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Lactylation of Hdac1 regulated by Ldh prevents the pluripotent-to-2C state conversion

Stem Cell Research & Therapy volume 15, Article number: 415 (2024) Cite this article

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Background

Embryonic stem cells (ESCs), derived from the inner cell mass (ICM) of the blastocysts, are functionally equivalent to the ICM, lacking the extraembryonic developmental potential. However, a sporadic population of ESCs, expressing high levels of 2-cell stage (2C) specific genes, known as 2-cell-like cells (2CLCs), possess expanded developmental potential, contributing to both embryonic and extraembryonic lineages when injected into early embryos [1]. Thus, 2CLCs provide a valuable tool to study totipotency in vitro, as well as zygotic genome activation (ZGA) [1,2,3,4,5].

Cellular metabolism plays an important role in pluripotency maintenance and establishment [6,7,8]. ESCs favor glycolysis for energy production, while differentiated cells preferentially utilize oxidative phosphorylation (OXPHOS) [9,10,11,12,13]. Suppression of glycolysis impairs the self-renewal of naive and primed human ESCs [14]. Stimulation of glycolysis promotes, while inhibition of glycolysis reduces, reprogramming efficiency by Yamanaka factors [7]. Comparing with ESCs, 2CLCs exhibit decreased glycolytic activity and oxygen consumption [15, 16]. Consistently, suppression of glycolysis by 2-deoxy-D-glucose (2-DG) activates the 2C transcriptional program in ESCs [3, 17]. Yet, how glycolysis regulates the transition between ESCs and 2CLCs 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. For example, histone lysine lactylation (Kla) regulates gene expression in immune cells, ocular melanoma, Alzheimer’s disease, vascular calcification, drug resistance and induction of pluripotency [18,19,20,21,22,23,24,25,26]. Histone lactylation marks active promoters and enhancers [27, 28]. In addition, the functions of Kla on non-histone proteins have been demonstrated, such as Kla of METTL3, METTL16, Nucleolin, Ikzf1, and MRE11, in tumor-infiltrating myeloid cells, cuproptosis of gastric cancer cells, intrahepatic cholangiocarcinoma, TH17 differentiation and DNA damage response of cancer cells, respectively [29,30,31,32,33]. Recently, the alanyl-tRNA synthetase 1 (AARS1) was identified as a lactyl-transferase catalyzing protein lactylation [34, 35]. Thus, we speculated that glycolysis regulates the conversion between ESCs and 2CLCs through protein lactylation.

In this study, we first demonstrated that inhibition or knockout (KO) of Ldha, the enzyme catalyzing the conversion of pyruvate to lactate, leads to the activation of 2C genes in ESCs. Next, through profiling the lactylome of ESCs with and without Ldha inhibition, 713 proteins with reduced lactylation upon Ldha inhibition, including Hdac1, were identified. Lactylation of Hdac1 promotes its binding at 2C genes and increases the deacetylase activity, resulting in reduced H3K27ac level and the suppression of 2C genes. In summary, our data provides mechanistic insights on how glycolytic metabolism regulates the transition between pluripotency and the 2C-like state.

Methods

Cell lines

Mouse ESC line E14 (RRID: CRL-1821) is a gift from Deqing Hu lab. Mouse ESC line J1 (RRID: CRL-1821) is a gift from Lin Liu lab. All ESC lines are cultured as follows: 85% Dulbecco’s Modified Eagle Media (DMEM) (Gibco, Waltham, Massachusetts, USA), 15% fetal bovine serum (HyClone, Logan, Utah, USA), 2 mM L-glutamine (Invitrogen, Carlsbad, CA, USA), 100 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, United States), and 1000 units/ml LIF. Mouse ESCs were plated on tissue culture dishes which were pre-coated with 0.2% gelatin (Sigma-Aldrich) for at least 20 min and grown in a humidified 5% CO2 at 37 °C incubator. The medium was changed every day and ESCs were passaged every 2–3 days. All ESC lines were tested for mycoplasma contamination every 2 weeks.

Transfection

Transfection was carried out with Lipo8000™ (Beyotime, Shanghai, China) according to the manufacturer’s recommendations.

