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RNAi screens identify HES4 as a regulator of redox balance supporting pyrimidine synthesis and tumor growth

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

NADH/NAD+ redox balance is pivotal for cellular metabolism. Systematic identification of NAD(H) redox regulators, although currently lacking, would help uncover unknown effectors critically implicated in the coordination of growth metabolism. In this study, we performed a genome-scale RNA interference (RNAi) screen to globally survey the genes involved in redox modulation and identified the HES family bHLH transcription factor HES4 as a negative regulator of NADH/NAD+ ratio. Functionally, HES4 is shown to be crucial for maintaining mitochondrial electron transport chain (ETC) activity and pyrimidine synthesis. More specifically, HES4 directly represses transcription of SLC44A2 and SDS, thereby inhibiting mitochondrial choline oxidation and cytosolic serine deamination, respectively, which, in turn, ensures coenzyme Q reduction capacity for DHODH-mediated UMP synthesis and serine-derived dTMP production. Accordingly, inhibition of choline oxidation preserves mitochondrial serine catabolism and ETC-coupled redox balance. Furthermore, HES4 protein stability is enhanced under EGFR activation, and increased HES4 levels facilitate EGFR-driven tumor growth and predict poor prognosis of lung adenocarcinoma. These findings illustrate an unidentified mechanism, underlying pyrimidine biosynthesis in the intersection between serine and choline catabolism, and underscore the physiological importance of HES4 in tumor metabolism.

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Fig. 1: HES4 negatively regulates the NADH/NAD+ ratio.
Fig. 2: HES4 sustains DHODH activity and UMP synthesis.
Fig. 3: HES4 regulates choline and serine catabolism.
Fig. 4: The vital metabolic role of transcriptional activity of HES4.
Fig. 5: EGFR activation enhances HES4 protein stability.
Fig. 6: The metabolic effect of HES4 contributes to tumor growth.

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Data availability

RNA sequencing datasets are available under accession number GSE234739. Other data in this work will be available upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key R&D Program of China (2020YFA0803602 to Y.J. and 2019YFA0904800 to Y. Zhao); the National Nature Science Foundation of China (32150030, 32030065, 32121005 and 92049304 to Y. Zhao and 81972586 to Y.J.); the Shanghai Municipal Education Commission (Gaofeng Clinical Medicine grant 20161319 to Y.J.); the Research Unit of New Techniques for Live-Cell Metabolic Imaging (Chinese Academy of Medical Sciences, 2019-I2M-5-013, to Y. Zhao); the Innovative Research Team of High-Level Local Universities in Shanghai; the State Key Laboratory of Bioreactor Engineering; and the Fundamental Research Funds for the Central Universities.

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Author notes

  1. These authors contributed equally: Jing He, Aoxue Wang.

Authors and Affiliations

  1. Department of Liver Surgery and Shanghai Cancer Institute, State Key Laboratory of Systems Medicine for Cancer, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Jing He, Qin Zhao, Nannan Sha, Guofang Hou, Bei Zhou & Yuhui Jiang

  2. Optogenetics & Synthetic Biology Interdisciplinary Research Center, Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China

    Aoxue Wang, Yejun Zou, Zhuo Zhang, Yi Yang & Yuzheng Zhao

  3. Research Unit of New Techniques for Live-Cell Metabolic Imaging, Chinese Academy of Medical Sciences, Beijing, China

    Aoxue Wang, Yejun Zou, Zhuo Zhang & Yuzheng Zhao

  4. Department of Gynecology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, China

    Yejun Zou

  5. Endoscopy Center, Department of Gastroenterology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, China

    Tao Chen

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Contributions

This study was conceived by Y.J. and Y. Zhao. Y.J., Y. Zhao and J.H. designed the study. J.H., A.W., Q.Z., Y. Zou, Z.Z., N.S., G.H. and B.Z. performed experiments. Y.Y. and T.C. provided support for reviewing the paper. Y.J. wrote the paper, with comments from all authors.

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Correspondence to Yuzheng Zhao or Yuhui Jiang.

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Nature Structural & Molecular Biology thanks Francois Schweisguth and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: D. Typas, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 HES4 negatively regulates NADH/NAD+ ratio.

