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E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development - PubMed

  • ️Sun Jan 01 2023

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development

Maria Paula Zappia et al. Proc Natl Acad Sci U S A. 2023.

Abstract

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Keywords: Drosophila; E2F transcription factor; Phosphoglycerate kinase (Pgk); Retinoblastoma protein; metabolism.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

E2F target genes involved in metabolism. (A) Simplified illustration of the enzymes Ald1, Pgk, and Pyk in the glycolytic pathway, Gpdh connecting triglyceride synthesis with glycolysis, kdn in the TCA cycle, and Cyt-c-p in the electron transport chain. (B) Protein levels quantified by TMT-MS in flight muscles of Mef2>mCherry-RNAi and Mef2>Dp-RNAi, scatter dot plots with bars, mean ± SD, Mann–Whitney U test, *P < 0.05, n = 4. (C) Heatmap depicting selected transcripts levels measured by RNAseq in Mef2>mCherry-RNAi and Mef2>Rbf-RNAi. Abs count normalized and scaled, n = 2 samples/genotype. (D–J) Left panel: ChIPseq for Rbf, Dp, and input visualized with Integrative Genomics Viewer browser for the genomic regions surrounding the genes. The most predominantly expressed transcript in adult flies, based on the FlyAtlas 2 profile (25), is displayed; n = 2 samples/condition. Read scales and genomic scales included on top left and top right, respectively. GroupAuto scale. Right panel: Dual luciferase reporter assay in S2R+ cells for (D) PCNA-Luc, (E) Ald1-Luc, (F) Pgk-Luc, (G) Pyk-Luc, (H) Gpdh-Luc, (I) kdn-Luc, and (J) Cyt-c-p-Luc. Values for Firefly Luciferase luminescence normalized to Renilla luminescence. Fold change relative to control (no E2f1 expression). Mutated E2F sites are indicated as ΔE2F. Scatter dot plots with bars, mean ± SD, (F, H, and I) One-way ANOVA followed by Tukey’s multiple comparisons test, (D, E, G, and J) Unpaired t test, ns P > 0.05, ****P < 0.0001, n = 2 replicates/group, N = 2 independent experiments. Bottom panel: E2F binding sites identified using degenerated motif WKNSCGCSMM. Mutations on the core are indicated in blue as ΔE2F. Blue rectangles indicate regions amplified and cloned upstream luciferase reporter. Positions are relative to transcription start site (TSS). Green bars indicate different sites amplified by ChIP-qPCR in Fig. 2 C–F and

SI Appendix, Fig. S1A

. Full genotypes: (B) w-;Mef2-GAL4/UAS-mCherry-RNAi (white bar) and w-;UAS-Dp-RNAi;Mef2-GAL4 (gray bar) (C) w-, UAS-Dicer2;+;Mef2-GAL4/UAS-mCherry-RNAi, and w-, UAS-Dicer2;+;Mef2-GAL4/UAS-Rbf-RNAi

Fig. 2.
Fig. 2.

The requirement of the E2F binding sites for the recruitment of E2F/Dp/Rbf and gene expression regulation in vivo. (A–H) Chromatin from third instar larvae immunoprecipitated with anti-Dp (Left), anti-Rbf (Right), and nonspecific control antibodies (IgG and anti-Myc on the Left and Right panel, respectively). Recruitment measured by qPCR flanking the E2F binding sites of the following genes (A and B) Gpdh, (C and D) Pgk, (E and F) kdn, and (G and H) Ald1. The negative site (NS) does not contain predicted E2F-binding sites. Scatter dot plots with bars, mean ± SD, fold enrichment relative to the positive site, Apr53D, for each ChIP sample. N = 2 independent experiments. Genomic location of primers amplifying site1 and site2 are indicated in Fig. 1 F, I, and J. (I–L) The expression of the genes (I) Gpdh, (J) Ald1, (K) kdn, and (L) Pgk measured by RT-qPCR in whole animals staged at third instar larva, mid pupa, pharate, and 1-d-old adults. Fold change relative to control. Scatter dot plots with bars, mean ± SEM, N = 3 samples/group, multiple unpaired t tests followed by corrected FDR method (Benjamini and Yekutieli), q-value for each comparison is indicated in the plot. Full genotypes: (A–L) w1118, (A, B, and I) w1118;GpdhΔE2F;+, (C, D, and L) w1118;PgkΔE2Fline12;+, (E, F, and K) w1118,kdnΔE2F;+; +, (G, H, and J) w1118;+;Ald1ΔE2F

Fig. 3.
Fig. 3.

Metabolic changes in PgkΔE2F. (A) Glucose flux in the stable isotope tracing experiment showing the fate of 13C from 13C-glucose through glycolysis and TCA cycle. (B) Steady-state levels of lactate, dihydroxyacetone phosphate (DHAP), citrate, alpha-ketoglutarate (alpha-KG), succinate, fumarate, and malate measured by GC-MS in w1118 and PgkΔE2F 5-d-old males. Scatter dot plots with bars, mean ± SD, unpaired t test: *P < 0.05, ***P < 0.01. (C) Contribution of 13C glucose to DHAP, lactate, citrate, α-ketoglutarate, and malate after 12 h of treatment. M + 3 and M + 2-labeled fractions of pool representing direct flux of 13Cglucose into glycolysis and TCA cycle, respectively. Box plot, whiskers min to max values, line at mean, unpaired t test: *P < 0.05, **P < 0.01. n = 3 samples/group, N = 2 independent experiments. (D) ATP levels measured in 5-d-old males by luminescence. Total µmol ATP normalized to g protein. Scatter dot plot with bars, mean ± SEM, unpaired t test, **P < 0.01, n = 3 samples/genotype, N = 2 independent experiments. Genotypes: w1118 and w1118;PgkΔE2Fline12;+

Fig. 4.
Fig. 4.