Plasmid construction

pLentil-Cas9-puro was used to construct gene knockout plasmids targeting Ldha, Ldhb, and Hdac1. The sgRNA oligonucleotides were designed using the website (https://www.zlab.bio/guide-design-resources). The sgRNA sequence targeting Ldha, Ldhb, and Hdac1 were 5′-TACCTTCATTAAGATACTGA-3′, 5′-AAATTGTCTCCAGCAAAGGT-3′, 5′-AAAATTGTGGCCGATAAAGG-3′, and 5′-TTGGCTTTGTGAGGACGCTA-3′, respectively. PB-CAG-3xFLAG was used to construct overexpression plasmids. Hdac1 coding region was amplified from complementary DNA (cDNA) of ESCs and inserted into PB-CAG-3xFLAG by seamless cloning (Beyotime). KQ and KR Hdac1 mutants were constructed by PCR mutagenesis. The primers are listed in Table S2.

Quantitative reverse-transcription PCR (qRT-PCR)

The total RNA was extracted from cells using TRIZOL (Invitrogen) according to the manufacturer’s instruction. 1 μg RNA was used for cDNA synthesis with a Reverse Transcription kit (GenStar, Beijing, China). The PCR reaction was performed with 2 × RealStar Power SYBR qPCR Mix (GenStar) and run on a Quantitative PCR machine (Bio-Rad, Hercules, CA, USA). The primers are listed in Table S2.

RNA-seq

Total RNA was extracted by RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Library construction and sequencing were performed by BGI Tech (Shenzhen, China).

Western blot

Cells were harvested by trypsin digestion and resuspended in lysis buffer (20 mM Tris–HCl pH 8.0, 60 mM NaCl, 0.2% glycerol, 0.02% NP-40, 0.04 mM EDTA, 1 mM PMSF, and 1.52 mg/ml protease inhibitor cocktail). Cell lysates were sonicated followed by centrifugation at 12,000 g for 15 min at 4 °C. Supernatants were transferred to fresh tubes. Protein concentrations were measured using BCA Protein Assay Kit (Beyotime). Samples were resolved in SDS-PAGE gel and transferred onto a PVDF membrane. Then, the PVDF membrane was incubated in blocking buffer for 1 h at room temperature, and incubated with primary antibodies overnight at 4 °C. Primary antibodies used were anti-Kla (PTM BIO, PTM-1401, 1:1000, Hangzhou, China), anti-β-Tubulin (Abmart, M20005H, 1:5000, Shanghai, China), anti-Ldha (Cell Signaling Technology, 2012S, 1:1000, Danvers, Massachusetts, USA), anti-Ldhb (Abcam, ab240482, 1:1000, Cambridge, UK), anti-Zscan4 (Sigma-Aldrich, AB4340,1:1000), anti-FLAG (Sigma-Aldrich, F1804, 1:3000), anti-Hdac1 (Abclonal, A0238,1:1000, Wuhan, China). After washing with TBS 3 times, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Secondary antibodies used were donkey anti-Rabbit (Cytiva, NA934V, 1:5000, Marlborough, Massachusetts, USA), mouse anti-Goat (Santa Cruz, sc-2354, 1:1000), sheep anti-Mouse (Cytiva, NA931V, 1:5000). Immunoreactivity was detected by ECL Plus (Shanghai Epizyme Biomedical Technology Co., Ltd, Shanghai, China). Digital images were taken with an automatic chemiluminescence imaging analysis system (Tanon, Shanghai, China). All full-length WB images are provided in Additional file 2.

Immunofluorescence (IF)

Cells were fixed with 4% PFA for 30 min at room temperature, permeabilized with 0.2% Triton X-100 for 30 min, and blocked in 5% Goat FBS (Beyotime) for 1 h at room temperature. Cells were then incubated with primary antibody anti-Zscan4 (Sigma, AB4340, 1:200) overnight at 4 °C. After three washes, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, A11034, 1:1000) for 1 h at room temperature. Cells were mounted with ProLong Gold Antifade Mountant with DNA Stain DAPI (Invitrogen) and imaged using a Zeiss Axio Imager Z1 microscope.

