E2F transcription factor-1 regulates oxidative metabolism

Abstract

Cells respond to stress by coordinating proliferative and metabolic pathways. Starvation restricts cell proliferative (glycolytic) and activates energy productive (oxidative) pathways. Conversely, cell growth and proliferation require increased glycolytic and decreased oxidative metabolism levels. E2F transcription factors regulate both proliferative and metabolic genes. E2Fs have been implicated in the G1/S cell-cycle transition, DNA repair, apoptosis, development and differentiation. In pancreatic β-cells, E2F1 gene regulation facilitated glucose-stimulated insulin secretion. Moreover, mice lacking E2F1 (E2f1(-/-)) were resistant to diet-induced obesity. Here, we show that E2F1 coordinates cellular responses by acting as a regulatory switch between cell proliferation and metabolism. In basal conditions, E2F1 repressed key genes that regulate energy homeostasis and mitochondrial functions in muscle and brown adipose tissue. Consequently, E2f1(-/-) mice had a marked oxidative phenotype. An association between E2F1 and pRB was required for repression of genes implicated in oxidative metabolism. This repression was alleviated in a constitutively active CDK4 (CDK4(R24C)) mouse model or when adaptation to energy demand was required. Thus, E2F1 represents a metabolic switch from oxidative to glycolytic metabolism that responds to stressful conditions.

Figure 1. Energy expenditure, adaptative thermogenesis, mitochondrial function, and physical activity

(a) Energy expenditure (VO2). n=4 animals/group. (b) Respiratory exchange ratio (V_CO2/V_O2) during total, light and dark phase of the experiment. n=5 animals/group. (c) Rectal temperatures under room temperature, fed (control), cold, or fasted conditions. n=4 animals/group. (d) Mitochondrial DNA (mtDNA) measured relative to nuclear DNA in BAT tissues of E2f1^+/+ and E2f1^−/− mice. n=5 animals/group. (e) Mitochondrial oxygen consumption without (−) and with (+) succinate. n=4 animals/group. (f) Mitochondrial DNA (mtDNA) content in E2f1^+/+ and E2f1^−/− muscles. n=5 animals/group. (g) GN mitochondrial O₂ consumption measured after electrotransfer of pCMV or pCMV-E2F1, without (−) and with (+) ADP and succinate. n=4 animals/group. (h) Relative gene expression of gastrocnemius myosin heavy chain (MyHC) type I, IIa, IIX, and IIb. Results were normalized to the expression of mouse 18S RNA. n=7 animals/group. (i) Immunofluorescence analysis of serial gastrocnemius sections showing expression of MyHCI, MyHCIIa and MyHCIIb (red) in fibers. Nuclei are stained with Hoechst reagent. n=4 animals/group. Percentage of positive stained fibers is indicated. Scale bars, 100μm. (j) E2f1^+/+ and E2f1^−/− mice were tested for physical endurance. Individual animal performances are shown. n= 11 E2f1^+/+ animals; n= 14 E2f1^−/− animals. (k) The effect of E2F1 rescue in E2f1^−/− mice was evaluated with an endurance test. Individual performances are represented for E2f1 wild type electroporated with empty vector (E2f1^+/+ pCMV), knock-out (E2f1^−/− pCMV) and knock-out-rescued animals (E2f1^−/− pCMV-E2F1). n= 4 animals for E2f1^+/+ pCMV group; n= 3 animals for E2f1−/− pCMV group; n= 3 animals for E2f1−/− pCMV-E2F1 group. Values represent means ± SEM. *p<0.05. **p<0.01.

Figure 2. Increased E2f1^−/− oxidative metabolic gene expression

(a, b) Relative expression of relevant mitochondrial genes in (a) brown adipose tissue (BAT) and (b) gastrocnemius (GN). Results were normalized to mouse 18S RNA expression. n=7 animals/group. (c) Relative expression levels of oxydative genes in E2f1^−/− GN electroporated with an empty vector (pRNAT-control) or a plamid expressing shRNA targeting Ucp2, Ppargc1a, Esrra or Pdk4. mRNA levels are represented relative to 18S mRNA. n=3 animals. (d) 72h post-electroporation, O₂ consumption was measured in isolated mitochondria from GN using a Clark electrode. O₂ was measured in the absence and presence of succinate and ADP. n=3 animals. Values represent means ±SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 3. Cold and fasting modulate gene expression through the pRb-E2F1 complex