Mitochondrial defects in PgkΔE2F led to dysfunctional muscles. (A) The expression of the glycolytic genes Hex-A, Pgi, Pfk, Ald1, Pgk, Gpdh, Eno, and Pyk measured by RT-qPCR in dissected ovaries, head, flight muscles, and dorsal abdomen containing fat body tissue in 2 to 3-d-old females. Fold change relative to control. Scatter dot plots with bars, mean ± SD, N = 3 samples/group. (B) Flight ability scored by quantifying the percentage of flies landing on top section of the column; 5- to 7-d-old males were tested. Scatter dot plots with bars, mean ± SEM, unpaired t test, *P < 0.05, N = 196 (w1118) and 158 (PgkΔE2F), N = 2 independent experiments. (C) Confocal section images of flight muscles in a sagittal view. Hemithorax sections of 5-d-old males stained with Phalloidin (blue), anti-kettin (red), and anti-ATP5A (green). (D) Quantification of percentage of hemithoraces displaying either normal morphology of mitochondria or fragmented shape (i.e., round). Stacked bars, N = 11 to 13 hemithoraces/group, (E) same as C, (F) same as D, N = 11 to 12 hemithoraces/group. Scale 10 μm. Genotypes: (A–D) w1118, (B–D) w1118;PgkΔE2Fline12;+, (E and F) Act88F-GAL4/Y;UAS-GFP-RNAi;+, and Act88F-GAL4/Y;+;UAS-Pgk-RNAiGL00101.

Fig. 5.
Fig. 5.

Reduction in chromatin accessibility and gene expression in PgkΔE2F (A) MA plot representing differential chromatin accessibility analyzed using DiffBind. The common 23,315 sites for ATAC-seq between PgkΔE2F and control animals plotted as a blue cloud. FDR < 0.05 cutoff (pink dots). The x axis values (“log concentration”) represent logarithmically transformed, normalized counts, averaged for all samples, for each site. The y axis values represent log 2 (fold change) values in PgkΔE2F relative to w1118. (B) Heatmaps showing the enrichment of ATAC reads in a 3,000 bp window centered on the summit for each peak. Scale is as indicated in the signal. Only 520 sites with differential chromatin accessibility are included, in which seven sites show a gain in enrichment in PgkΔE2F and 513 show a loss (reduction). (C) Distribution of reads for ATAC-seq data in a 6,000 bp window centered on the TSS of the gene for each peak. Only the 520 sites with differential chromatin accessibility in PgkΔE2F are included. (D) Peak annotation pie chart for the 520 sites with differential chromatin accessibility in PgkΔE2F. (E and F) Differential chromatin accessibility determined by ATAC-seq visualized with Integrative Genomics Viewer browser for the genomic regions surrounding (E) the glycolytic genes: Pgk, Hex-A, and Ald1, and (F) the lipid metabolism genes: Agpat3, puml, and mino. The most predominantly expressed transcript in adult flies, based on the FlyAtlas 2 profile (25), is displayed; n = 2 samples/genotype, each track is shown separately for each replicate. Read scales and genomic scales included on top right and top left, respectively. GroupAuto scale. **FDR < 0.05 in chromatin accessibility. (G–I) The expression of the metabolic genes measured by RT-qPCR in whole animals staged at pharate. (F) glycolytic genes: Hex-A, Hex-C, Pgi, Pfk, Ald1, Gapdh2, Pglym78, Eno, Pyk, and Ldh (G) TCA genes: kdn, mAcon1, Idh, Oxoglutarate dehydrogenase (Nc73EF), Succinyl-coenzyme A synthase, alpha subunit 1 (Scsalpha1), Succinate dehydrogenase, subunit C (SdhC), and Fumarase 1 (Fum1) (H) lipid metabolism genes: ACC1, Acylglycerol-3-phosphate O-acyltransferase 2 (Agpat2), Agpat3, Glycerol-3-phosphate acyltransferase 4 (Gpat4), Lpin, mino, Lipid storage droplet-1 (Lsd-1), Lsd-2, Seipin, Hormone-sensitive lipase (Hsl), and Puml. Fold change relative to control. Scatter dot plots with bars, mean ± SEM, N = 3 samples/group, multiple unpaired t tests followed by corrected Holm–Šídák method for multiple comparison correction. *P < 0.05. Full genotypes: w1118 and w1118; PgkΔE2F line12;+.

Fig. 6.
Fig. 6.

Impact on adult physiology and development in PgkΔE2F. (A) Adult life span determined as survival curves in male flies. Kaplan–Meier analysis, P < 0.0001, median survival = 77 (w1118) and 50 (PgkΔE2F). N = 97 and 84 flies/genotype. (B) Percentage of hatched eggs quantified as number of first instar larva over number of laid eggs. Box plot and whiskers represent 5 to 95 percentile. Mann–Whitney U test, **P < 0.01. N = 533 eggs for w1118 and 496 for PgkΔE2F. (C) Representative images of the ovaries found in ~20 to 40% of females, scale 500 μm. (D) Confocal section images of ovarioles dissected out of 2 to 3-d-old females. Ovaries were stained with Phalloidin (green) and DAPI (magenta). Condensed and fragmented nurse cell nuclei, indicating degenerated egg chambers (white arrows), found in ~30% ovaries at midoogenesis (around st8), scale 100 μm. (E) Measurement of total whole-body glycogen content in 5-d-old females and males normalized to protein content. Box plot and whiskers represent 5 to 95 percentile, n = 6 sample/group, N = 2 independent experiments, unpaired t test, **P < 0.01. One representative experiment shown. Full genotypes: w1118 and w1118;PgkΔE2Fline12;+.

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