Quantitative lactylome analysis

The basic medium was prepared as following: 85% DMEM for SILAC (Thermo Fisher, Waltham, Massachusetts, USA), 15% dialyzed fetal bovine serum (GEMINI, New York, NY, USA), 2 mM L-glutamine (Invitrogen), 100 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), and 1000 units/ml LIF. The light and heavy media were the basic medium supplemented with 0.458 mM 12C614N4-arginine (Thermo Fisher) and 0.966 mM 12C614N2-lysine (Thermo Fisher), and 0.458 mM 13C615N4-arginine (Thermo Fisher) and 0.966 mM 13C614N2-lysine (Thermo Fisher), respectively. ESCs were cultured in light or heavy medium for 7 passages. ESCs cultured in heavy medium were treated with 20 mM SO for 24 h before harvesting. Frozen cell pellets were sent to PTM Biolabs, and the following steps were carried out by PTM Biolabs. Protein lysates of control and SO treated ESCs were prepared and mixed equivalently. Subsequently, trypsin digestion, high-performance liquid chromatography (HPLC) fractionation, Kla peptide enrichment with immobilized anti-Kla antibody, and high-resolution liquid chromatography–tandem MS (LC–MS/MS) were implemented. The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against SwissProt Mouse database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification and lactylation on Lys were specified as variable modifications. False discovery rate (FDR) was adjusted to < 1% and minimum score for modified peptides was set > 40. The amount of lactylated peptides was normalized to the input peptides, and unnormalized data due to missing values were not included for further quantitative analysis. 1.3-fold threshold was applied to select up- and down-regulated lactylation sites, and proteins with up- or down-regulated lactylation sites are called as up- or down-regulated lactylation proteins.

Immunoprecipitation

Cells were harvested by trypsin digestion. Cell pellets were resuspended in lysis buffer, and rotated for 1 h at 4 °C followed by centrifugation at 12 K g for 30 min at 4 °C. The supernatants were transferred to a fresh tube. Protein concentrations were measured using BCA Protein Assay Kit (Beyotime). 2% cell lysates were taken as Input. For immunoprecipitation, equivalent protein lysates were incubated with anti-FLAG M2 Magnetic Beads at 4 °C, or specific antibodies followed by precipitation with 10 μl of 50% protein A or G agarose beads (Cytiva) on a rotor overnight. After three 5 min washes with lysis buffer, 30 μl 2 × SDS-PAGE Plus Sample Buffer (Genstar) was added and the beads were boiled for 5 min at 100 °C to elute. Then the samples were resolved with SDS-PAGE gel and subjected to Western blot.

Mice and care

ICR females (age 3 weeks old) and males (age 8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd and were housed and cared in individually ventilated cages (IVCs) on a standard 12 h light:12 h dark cycles at 50–60% humidity in sterile Animal Resources Center at Nankai University. The animal experiments that were conducted as part of this research were completed according to the guidelines provided by the Nankai University Animal Care and Use Committee. Mice were quickly euthanized by standard cervical dislocation method without using chemical drugs. The work has been reported in line with the ARRIVE guidelines 2.0.

Generation of chimeric embryos

30 ICR females (age 4 weeks old) were intraperitoneally injected with 7.5 IU pregnant mare serum, followed by intraperitoneal injection of 7.5 IU human chorionic gonadotropin 46–48 h later for super-ovulating. These female mice were mated with ICR male mice (age 9 weeks old) at 1:1 pairing. Vaginal plugs were checked the next morning, which is designated as 0.5 d post coitum (dpc). 2-cell embryos were collected at 1.5 dpc, washed with M2 medium, and cultured in KSOM under sterile paraffin oil overnight in a humidified incubator at 37 °C with 5% CO2 until the 8-cell stage. ~ 250 8-cell stage embryos were obtained. ESCs were treated with 15 μg/ml Tat-PFBD nanodots [36] for 5 h, and then 6–10 cells were micro-injected into 8-cell stage mouse embryos. After a 1-day in vitro culture, the images of the resulting blastocysts were captured using a LSM710 (Zeiss, Germany).

