Ldha positively regulates the lactylation of Hdac1 to hinder the 2-cell state of embryonic stem cells [RNA-seq]

Protein lactylation is a process that is fueled by lactate, generated by the enzyme lactate dehydrogenase (Ldh) from pyruvate. Despite prior research, the precise role of protein lactylation in controlling the identity of mouse embryonic stem cells (ESCs) is still not fully understood. We observed that inhibiting or eliminating Ldha causes a reduction in global protein lactylation in ESCs, and RNA-seq analysis suggests that Ldha inhibition induces a 2-cell-like cell (2CLC) signature in ESCs. To probe the underlying mechanisms, we performed quantitative lactylation proteomics analysis, we discovered that Hdac1, a gene with significant regulatory roles during the 2-cell stage (2C), undergoes lactylation modification. Additionally, we observed that treatment with an Ldh (lactate dehydrogenase) inhibitor can decrease the lactylation levels of Hdac1. Mechanistically, we discovered that Ldha positively regulates the lactylation of Hdac1, promoting its direct binding to zygotic genome activation (ZGA) gene promoters and has stronger deacetylase activity. This leads to the removal of acetyl groups from H3K27 on these loci, effectively suppressing the expression of 2C genes. Our study presents novel evidence supporting protein lactylation's potential as a means of inhibiting the generation of 2CLCs and modulating acetylation activity. Comparative gene expression profiling analysis of RNA-seq data for mESCs and its KO (Ldha KO, and Ldhb KO) or inhibitor treatment (SO treatment) derivatives.

蛋白质乳酰化（Protein lactylation）是一种由乳酸供能的过程，而乳酸由乳酸脱氢酶（lactate dehydrogenase，Ldh）催化丙酮酸生成。尽管已有相关研究，但蛋白质乳酰化在调控小鼠胚胎干细胞（embryonic stem cells，ESCs）身份特性中的精确作用仍未完全阐明。我们观察到，抑制或敲除Ldha会导致小鼠胚胎干细胞内整体蛋白质乳酰化水平下降，而RNA测序（RNA-seq）分析显示，抑制Ldha可在小鼠胚胎干细胞中诱导出2细胞样细胞（2-cell-like cell，2CLC）特征谱。为探究其潜在分子机制，我们开展了定量乳酰化蛋白质组学分析，发现组蛋白去乙酰化酶1（Hdac1）——一种在2细胞阶段（2-cell stage，2C）发挥重要调控功能的基因——发生了乳酰化修饰。此外，我们发现使用乳酸脱氢酶抑制剂处理可降低Hdac1的乳酰化水平。从机制层面来看，我们发现Ldha可正向调控Hdac1的乳酰化修饰，增强其与合子基因组激活（zygotic genome activation，ZGA）基因启动子的直接结合能力，并使其具备更强的去乙酰化酶活性。这一过程会去除这些基因位点上H3K27的乙酰基团，有效抑制2C基因的表达。本研究提供了全新证据，证实蛋白质乳酰化具备抑制2CLC产生、调控乙酰化活性的潜在功能。本研究还针对小鼠胚胎干细胞（mouse embryonic stem cells，mESCs）及其Ldha敲除（Ldha KO）、Ldhb敲除（Ldhb KO）或抑制剂处理（SO treatment）的衍生样本的RNA-seq数据，开展了比较基因表达谱分析。