(a, b) Fluorescence measurement of SoNar (a) and iNapc (b) in H1299 cells treated with 5 mM oxamate, 2 mM lactate, 10 μM rotenone or 0.5 mM pyruvate respectively at the indicated time. Scale bars, 10 μm. Fluorescence images (left) and quantification (right) of SoNar in H1299 cells (a). Fluorescence images (left) and quantification (right) of iNapc in H1299 cells (b). (c) Pie chart analysis of genome-wide RNAi screen. Genes were classified as ribosomal proteins, transcriptional factors, metabolic enzymes and others based on functional annotation. 128 and 21 genes were identified as negative and positive regulators of NADH/NAD+ ratio, respectively, where only HES1 and HES4 belong to the same gene family. (d) A549 or H1299 cells were transfected with or without two independent shRNA targeting HES1 (shHES1) or HES4 (shHES4) as indicated. Immunoblotting analyses were performed using the indicated antibodies. #2 of shHES1 and #2 of shHES4 were selected to be utilized in the subsequent experiments. (e, f) NADH/NAD+ ratio (e) and cell viability (f) were measured in shCtrl, shHES1 and shHES4 A549 (left) or H1299 (right) cells. (g, h) KEGG analysis of non-targeted metabolite profiling of shCtrl, shHES1 (g) or shHES4 (h) A549 cells. Unpaired, two-tailed t-test. (i) Indicated metabolites were measured in shCtrl and shHES1 A549 cells reconstituted with or without expression of WT rHES1. (j) Indicated metabolites were measured in shCtrl and shHES4 A549 cells reconstituted with or without expression of WT rHES4. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 2 HES4 deficiency promotes glycolysis reprograming.

(a) Lactate/pyruvate ratio was measured in shCtrl and shHES4 A549 (left) or H1299 (right) cells reconstituted with or without expression of WT rHES4. Lac, lactate; pyr, pyruvate. (b, c) Cell viability in day 2 (b), NADH/NAD+ ratio (c) were examined in shCtrl and shHES4 A549 or H1299 cells supplemented with or without 200 μM choline chloride, 50 μM betaine, 2 mM serine, 3 mM sodium pyruvate and 100 μM each of uridine (U) and thymidine (T) for 24 h. (d) NADH/NAD+ ratio was examined in shCtrl and shHES4 A549 (left) or H1299 (right) cells supplemented with or without 1 mM α-KB and 100 μM each of uridine (U) and thymidine (T) for 24 h. (e) UMP and dTMP levels were examined in shCtrl and shHES4 H1299 cells supplemented with or without metabolites as indicated for 24 h. (f) UMP and dTMP levels were examined in shCtrl and shHES4 A549 (left) or H1299 (right) cells supplemented with or without metabolites as indicated for 24 h. (g) Cell viability in day 2 were examined in shCtrl and shHES4 A549 (left) or H1299 (right) cells supplemented with or without metabolites as indicated for 24 h. (h) Glucose consumption was examined in shCtrl and shHES4 A549 (left) or H1299 (right) cells. (i) Lactate secretion was examined in shCtrl and shHES4 A549 (left) or H1299 (right) cells. (j, k) Extracellular acidification rate (ECAR) was measured in shCtrl and shHES4 A549 (j) or H1299 (k) cells (left). Glycolytic capacity was quantified (right). (l) mRNA levels of glycolytic and relevant genes were analyzed in shCtrl and shHES4 A549 cells. (m) mRNA levels of pentose phosphate pathway enzymes were analyzed in shCtrl and shHES4 A549 cells. (n) Schematic of isotopomer distribution of indicated metabolites derived from [U-13C] Glucose. 13C atoms are depicted in blue. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 3 HES4 sustains CoQ reduction capacity and DHODH activity.

(a–e) Overexpression of ectopic PHGDH in shCtrl and shHES4 A549 cells. Immunoblotting analyses were performed using the indicated antibodies (a). En, endogenous PHGDH; OE, overexpression of ectopic PHGDH. NADH/NAD+ ratio (b), UMP and dTMP levels (c), cell viability (d), FBP and serine levels (e) were measured. (f, g) UMP and dTMP levels (f) and cell viability in day 2 (g) were measured in shCtrl and shHES4 A549 or H1299 cells supplemented with or without 2 mM serine for 24 h. (h, i) Aspartate (h) and R5P (i) levels were examined in shCtrl and shHES4 A549 (left) or H1299 (right) cells. (j) mRNA levels of indicated genes were analyzed in shCtrl and shHES4 A549 cells. (k) Schematic of conversion of DHO to orotate by DHODH (top). DHODH activity indicated by Orotate/DHO ratio was measured in A549 (left) or H1299 (right) cells. (l, m) Oxygen consumption rate (OCR) was measured in real time in XFe96 analyzer after injection of oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) and Rot/AA in shCtrl and shHES4 A549 (c) or H1299 (d) cells. Maximal respiration and spare respiration capacity were quantified as indicated. (n) CoQH2/CoQ ratio was examined in shCtrl and shHES4 H1299 cells treated with or without 1 mM brequinar (BQR) for 4 h. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 4 HES4 maintains ETC integrity for UMP synthesis.