Chromatin immunoprecipitation (ChIP)

4 × 106 ESCs were collected and resuspended in 10 ml PBS. 270 μl 37% formaldehyde (Sigma-Aldrich) was added. After 10 min incubation at room temperature, the reaction was quenched with 1 ml 1.25 M of glycine buffer for 5 min at room temperature. Then the cells were centrifuged at 1500 rpm for 5 min and washed three times with 10 ml ice-cold PBS. Chromatin fragmentation by MNase and immunoprecipitation were carried out with the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer’s instruction. Anti-H3K27ac (Cell Signaling Technology, 8173S, 1:100) and anti-FLAG (Sigma-Aldrich, F1804, 1:300) were used for immunoprecipitation. The ChIP–qPCR primers used are listed in Table S2.

ChIP–seq

Purified ChIP DNA was sent to Novogene (Tianjin, China) for library construction and sequencing.

In vitro deacetylation assay

In vitro deacetylation assay was performed as described elsewhere [37]. FLAG-tagged WT, KQ and KR Hdac1 proteins were immunoprecipitated from ESCs using anti-FLAG M2 Magnetic Beads at 4 °C on a rotor overnight. For the deacetylation assay, Hdac1 and acid-extracted histone protein were incubated in deacetylation buffer (25 mM Tris, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2) at 30 °C for 30 min. SDS-PAGE Plus Sample Buffer was added and the samples were boiled for 5 min at 100 °C. Then the samples were resolved in SDS-PAGE gel and subjected to Western blot.

Lactate analysis

Lactate level was measured by CheKine™ Micro Lactate Assay Kit (Abbkine Inc.) according to the manufacturer’s protocol.

Data analysis

RNA-seq reads were aligned to the mouse genome (mm9) using STAR (v2.7.6a) with default parameters for accurate mapping. Transcript assembly and differential expression analysis were conducted using Cufflinks (v2.2.1), with novel transcript assembly disabled (-G) and default settings applied elsewhere. FPKM values for transcripts sharing the same gene_id were summed and exported using Cuffdiff. Differentially expressed genes (DEGs) were identified using fold-change > 2, or fold-change ≤ 0.5.

ChIP-seq reads were pre-processed with Trim Galore (v0.6.3) for adapter trimming and quality filtering, and aligned to the mm9 mouse genome using Bowtie2 (v2.3.4). Mapped reads were exported, sorted, and duplicates were removed with MarkDuplicates from PICARD (v2.14.0). The resulting BAM files were converted to TDF format for visualization in IGV (v2.7.2). MACS2 (v2.2.7) was used for peak calling with default parameters. Peaks were annotated against the mm9 genome using the annotatePeaks module in HOMER (v4.11). Heatmaps and mean intensity curves of ChIP-seq data at specific genomic regions were plotted by the NGSplot program (v2.61).

Quantification and statistical analysis

Statistical analysis of all experiments was carried out using GraphPad Prism version 8. At least 3 independent experiments were analyzed, and data were presented as average ± SD. Statistical analysis was performed with unpaired two-tailed Student’s t test, one-way ANOVA or two-way ANOVA, which were indicated in the figure legends. Statistically significant p values were indicated in figures as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Results

Inhibition of lactate dehydrogenase induces 2CLC signature

Previous studies have demonstrated that suppression of glycolysis in ESCs promotes the entry to the 2C-like state [3, 17]. To test whether the glycolytic product lactate regulates the pluripotent-to-2C state conversion, we employed sodium oxamate (SO) as a pharmacological inhibitor to suppress lactate dehydrogenase (Ldh), which catalyzes the conversion of pyruvate to lactate. Upon SO treatment, the extracellular lactate level decreases (Fig. S1A), suggesting that SO treatment indeed suppresses the production of lactate. However, the intracellular lactate concentration is only slightly, but statistically insignificantly, reduced, when treated with SO (Fig. S1B). It is likely that the intracellular lactate level is tightly regulated. Even though SO treatment reduces the production of lactate from the glycolysis pathway, the consumption of intracellular lactate, such as lactylation and lactate exportation, might be suppressed. Thus, the overall level of intracellular lactate does not change significantly.

