Extended Data Fig. 4: FABO is required for acetylation in the rim of the wing disc.
From: Nuclear position and local acetyl-CoA production regulate chromatin state
(a-c) Nejire (nej) transcript is uniform in the wing disc, detected by fluorescence in situ hybridization (FISH) in wildtype discs (a), or in nej knockdown discs (b) to validate specificity of the nej FISH probe. Temperature-sensitive knockdown of nej was induced for only 1 day at 29 °C because longer knockdown causes tissue loss. Representative images in (b), quantified in (c). Statistical significance by Mann-Whitney test (two-sided). (d-f) Uniform distribution of nejire (nej) transcript in the wing disc was validated with a second FISH probe (d). Specificity of the second nej FISH probe was validated by nej knockdown in the posterior compartment (IFP+), decreasing the nej FISH signal. Temperature-sensitive knockdown of nej was induced for 1 day at 29 °C. Representative images in (e), quantified in (f). Statistical significance by Mann-Whitney test (two-sided). (g-j) Glucose-derived acetyl-CoA is not required for acetylation of H3K18. Knockdown of pyruvate dehydrogenase α (PDHa, which should reduce glycolytic acetyl-CoA synthesis) or of pyruvate dehydrogenase kinase (PDK, which should increase glycolytic acetyl-CoA synthesis), each validated by pPDH S293 immunostaining (g and i, respectively), does not impact levels of H3K18ac (h and j, respectively). Temperature-sensitive knockdown of PDHa and of PDK was induced for 1 day at 29 °C in the posterior compartment (IFP+). (g, n = 9 discs; h, n = 10 discs; i, n = 9 discs; j, n = 8 discs). (k-l) Etomoxir-induced decrease in H3K18ac is concentration dependent. Etomoxir treatment shows already at low concentrations (50 μM) a decrease in H3K18ac levels. H3K18ac immunosignal further decreases with increasing levels of etomoxir (100 μM, 500 μM). Discs incubated with the indicated concentrations for 2 h in explant cultures. Representative images in (k), quantified in (l). Statistical significance by Kruskal-Wallis test with Dunn’s multiple comparisons test. (m-n) Decrease in H3K18ac upon etomoxir treatment is not due to inhibition of respiratory chain complex I. Inhibition of complex I with rotenone (10 μM) only mild decreases H3K18ac whereas etomoxir treatment (500 μM) causes strong loss of the acetylation mark. Discs incubated with the indicated compounds for 2 h in explant cultures. Representative images in (m), quantified in (n). Statistical significance by Kruskal-Wallis test with Dunn’s multiple comparisons test. (o-p) Rotenone activity is already maximal at 2.5 µM. Discs incubated for 2 h with various concentrations of rotenone (2.5-25 μM) and immunostained for H3K18ac. Representative images in (o), quantified in (p). Statistical significance by Kruskal-Wallis test with Dunn’s multiple comparisons test. (q-r) The decrease in H3K18ac caused by inhibition of FABO (500 μM etomoxir) is blunted by HDAC1 knockdown in the posterior compartment (GFP+). Discs incubated in the presence or absence of etomoxir for 1 h in explant cultures. Representative images in (q), quantified in (r). Statistical significance by two-way ANOVA with Šídák’s multiple comparisons test. Box plots: center line (median), box limits (1st and 3rd quartiles), whiskers (outer data points). A, anterior; P, posterior.