Nutritional vitamin B12 regulates RAS/MAPK-mediated cell fate decisions through one-carbon metabolism – PubMed Black Hawk Supplements
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Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin…
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Nutritional vitamin B12 regulates RAS/MAPK-mediated cell fate decisions through one-carbon metabolism
Ana Cristina Laranjeira et al. Nat Commun. .
Abstract
Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin B12 enhances via the choline/phosphatidylcholine metabolism vulval induction by down-regulating fat biosynthesis genes and increasing H3K4 tri-methylation, which results in increased expression of RAS/MAPK target genes. Furthermore, the nucleoside metabolism and H3K4 tri-methylation positively regulate germ cell apoptosis and oocyte production. Using mammalian cells carrying different activated KRAS and BRAF alleles, we show that the effects of methionine on RAS/MAPK-regulated phenotype are conserved in mammals. Our findings suggest that the vitamin B12-dependent one-carbon metabolism is a limiting factor for diverse RAS/MAPK-induced cellular responses.
© 2024. The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
a Schematic representation of one-carbon metabolism. b Illustrations of the observed phenotypes: germ cell apoptosis (corpses), oocyte differentiation, or vulval induction. Scale bar: 10 µm. Number of corpses (c) or oocytes (d) in wild-type animals. e Vulval induction index of let-60(gf) animals. c–e Animals were fed with E. coli OP50 (gray), Comamonas DA1877 (blue), or E. coli OP50 supplemented with 64 nM B12 (pink). Number of corpses (f), oocytes (g) and vulval induction index (h) for indicated genotypes fed either with E. coli OP50 (gray) or Comamonas DA1877 (blue). Number of corpses (i), oocytes (j) and vulval induction index (k) for indicated genotypes fed with E. coli OP50 and supplemented with 64 nM B12. Dots (in c, d, f, g, i, j) represent individual corpses/oocytes or animals (in e, h, k) from two independent biological replicates; number of animals in brackets. Boxplots show 25-75 percentiles and median; whiskers represent min and max values. **P < 0.005, ***P < 0.0005, ****P < 0.0001 using one-way ANOVA followed by Dunnett’s multiple comparison tests in c, d, i and j, two-way ANOVA followed by a Tukey’s multiple comparison test in f, g and h and a Kruskal-Wallis test for non-parametric data followed by a Dunn’s multiple comparison test in e and k. Source data are provided as a Source Data file.
a Oocyte number for indicated genotypes. Dots represent individual oocytes. b Mitotic index for indicated genotypes. Dots represent individual animals. c Fluorescence images showing the pachytene-to-diplotene (P-D) transition (yellow dashed lines) for indicated genotypes; germ cell nuclei are marked with H2B::GFP. Gonads are outlined by white dashed lines. Scale bar: 10 µm. d Number of cells inside an area starting from the P-D and ending 15 cell-rows distally of indicated genotypes; P-D was determined based on the nuclear marker shown in c; in let-60(gf) animals the loop region was used as the starting point. e Length of 15-cell rows for indicated genotypes. f Distance from the P-D to the loop region for indicated genotypes. d–f dots represent individual animals. g Speed of germ cell progression through the pachytene region for indicated genotypes; for individual tracking see Supplementary Fig. 2. Dots represent the speed of individual cells. h Ovulation time for indicated genotypes. Dots represent individual oocytes. Bars represent mean ± SD. a–h Animals were fed with E. coli OP50 (gray) or Comamonas DA1877 (blue). Number of animals in brackets. Boxplots show 25-75 percentiles and median; whiskers represent min and max values. ****P < 0.0001 using two-way ANOVA followed by uncorrected Fisher’s LSD multiple comparison test in a, one-way ANOVA followed by Dunnett’s multiple comparison tests in b, d–h. Source data are provided as a Source Data file.