RNA-seq analysis showed that SO treatment activates the expression of 2C genes in ESCs, such as the Zscan4 gene family and the MERVL retroelements (Fig. 1A, B). Gene set enrichment analysis (GSEA) also revealed the upregulation of 2C signature gene set upon SO treatment (Fig. 1C). Quantitative reverse-transcription PCR (qRT-PCR) analysis validated the elevated expression of 2C genes, MT2/MERVL, Tcstv3, Gm2016, Zscan4, Pramel7, and Nfam1, in SO treated ESCs (Fig. 1D). The enhanced expression of Zscan4 was further confirmed by Western blot and immunofluorescence (IF) (Fig. 1E–G).

Fig. 1
figure 1

Inhibition of Ldha induces 2CLC signature. A–G ESCs were treated with or without 20 mM SO for 24 h, and subjected to RNA-seq (A–C), qRT-PCR (D), Western blot (E), and IF (F, G). A Scatterplot of gene expression in ESCs with or without SO treatment. B Scatterplot of retroelement expression in ESCs with or without SO treatment. Parallel diagonal lines indicate the two-fold threshold in expression difference. Red dots mark genes with more than twofold upregulation upon SO treatment, while green indicates genes with a greater than twofold reduction in expression. C Gene set enrichment analysis (GSEA) shows the activation of 2C signature gene set in SO treated ESCs. NES: normalized enrichment score. D qRT-PCR analysis of 2C genes in ESCs with or without SO treatment. E and F Western blot E and IF F indicate enhanced Zscan4 expression in SO-treated ESCs. G The fraction of Zscan4+ cells quantified from the IF data (F). H ESCs were treated with 0, 5, 10, and 20 mM SO for 24 h, and subjected to Western blot to detect Kla levels. For qRT-PCR and IF, n = 3. Data are presented as average ± SD. Statistical analysis was performed with unpaired two-tailed Student’s t test. ***, p < 0.001. Scale bar: 50 μm

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To rule out the off-target effect of SO, we also tried to knock out Ldh. There are four homologous Ldh genes, Ldha, Ldhb, Ldhc, and Ldhd, in the mouse genome. Through analyzing RNA-seq data, we found that Ldha is most abundantly expressed in mouse ESCs, while Ldhc and Ldhd are almost undetectable in ESCs (Fig. S2A). Thus, we only established Ldha KO ESCs (Ldha KO-1 and Ldha KO-2) and Ldhb KO ESCs (Ldhb KO-1 and Ldhb KO-2), using CRISPR/Cas9 (Fig. S2B–G). Consistent with the expression levels of Ldha and Ldhb, KO of Ldha, but not Ldhb, activates the 2C transcriptional program in ESCs (Fig. 2A–G and S3). In addition, both SO treatment and Ldha KO activates 2C genes in another ESC line, J1 (Fig. S4).

Fig. 2
figure 2

Deletion of Ldha activates 2C gene expression. A–G E14 and Ldha KO ESCs were subjected to RNA-seq (A–C), qRT-PCR (D), Western blot (E), and IF (F, G). A Scatterplot of gene expression in E14 and Ldha KO ESCs. B Scatterplot of retroelement expression in E14 and Ldha KO ESCs. Parallel diagonal lines indicate the two-fold threshold in expression difference. Red dots mark genes with more than twofold upregulation upon Ldha KO, while green indicates genes with a greater than twofold reduction in expression. C GSEA shows the activation of 2C gene set in Ldha KO ESCs. D qRT-PCR analysis of 2C genes in E14 and Ldha KO ESCs. E and F Western blot (E) and IF (F) indicate enhanced Zscan4 expression in E14 and Ldha KO ESCs. G The fraction of Zscan4+ cells quantified from the IF data (F). H E14 and Ldha KO ESCs were subjected to Western blot to detect Kla levels. For qRT-PCR and IF, n = 3. Data are presented as average ± SD. Statistical analysis was performed with one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar: 50 μm

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In conclusion, the activation of 2C genes by suppression of Ldh implicates that lactate, the metabolic product of glycolysis, plays a role in silencing 2C genes and preventing the transition from ESCs to the 2C-like state.