(a) Mitochondrial ROS was examined by MitoSOX Red in shCtrl and shHES4 A549 (left) or H1299 (right) cells treat with or without 2 mM NAC for 24 h. (b) shCtrl and shHES4 H1299 cells were transfected with or without Flag-tagged Ciona intestinalis alternative oxidase (Flag-CiAOX). Immunoblotting analyses were performed using the indicated antibodies (top). Mitochondrial ROS was examined in shCtrl and shHES4 H1299 cells transfected with or without Flag-CiAOX and treated with or without 1 mM brequinar (BQR) for 4 h (bottom). (c) Detection of mitochondrial complex I and supercomplexes in isolated mitochondria by blue native electrophoresis in A549 (left) or H1299 (right) cells treated with or without 2 mM NAC for 24 h or transfected with or without Flag-CiAOX. Immunoblotting analyses were performed using the indicated antibodies against complex I component NDUFA9. VDAC1 was used as a loading control. sl, slow-migrating complex I and III supercomplex. (d–f) Orotate/DHO ratio (d), UMP levels (e) and cell viability in day 2 (f) were examined in shCtrl and shHES4 H1299 cells transfected with or without Flag-CiAOX and treated with or without 1 mM brequinar (BQR) for 4 h. (g–i) Orotate/DHO ratio (g), UMP levels (h) and cell viability in day 2 (i) were examined in shCtrl and shHES4 A549 (left) or H1299 (right) cells treated with or without 2 mM NAC for 24 h. (j) Glutamine consumption was examined by glutamine assay kit in shCtrl and shHES4 A549 (top) or H1299 (bottom) cells. (k, l) Schematic of isotopomer distribution of indicated metabolites derived from [U-13C] Glutamine (k) or [1-13C] Glutamine (l). 13C atoms are depicted in blue. n = 3 independent repeats. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

Source data

Extended Data Fig. 5 HES4 regulates choline and serine catabolism.

(a) RNA-seq analysis of shCtrl, shHES1 and shHES4 A549 cells. Differentially metabolic enzymes are presented in heatmap base on z-score. Genes belonged to serine and choline catabolism are depicted in red. (b) GO analysis of shCtrl, shHES1 and shHES4 A549 cells. Processes belonged to serine and choline catabolism are depicted in red. Unpaired, two-tailed t-test. (c) mRNA levels of serine and choline catabolism enzymes were analyzed in shCtrl and shHES4 A549 (left) or H1299 (right) cells. (d) mRNA levels of serine and choline catabolism enzymes were analyzed in shCtrl and shHES1 A549 (left) or H1299 (right) cells. (e) Schematic of isotopomer distribution of indicated metabolites derived from [2,3,3-2H] Serine. 2H atoms derived from cytosolic or mitochondrial [2,3,3-2H] Serine were depicted in red or blue, respectively. (f) Isotopomer distributions of dTMP from [2,3,3-2H] Serine in shCtrl and shHES4 A549 cells. (g–j) shCtrl and shHES4 A549 or H1299 cells were transfected with or without SDS sgRNA (sgSDS). Immunoblotting analyses were performed using the indicated antibodies in A549 (left) or H1299 (right) cells (g). mRNA levels of PHGDH, SHMT1, SHMT2 and SFXN1 were analyzed by quantitative real-time PCR in H1299 cells (h). Cellular dTMP concentration was examined in A549 cells (i). CoQH2/CoQ ratio was examined in A549 cells (j). (k) Glycerol-3-phosphate (Gro3P), palmitate and choline were examined in shCtrl and shHES4 A549 cells. (l) Cells were incubated with 100 μM 2H-choline for 2 h, followed by replaced with fresh medium without 2H-choline. Cells were harvest at indicated time. 2H labeled phosphocholine (PCho) were measured by LC-MS. Consumption was calculated and normalized to time 0. n = 3 independent repeats. (m) Schematic of isotopomer distribution of indicated metabolites derived from D9-choline. 2H atoms derived from D9-choline were depicted in red. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

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Extended Data Fig. 6 Depletion of HES4 facilitates choline catabolism.