a, b Corpse and oocyte numbers for indicated genotypes grown at 15 °C and transferred to 25 °C 4 h before quantification, or grown at 20 °C and transferred to 25 °C 18 h. c DIC images of gonads of indicated genotypes grown at 20 °C and transferred to 25 °C 18 h before quantification. Numbers indicate mature oocytes. Scale bar: 10 µm. d Vulval induction index for indicated genotypes. a–d Animals were fed with E. coli OP50. e, f Corpse and oocyte numbers for indicated genotypes grown at 15 °C and transferred to 25 °C 4 h before quantification. g, h Corpse and oocyte numbers for indicated genotypes grown at 20 °C and transferred to 25 °C 5 h before quantification. e–h Animals were fed with E. coli OP50 (gray) or Comamonas DA1877 (blue). i dpERK intensity profiles in gonads of wild-type fed with E. coli OP50 (gray) or Comamonas DA1877 (blue), and let-60(gf ts) fed with E. coli OP50 (orange). x-axis represents normalized gonad length. Average intensities ± SD (shaded) are shown. Data from three independent biological replicates. j Western blot quantification of the somatic and germline isoform of dpERK for indicated genotypes. Bars represent mean ± SD normalized to the control (wild-type E. coli OP50) from three independent biological and technical replicates. See Supplementary Fig. 4 for the individual WB. k ERK-nKTR biosensor quantification. Dots represent individual animals from 2 independent biological replicates. Data show median ± min and max. l–m Normalized intensity of LIN-1::GFP or LIN-39::GFP at the Pn.px stage in 1° and 2° VPCs of let-60(gf) animals. Data were normalized to the control (E. coli OP50). Dots represent individual animals from two independent biological replicates. j–m Animals were fed with E. coli OP50 (gray) or Comamonas DA1877 (blue). Number of animals is in brackets. In a–h, j, l, m, boxplots show 25–75 percentiles and median; whiskers represent min and max values. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 using one-way ANOVA followed by Dunnett’s multiple comparison tests in a and b, a Kruskal-Wallis test for non-parametric data in d, two-way ANOVA followed by a uncorrected Fisher’s LSD multiple comparison tests in e–h and j and unpaired, two-tailed t-tests in l and m. Source data are provided as a Source Data file.
Number of corpses (a, d), oocytes (b, e), and vulval induction index (c, f) for indicated genotypes fed with E. coli OP50 and supplemented with the following metabolites; Met – 5 mM methionine; FA – 100 µM folinic acid; Chol – 40 mM choline; 1 mM nucleosides. Oocyte number of wild-type animals (g, i) and vulval induction index of let-60(gf) animals (h, j) fed with indicated RNAi clones or control (EV – empty vector). NGM was supplemented with 64 nM of B12; animals were exposed to RNAi from L1 except where indicated (L4). a–j Dots represent individual corpses/oocytes or animals from two independent biological replicates. Boxplots show 25–75 percentiles and median; whiskers represent min and max values. Number of animals in brackets. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 using two-way ANOVA followed by a Tukey’s multiple comparison test in a–c, two-way ANOVA followed by uncorrected Fisher’s LSD multiple comparison test in d, e, and f, one-way ANOVA followed by Dunnett’s multiple comparison test in g and i (WT since L1), an unpaired, two-tailed t-test in i (since L4) and a Kruskal-Wallis test for non-parametric data followed by a Dunn’s multiple comparison test in h and j. Source data are provided as a Source Data file.