Global overview of Ldh-regulated lactylome in mouse ESCs

It has been shown that protein lactylation, using lactate as a substrate, regulates various biological events [18,19,20,21,22, 29,30,31]. We then tested the possibility that lactate suppresses the expression of 2C genes and the entry to the 2C-like state through protein lactylation. Indeed, both SO treatment and Ldha KO reduce the global level of Kla (Figs. 1H and 2H), implying a functional role of protein lactylation. As expected, Ldhb KO hardly affects protein lactylation (Fig. S2H).

To investigate how protein lactylation regulated by Ldh controls the 2C transcriptional program, stable isotope labeling by amino acids in cell culture (SILAC) coupled with lactylated peptide enrichment and quantitative mass spectrometric analysis was carried out to characterize the lactylome profiles of ESCs with and without SO treatment (Fig. 3A). ESCs were labeled with heavy isotopes, 13C615N4-arginine (Arg10) and 13C614N2-lysine (Lys6), or light isotopes, 12C614N4-arginine (Arg0) and 12C614N2-lysine (Lys0), respectively, for a total of 7 passages to ensure efficient labeling (> 97%). The ESCs labeled with heavy isotopes were then treated with 20 mM SO for 24 h before harvesting cells. After coimmunoprecipitation with Kla antibody, 1716 lactylated proteins with 4187 lactylation sites were identified by mass spectrometry. Among these identified proteins, 1465 proteins containing 3577 lactylation sites are quantifiable after normalization (Table S1). Motif analysis of all identified lactylated proteins revealed that proline and serine were enriched at the -2 and -1 positions, respectively, and that glycine, leucine and alanine were enriched in the + 1 position (Fig. 3B).

Fig. 3
figure 3

Lactylome analysis of ESCs with or without SO treatment. A Schematic illustration of the lactylome analysis procedures. B Icelogo presentation of flanking sequence preferences for all Kla sites. C The number of differentially expressed lactylated proteins and lactylation sites in control and SO treated ESCs. D Pie chart showing the subcellular localization of down-regulated Kla proteins. E GO analysis of down-regulated Kla proteins. F Pie chart showing the subcellular localization of up-regulated Kla proteins. G GO analysis of up-regulated Kla proteins

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With a threshold of 1.3-fold change, 690 proteins and 1270 sites with reduced Kla levels upon SO treatment were identified, while Kla levels of 325 proteins and 508 sites are increased in SO treated ESCs (Fig. 3C and Table S1). Notably, the number of down-regulated lactylated proteins was twice of the up-regulated lactylated proteins, aligning with the inhibitory effect of SO on protein lactylation. About two thirds and one sixth of the down-regulated Kla proteins are localized in the nucleus and in the cytoplasm, respectively, whereas the numbers of the up-regulated Kla proteins in the nucleus and in the cytoplasm are about the same (Fig. 3D, F), suggesting that lactylation promoted by Ldh is biased to regulate nuclear proteins. Gene Ontology (GO) analysis revealed that the down-regulated lactylated proteins are involved in various chromatin-associated biological processes, including chromatin organization, chromatin remodeling, and histone deacetylation (Fig. 3E). In contrast, the up-regulated lactylated proteins are enriched in RNA processing, such as cytoplasmic translation and RNA splicing (Fig. 3G).

Hdac1 is lactylated in mouse ESCs

Among the down-regulated Kla proteins, Hdac1, a member of Class I histone deacetylases, which is associated with GO terms of chromatin organization, chromatin remodeling, negative regulation of transcription, and histone deacetylation, drew our attention. Histone acetylation is associated with the activation of 2C genes in ESCs [38]. Moreover, Hdac1 has been identified as an epigenetic repressor for MERVL in ESCs [39, 40].

The lactylome analysis revealed that upon SO treatment, the levels of Hdac1 K412 and K438 lactylation are reduced by 0.506- and 0.460-fold, respectively (Fig. 4A, and Table S1). To validate the lactylome data, immunoprecipitation (IP) assays were performed. Kla signal was detected in IP sample with an anti-Hdac1 antibody (Fig. 4B). Reciprocally, IP with an anti-Kla antibody was able to pull down Hdac1 (Fig. 4C). These data validate that Hdac1 is lactylated. Both SO treatment and Ldha KO reduce the lactylation level of Hdac1 (Fig. 4D, E). Conversely, L-lactate promotes Hdac1 lactylation (Fig. 4F). These data suggest that lactylation of Hdac1 is stimulated by glycolysis. To map the lactylation sites on Hdac1, K412 and K438 of Hdac1 were mutated to arginine individually or simultaneously. Replacing K412 or K438 with arginine (K412R or K438R) reduces the lactylation of Hdac1. Simultaneous mutation of K412 and K438 to arginine (KR) further suppresses the lactylation of Hdac1 (Fig. 4G), confirming that K412 and K438 are major lactylation sites on Hdac1.