(a, b) shCtrl and shHES4 A549 or H1299 cells were single or double transfected with or without SDS sgRNA (sgSDS) or SLC44A2 sgRNA (sgSLC44A2). Immunoblotting analyses were performed using the indicated antibodies in A549 (left) or H1299 (right) cells (a). mRNA levels of indicated genes were examined (b). (c) Mitochondrial serine and glutamate uptake were measured. n = 3 independent repeats. (d) Complex I independent OCR were examined in shCtrl and shHES4 A549 (top) or H1299 (bottom) cells single or double transfected with or without SDS sgRNA (sgSDS) or SLC44A2 sgRNA (sgSLC44A2). Complex I independent OCR was depicted in gray shadow (left). Complex I independent OCR was calculated (right). (e–i) shCtrl and shHES4 A549 or H1299 cells were transfected with or without SLC44A2 sgRNA (sgSLC44A2), CHDH sgRNA (sgCHDH) or SDS sgRNA (sgSDS). Immunoblotting analyses were performed using the indicated antibodies in A549 (left) or H1299 (right) cells (e). CoQH2/CoQ ratio (f), Orotate/DHO ratio (g) and UMP level (h) were examined. Isotopomer analyses of betaine and DMG from D9-Choline was examined (i). n = 3 independent repeats. (j) Cell viability was examined in shCtrl and shHES4 H1299 cells single or double transfected with or without SDS sgRNA (sgSDS) or SLC44A2 sgRNA (sgSLC44A2) for indicated days. (k–p) A549 or H1299 cells were single or double transfected with or without SDS or SLC44A2. Immunoblotting analyses were performed using the indicated antibodies (k). The intensity was measured using ImageJ and normalized to mock vector. UMP and dTMP levels (l), isotopomer distributions of dTMP from [2,3,3-2H] Serine (m), CoQH2/CoQ ratio (n), Orotate/DHO ratio (o) and cell viability in day 2 supplemented with or without indicated metabolites were examined. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

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Extended Data Fig. 7 Serine-choline catabolic genes are inversely related.

(a) Immunoblotting analyses of basal protein expression in various cell lines as indicated (left). Two-tailed Pearson correlation (r) between indicated protein and mRNA levels were analyzed (right). (b) Two-tailed Pearson correlation (r) between HES4 protein and SDS or SLC44A2, SDS and SHMT1 or SLC44A2 SFXN1 as indicated were analyzed in various cell lines as displayed in Extended Data Fig. 7a. (c, d) Two-tailed Pearson correlation of SLC44A2 and SFXN1 (c) or SDS and SHMT1 (d) across distinct types of cancers by TCGA data analysis. The datasets show the gene-level transcription estimates, as in log2(x + 1) transformed RSEM normalized count. BRCA, breast invasive carcinoma; ESCA, esophageal carcinoma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; SKCM, skin cutaneous melanoma; THCA, thyroid carcinoma.

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Extended Data Fig. 8 The vital metabolic role of transcriptional activity of HES4.

(a) NADH/NAD+ ratio was measured in shCtrl and shHES4 A549 (left) or H1299 (right) cells transfected with or without CiAOX. (b) NADH/NAD+ ratio was measured in shCtrl and shHES4 H1299 cells single or double transfected with or without SDS sgRNA (sgSDS) or SLC44A2 sgRNA (sgSLC44A2). (c) NADH/NAD+ ratio was measured in A549 (left) or H1299 (right) cells single or double transfected with or without SDS or SLC44A2. (d, e) A549 or H1299 cells were transfected with or without SDS sgRNA (sgSDS) or ALDH3B2 sgRNA (sgALDH3B2). Immunoblotting analyses were performed using the indicated antibodies (d). NADH/NAD+ ratio was measured (e). (f) ChIP analyses were performed in H1299 cells expressing Flag-HES4. The y axis shows the value normalized to IgG. (g) Putative HES family binding consensus sequences in promoter of SDS, SLC44A2 and ALDH3B2 (top) were compared with motif predicted using JASPAR database (bottom). (h, i) shCtrl and shHES4 A549 or H1299 cells were reconstituted with or without shRNA-resistant wild-type HES4 (WT rHES4) or HES4 mutant lacking of DNA binding domain (rHES4 ΔDBD). Mutation sites were depicted in red (top). Immunoblotting analyses were performed using the indicated antibodies (bottom) in A549 (left) or H1299 (right) cells (h). mRNA levels of SDS, SLC44A2 and ALDH3B2 were analyzed in H1299 cells (i). (j–o) shCtrl and shHES4 A549 or H1299 cells were transfected with or without WT rHES4 or rHES4 ΔDBD (left), and WT rHES4 and rHES4 ΔDBD A549 or H1299 cells were co-transfected with or without sgSDS and sgSLCAA42 (sgSDS/SLC) (right). Orotate/DHO ratio (j, k), UMP and dTMP levels (l), cell viability in day 2 (m) and NADH/NAD+ (n, o) ratio were measured. (p) Immunoblotting analyses were performed using the indicated antibodies in WT rHES4 and rHES4 ΔDBD A549 (left) or H1299 (right) cells co-transfected with or without sgSDS and sgSLC44A2 (sgSDS/SLC44A2). Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

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Extended Data Fig. 9 EGFR signaling promotes protein stability of HES4.