(a,b) Q-PCR quantification of the expression of relevant genes involved in oxidative metabolism in (a) BAT and (b) gastrocnemius of E2f1^+/+ and E2f1^−/− mice in cold/room temperature and fasted/refed conditions, respectively (See Supplementary Table 1 for sequences). Results were normalized to the expression of mouse 18S RNA and are expressed as means ± SEM of three independent experiments. (c, d, e) Immunohistochemistry analysis of pRb phosphorylation on serine 780 (^S780p-pRb). (c) BAT was obtained from mice placed at +23° or +4°C (d) GN was obtained from mice fasted for 24hr or fasted for 20hr and refed for 4hr. (e) Mice were fasted for 4hr, injected i.p. with 0.9% NaCl or 0.9% NaCl+isoproterenol and GN were harvested 30 min after injection. Tissues were subsequently processed for immunohistochemistry as described in the methods section. Scale bars, 100μm. (f) Mitochondrial activity, measured as oxygen consumption in E2f1+/+ embryonic cells in the presence or absence of isoprotenerol and the cdk4 inhibitor PD0332991 as indicated. Results were normalized to protein levels and are expressed as means ± SEM of four independent experiments per conditions. (g,h,i) Chromatin immunoprecipitation demonstrates binding of E2F1 and pRB to oxidative metabolic gene promoters in (g) BAT obtained at room temperature (23°) or after 4-h cold exposure (4°C) or in (h-i) muscle in 24hr fasting (F) and 4hr refed (R) conditions. Values represent means ± SEM of six independent experiments. Immunoprecipitates (IP) were analyzed by Q-PCR (g, i) or classical PCR (h) with specific primers for the E2F-RE identified in these promoters (See Supplementary Table 1 for sequences). Values represent means ±SEM. *p<0.05; **p<0.01; ***p<0.001. (*)p<0.05 E2f1^+/+ fast (24 hours) vs refed (4 hours); (£)p<0.05 E2f1^+/+ fast vs E2f1^−/− fast; and (#) p<0.05 E2f1^+/+ refed vs E2f1^−/− refed.

Figure 4. Increased O2 consumption, running time and expression of oxidative genes in CDK4^R24C/R24C mice

(a) Mitochondrial oxygen consumption without (−) and with (+) succinate. n=6 animals. (b) WT and CDK4^R24/R24C mice were tested for physical endurance. Average running time to exhaustion is shown. n= 6 animals/group. (c) Q-PCR quantification of the expression of relevant genes involved in oxidative metabolism in gastrocnemius of WT versus CDK4^R24/R24C mice in fasted /refed conditions. Results were normalized to the expression of mouse 18S RNA and are expressed as means ± SEM of three independent experiments. n= 3 animals/group. (d) Chromatin immunoprecipitation demonstrates binding of E2F1 and pRB to oxidative metabolic gene promoters in muscle in 24hr fasting and 5hr refed conditions. n=3 animals. Values represent means ± SEM *p<0.05; **p<0.01; (*)p<0.05 E2f1^+/+ fast (24 hours) vs refed (4 hours); (£)p<0.05 E2f1^+/+ fast vs CDK4^R24/R24C fast; and (#) p<0.05 E2f1^+/+ refed vs CDK4^R24/R24C refed.

Figure 5. DNA methylation of proliferative target genes modulates E2F1 transcriptional activity in muscle

(a) Q-PCR quantification of the expression of Dhfr and Tk-1 in WT mice in fasted /refed conditions (See Supplementary Table 1 for sequences). Results were normalized to the expression of mouse 18S RNA and are expressed as means ± SEM. n= 6 animals/group. (b) Chromatin immunoprecipitation demonstrates binding of E2F1 relative to IgG to proliferative gene promoters in muscle in 24hr fasting or 20hr fasting/4hr refed conditions. n=3 animals. (c) Methylation status of Dhfr, Tk-1, and Ppargc1a promoters on CpG island in fasted muscles. PCR analysis of methylated DNA-MBD2b complexes in genomic DNA from muscle. A sample corresponding to genomic DNA before incubation with MBD2b was included in the PCR (Input). As a positive control (+), a PCR reaction using A431 methylated DNA and Dhfr, Tk-1 and Ppargc1a promoter specific primers was performed. A negative control (−) was performed using unmethylated Hela genomic DNA as a template and Dhfr, Tk-1 and Ppargc1a promoter specific primers. (d) In basal (fed/room temperature) conditions, E2F1/pRb complexes can repress the oxidative gene program (Oxphos) by switching off those genes. External stimuli activate E2F1 transcriptional activity through the release of pRb, which induces the cell to switch to oxidative metabolism. This molecular mechanism enables the transcription of an oxidative metabolic gene program, allowing the cell to adapt to energy demands and triggers physiological processes such as thermogenesis in BAT or enhanced physical activity in muscle. On the other hand, classical proliferative E2F1 target genes are not subjected to E2F1 regulation due to epigenetic mechanisms.