a Differentially expressed genes in adult wild-type animals fed with E. coli OP50 or Comamonas DA1877. Genes with a p-value < 0.05 and a log2-fold change > 2.5 are highlighted. p-values were calculated as described in the methods. b Top 50 differentially expressed genes for the indicated conditions. c Fluorescence images of dsh-3::gfp marking intestinal lipid droplets (LD) in animals fed with E. coli OP50 (gray), Comamonas DA1877 (blue), or E. coli OP50 supplemented with 40 mM choline (yellow). Scale bar: 10 µm. c’ Area of intestinal LD; dots represent individual droplet areas of 100 droplets per animal, from two independent biological replicates. Bars represent mean with SD. vulval induction index of let-60(gf) (d) or oocyte number of wild-type animals (e) fed with indicated RNAi clones and control (EV – empty vector). f Vulval induction index of let-60(gf) and g, oocyte number of wild-type animals fed with E. coli OP50 (gray) or Comamonas DA1877 (blue), supplemented with indicated fatty acids. d–g dots represent individual gonads (e, g) or animals (d, f) from two independent biological replicates. Boxplots show 25–75 percentiles and median; whiskers represent min and max values. Number of animals in brackets. **P < 0.005, ***P < 0.0005, ****P < 0.0001 using one-way ANOVA followed by Dunnett’s multiple comparison test in c’ and e, two-way ANOVA followed by a Tukey’s multiple comparison test in f and g and a Kruskal-Wallis test for non-parametric data followed by a Dunn’s multiple comparison test in d, h, i. Source data are provided as a Source Data file.
a Western blot quantification of tri-methylated H3K4 for indicated genotypes fed with E. coli OP50 (gray), Comamonas DA1877 (blue), or E. coli OP50 supplemented with 40 mM choline (yellow); levels of tri-methylated H3K4 were normalized to total H3 levels. Bars represent mean ± SD, normalized to the control (wild-type E. coli OP50), from four independent biological and technical replicates; see Supplementary Fig. 8a for individual Western blots. b Western blot quantification of tri-methylated H3K4 in wild-type animals fed with indicated RNAi clones; levels of tri-methylated H3K4 were normalized to total H3 levels. Bars represent mean ± SD, normalized to the control (wild-type EV), from three independent biological and technical replicates; see Supplementary Fig. 8b for individual Western blots. Vulval induction of let-60(gf) animals (c) and oocyte number of wild-type animals (d) fed with indicated RNAi clones or control (EV – empty vector); non-supplemented NGM (gray) or supplemented with 64 nM B12 (pink). Dots represent individual animals (c) or oocytes (d) from two independent biological replicates. Boxplots show 25–75 percentiles and median; whiskers represent min and max values. Number of animals in brackets. e Model depicting the tissue-specific effects of one-carbon metabolism on nucleosides synthesis, fat metabolism and histone methylation and its effects on germ cell and VPCs differentiation; green arrows are interactions supported by this work; dashed arrows are hypothesized from literature (gray) or conclusions made in this work (green). *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001 using a Kruskal-Wallis test for non-parametric data followed by a Dunn’s multiple comparison test in a–c and one-way ANOVA followed by Dunnett’s multiple comparison test in d. Source data are provided as a Source Data file.
a Collective migration of A459 cells grown with MR (0 µM Met; 400 µM Hcy) (blue), without MR (200 µM Met; 400 µM Hcy) (gray), or treated with 1 µM MEK162 and 1 µM LGX818 MEK inhibitors (pink); yellow dashed lines represent the migrating front over time. a’ Normalized percentage of the area closed over time. Symbols indicate mean ± SD from four independent biological replicates. a”, Migration speed was calculated as the slope of graphs shown in a’. Bars represent the mean ± SD. b Staining of A431 cells with DAPI (blue) and phalloidin (green), with and without EGF stimulation or MR. Scale bar: 10 µm. For additional examples and the scoring criteria, see Fig. S9. b’ Percentage of stimulated cells with filopodia under indicated conditions. Bars represent mean ± SD from three independent biological replicates; total number of cells scored in brackets. c-d’ Quantification of collective migration in MEFs expressing different KRAS and BRAF mutations grown without c and with MR d. c, d Normalized percentage of area closed over time. Data represents mean ± SD from three independent biological replicates. c’, d’ Migration speed was calculated as the slope of graphs shown in c and d, respectively. Bars represent the mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005 using one-way ANOVA followed by Dunnett’s multiple comparison test in a”, c’ and d’. Source data are provided as a Source Data file.
References
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- Green, R. et al. Vitamin B12 deficiency. Nat. Rev. Dis. Primers. 3, 17041 (2017). – PubMed
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