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.

NADH/NAD ⁺氧化还原平衡对于细胞代谢至关重要。 NAD(H) 氧化还原调节剂的系统鉴定虽然目前缺乏，但将有助于发现与生长代谢协调密切相关的未知效应物。在这项研究中，我们进行了基因组规模的 RNA 干扰 (RNAi) 筛选，以全面调查参与氧化还原调节的基因，并确定 HES 家族 bHLH 转录因子 HES4 作为 NADH/NAD ⁺比率的负调节因子。从功能上来说，HES4 对于维持线粒体电子传递链 (ETC) 活性和嘧啶合成至关重要。更具体地说，HES4 直接抑制SLC44A2和SDS的转录，从而分别抑制线粒体胆碱氧化和胞浆丝氨酸脱氨，从而确保 DHODH 介导的 UMP 合成和丝氨酸衍生的 dTMP 生产的辅酶 Q 还原能力。因此，抑制胆碱氧化可保持线粒体丝氨酸分解代谢和 ETC 偶联的氧化还原平衡。此外，HES4蛋白稳定性在EGFR激活下增强，HES4水平升高促进EGFR驱动的肿瘤生长并预测肺腺癌的不良预后。这些发现说明了丝氨酸和胆碱分解代谢交叉过程中嘧啶生物合成的一个未确定的机制，并强调了 HES4 在肿瘤代谢中的生理重要性。