(a–e) A549 or H1299 cells treated with or without 10 μM CB-103 for 24 h. mRNA (a) or protein (b) levels of HES1 and HES4 were examined in H1299 cells treated with or without 10 μM CB-103 for 24 h. Immunoblotting analyses were performed using the indicated antibodies (b). Cell viability (c), NADH/NAD+ ratio (d) and UMP and dTMP level (e) were examined. (f, g) mRNA (f) or protein (g) levels of HES4, SDS and SLC44A2 were examined in shCtrl and shHES4 A549 cells transfected with or without Kras sgRNA (sgKras). Immunoblotting analyses were performed using the indicated antibodies (g). (h, i) mRNA (h) or protein (i) levels of HES4, SDS and SLC44A2 were examined in shCtrl and shHES4 A549 cells transfected with or without p53 sgRNA (sgp53). Immunoblotting analyses were performed using the indicated antibodies (i). (j, k) mRNA (j) or protein (k) levels of HES4, SDS and SLC44A2 were examined in shCtrl and shHES4 H1299 cells transfected with or without p53. Immunoblotting analyses were performed using the indicated antibodies (k). (l) A549 (left) or H1299 (right) cells were treated with 0, 10, 20, 40 μM gefitinib as indicated for 24 h. Immunoblotting analyses were performed using the indicated antibodies. (m) mRNA levels of HES1 and HES4 were examined in A549 (left) or H1299 (right) cells treated with or without 10 μM gefitinib for 24 h. (n) Time courses of HES4 protein in H1299 cells treated with 100 μg/ml cycloheximide (CHX) for 0, 4 and 8 h. Immunoblotting analyses were performed using the indicated antibodies (top). The intensity was measured using ImageJ and normalized to time 0 (bottom). (o) Protein levels of HES4 were examined in H1299 cells treated with or without 10 μM gefitinib, 20 μM proteasome inhibitor MG132, 50 mM lysosome inhibitor NH4Cl or 100 μM chloroquine (CQ) for 24 h. Immunoblotting analyses were performed using the indicated antibodies. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

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Extended Data Fig. 10 The metabolic effect of HES4 contributes to tumor growth.

(a–c) shCtrl and shHES4 H1299 cells were treated with or without 10 μM gefitinib for 24 h. mRNA levels of SDS and SLC44A2 were analyzed (a). Immunoblotting analyses were performed using the indicated antibodies (b). UMP and dTMP levels were examined (c). (d) ChIP analyses were performed in Flag-HES4 expressing HepG2 cells transfected with or without EGFR. DNA enrichment was examined by quantitative real-time PCR. The y axis shows the value normalized to IgG. (e–g) shCtrl and shHES4 HepG2 cells were transfected with or without EGFR. mRNA levels of SDS and SLC44A2 were analyzed (e). Immunoblotting analyses were performed using the indicated antibodies (f). UMP and dTMP levels were examined (g). (h) ChIP analyses were performed in Flag-HES4 expressing SW1116 cells treated with or without 10 μM gefitinib for 24 h. DNA enrichment was examined by quantitative real-time PCR. The y axis shows the value normalized to IgG. (i–k) SW1116 cells transfected with or without Flag-HES4 were treat with or without 10 μM gefitinib for 24 h. mRNA levels of SDS and SLC44A2 were analyzed (i). Immunoblotting analyses were performed using the indicated antibodies (j). UMP and dTMP levels were examined (k). (l, m) Representative tumor xenografts. shCtrl and shHES4 A549 (left) or H1299 (right) cells were co-transfected with or without sgSDS and sgSLCAA42 (sgSDS/SLC44A2) (l). WT rHES4 and rHES4 ΔDBD A549 (left) or H1299 (right) cells were co-transfected with or without sgSDS and sgSLC44A2 (sgSDS/SLC44A2) (m). n = 6 biologically independent animals. (n, o) Tumor lysates harvested from l or m respectively were subjected to immunoblotting analyses as indicated. Immunoblotting analyses were performed using the indicated antibodies. Data are presented as mean ± s.d., n = 3 independent repeats. Unpaired, two-tailed t-test.

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He, J., Wang, A., Zhao, Q. et al. RNAi screens identify HES4 as a regulator of redox balance supporting pyrimidine synthesis and tumor growth. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01309-3

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