****p < 0.0001. Tukey multiple comparisons test, ****p < 0.0001. (E) Log2fold (l2fc) of differentially expressed genes (DEGs) between SD/CD in w1118cs ± OSMI and Pclc429 SD/CD. (F) GO term analysis of the DEGs measured in the Gr5a+ neurons of flies fed a CD and SD + OSMI. (G) Taste responses (y-axis) to stimulation of the labellum with 30, 10, and 5% sucrose (x-axis) for a subset of DEGs in (E, purple circle) that show dependence on OGT and Polycomb Repressive Complex 2.1 (PRC2.1). n = 14–49. Purple, knockdown; red, overexpression; bold, direct OGT/PRC2.1 targets. Two-way repeated measure ANOVA, main effect of GAL4>wcs control genotype compared to each control genotype: ****p <0.0001. Tukey multiple comparisons test, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. ACL, ATP Citrate Lyase; Irk1, inwardly rectifier potassium channel 1; daw, dawdle; cbt, cabut; exex, extra extra; syt-a, synaptotagmin alpha. Data are shown as mean ± standard error of the mean (SEM).

Figure 4.

Figure 4—figure supplement 1. Pcl occupancy at PRE and Pcl peaks with inhibition of O-GlcNAc Transferase (OGT) activity.

Figure 4—figure supplement 1.

(A) Effects of OSMI and Tretmetibin treatments on survival, n = 3 vials of 30 flies each. One-way analysis of variance (ANOVA) with Dunnett’s test. (B) Design of the TaDa and CaTaDa experiment in flies fed a CD + OSMI and SD + OSMI. (C) Principal component analysis of normalized log2(Pcl::Dam/Dam) flies on CD + OSMI (light pink) or SD + OSMI (dark pink). (D) Overlap of log2(Dam::Pcl/Dam) chromatin occupancy peaks in flies with (green outline) or without (pink) OSMI (find_peaks, q < 0.01).
Figure 4—figure supplement 2. Pathway enrichment analysis of the effects of OSMI on Dam::Pcl peaks.

Figure 4—figure supplement 2.

iPAGE identification of pathways depleted (blue) or enriched (red) compared to background gene list from the Dam::Pcl peaks shared (left) or different (right) between SD/CD with or without OSMI. Scale represents over-representation (red) or under-representation (blue) of genes within a specific bin for the corresponding GO term. Black outlined boxes represent q < 0.05.
Figure 4—figure supplement 3. Transcriptional responses to the dietary environment when O-GlcNAc Transferase (OGT) activity was inhibited.

Figure 4—figure supplement 3.

(A) Principal component analysis of Gr5a>UAS-Rpl3-3XFLAG flies on a control (light gray) or sugar (dark gray) diet for 7 days supplemented with OSMI-1. (B) Log2fold change (l2fc) (Gr5a IP/Input) for Gr5a, Gr64f, Gr64a, Ir56D, Gr32a, and Gr66a genes. (C) Volcano plot representing differential expression in the Gr5a+ neurons of age-matched male Gr5a>UAS-Rpl3-3XFLAG flies on a control or sugar diet for 7 days supplemented with OSMI. n = 3 replicates per condition. Genes with q < 0.1 (Wald test) are in green. (D) Log2fold change (l2fc) for candidate gene targets of Cad and Ptx1 at SD7 (teal), Pclc429 (pink), and OSMI fed flies at SD7 (green).
Figure 4—figure supplement 4. Pathway enrichment analysis of genes reverted or unchanged by OSMI.

Figure 4—figure supplement 4.

iPAGE identification of pathways depleted (blue) or enriched (red) compared to background gene list from genes with positive or negative log2 fold changes on SD + OSMI. Scale represents over-representation (red) or under-representation (blue) of genes within a specific bin for the corresponding GO term. Black outlined boxes represent q < 0.05.