Institute of Translational Medicine, Tianjin Union Medical Center, State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Sciences, Frontiers Science Center for Cell Responses, National Demonstration Center for Experimental Biology Education and College of Life Sciences, Nankai University, Tianjin, 300071, China.
Stem Cell Res Ther. 2024 Nov 13;15(1):415. doi: 10.1186/s13287-024-04027-1.
Cellular metabolism regulates the pluripotency of embryonic stem cells (ESCs). Yet, how metabolism regulates the transition among different pluripotent states remains elusive. It has been shown that protein lactylation, which uses lactate, a metabolic product of glycolysis, as a substrate, plays a critical role in various biological events. Here we focused on that glycolysis regulates the conversion between ESCs and 2-cell-like cells (2CLCs) through protein lactylation.
RNA-seq revealed the activation of 2-cell (2C) genes by suppression of Ldh. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with lactylated peptide enrichment and quantitative mass spectrometric analysis was carried out to investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition. And we focused on Hdac1. Lactylation of Hdac1 required for silencing 2C genes was proved by quantitative reverse-transcription PCR (qRT-PCR), immunofluorescence (IF), Western blot and chimeric embryos. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and in vitro deacetylation assay confirmed lactylation of Hdac1 promoting its binding at 2C genes and enhancing its deacetylase activity, thereby facilitating the removal of H3K27ac and the silencing of 2C genes.
We found that inhibition or depletion of Ldha, the enzyme converting pyruvate to lactate, leads to the activation of 2C genes, as well as reduced global lactylation in ESCs. To investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition, quantitative lactylome analysis was performed, and 1716 lactylated proteins were identified. We then focused on Hdac1, a histone deacetylase involved in the silencing of 2C genes. Lactylation of Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes.
In summary, our study reveals a mechanistic link between cellular metabolism and pluripotency regulation through protein lactylation. Our research is the first time to reveal that quantitative lactylome analysis in mouse ESCs. We found that lactylated Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes.
细胞代谢调节胚胎干细胞（ESCs）的多能性。然而，代谢如何调节不同多能状态之间的转变仍然难以捉摸。已经表明，使用代谢产物乳酸作为底物的蛋白质乳酰化在各种生物事件中起着关键作用。在这里，我们专注于糖酵解通过蛋白质乳酰化调节 ESC 和 2 细胞样细胞（2CLC）之间的转化。
RNA-seq 显示通过抑制 Ldh 激活 2 细胞（2C）基因。通过细胞培养中的稳定同位素标记氨基酸（SILAC）与乳酰化肽富集和定量质谱分析相结合，研究了蛋白质乳酰化如何调节多能性向 2C 转变的机制。我们专注于 Hdac1。通过定量逆转录 PCR（qRT-PCR）、免疫荧光（IF）、Western blot 和嵌合胚胎证明了沉默 2C 基因所需的 Hdac1 乳酰化。染色质免疫沉淀结合测序（ChIP-seq）和体外去乙酰化测定证实了 Hdac1 的乳酰化促进了其在 2C 基因上的结合，并增强了其去乙酰化酶活性，从而促进了 H3K27ac 的去除和 2C 基因的沉默。
我们发现，抑制或耗尽将丙酮酸转化为乳酸的酶 Ldha 会导致 2C 基因的激活，以及 ESCs 中全局乳酰化水平降低。为了研究蛋白质乳酰化如何调节多能性向 2C 转变的机制，进行了定量乳酰组分析，鉴定出 1716 个乳酰化蛋白。然后，我们专注于 Hdac1，一种参与 2C 基因沉默的组蛋白去乙酰化酶。Hdac1 的乳酰化促进了其在 2C 基因上的结合，并增强了其去乙酰化酶活性，从而促进了 H3K27ac 的去除和 2C 基因的沉默。
总之，我们的研究通过蛋白质乳酰化揭示了细胞代谢与多能性调控之间的机制联系。我们的研究首次揭示了小鼠 ESCs 中的定量乳酰组分析。我们发现乳酰化的 Hdac1 促进了其在 2C 基因上的结合，并增强了其去乙酰化酶活性，从而促进了 H3K27ac 的去除和 2C 基因的沉默。