Fig. 4
figure 4

Hdac1 is lactylated on K412 and K438. A MS/MS spectra showing 2 lactylated sites (K412 and K438) of Hdac1. The b and y ions refer to the N-terminal and C-terminal parts of the peptide, respectively. B After IP with IgG or anti-Hdac1 antibody, using E14 ESC lysate, Kla and Hdac1 were detected by Western blot. C E14 ESCs were subjected to IP with IgG or anti-Kla antibody, and Western blot was performed to detect Hdac1. D Lysates of E14 ESCs treated with or without SO were immunoprecipitated with IgG or anti-Kla, followed by immunoblotting with anti-Hdac1. E E14 or Ldha KO ESCs were immunoprecipitated with anti-Hdac1 followed by immunoblotting with antibodies against with indicated proteins. F E14 ESCs treated with or without 20 mM L-lactate for 24 h, were immunoprecipitated with IgG or anti-Kla followed by immunoblotting with anti-Hdac1. G E14 ESCs expressing 3 × FLAG-tagged WT, K412R, K438R and KR Hdac1, were immunoprecipitated with anti-FLAG followed by immunoblotting with antibodies against with indicated proteins. Red asterisks mark the specific band of Hdac1

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Ldh inhibition activates 2C genes mainly through inactivating Hdac1

To prove the function of Hdac1 in regulating 2C gene expression, we constructed Hdac1 KO ESC lines (Hdac1 KO-1 and Hdac1 KO-2) using CRISPR/Cas9 (Fig. 5A, B and S5). The expression levels of Zscan4 in Hdac1 KO ESCs are higher than that in WT E14 ESCs (Fig. 5B, C). Moreover, IF revealed that Hdac1 KO increases the fraction of Zscan4+ ESCs (Fig. 5D). Other 2C genes are also activated in Hdac1 KO ESCs (Fig. 5E). These data demonstrate that Hdac1 is an epigenetic repressor not only for MERVL, but also for 2C genes. More importantly, SO treatment does not further elevate the expression levels of 2C genes in Hdac1 KO ESCs, while 2C genes are activated by SO in WT E14 ESCs (Fig. 5F), suggesting that the activation effect on 2C genes by Ldh inhibition is mainly mediated by Hdac1.

Fig. 5
figure 5

2C genes are activated when Hdac1 is knocked out. A A schematic illustration of the experimental design to knock out Hdac1. Blue rectangles represent the exons of Hdac1. The sgRNA targeting sequence is shown, and the protospacer-adjacent motif (PAM) is marked in red. B Western blot detecting the expression of Hdac1 and Zscan4 in E14 and Hdac1 KO ESCs. C IF showing the expression of Zscan4 in E14 and Hdac1 KO ESCs. D The fraction of Zscan4+ cells quantified from the IF data (C). E qRT-PCR analysis of 2C genes in E14 and Hdac1 KO ESCs. F qRT-PCR analysis of 2C genes in E14 and Hdac1 KO ESCs with or without SO treatment. For qRT-PCR, Western blot and IF, n = 3. Data are presented as average ± SD. Statistical analysis was performed with one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar: 50 μm

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Hdac1 lactylation facilitates silencing 2C genes

Subsequently, we aimed to elucidate the functional implications of Hdac1 lactylation. For this purpose, both K412 and K438 of Hdac1 were simultaneously mutated to either glutamine, referred to as KQ mutant, or arginine, referred to as KR mutant, to mimic lactylated and unlactylated status, respectively. Hdac1 KO ESCs stably expressing FLAG-tagged WT, KQ, and KR Hdac1, were established and denoted as WT, KQ, and KR ESCs (Fig. 6A). Even though WT, KQ and KR Hdac1 all suppress the expression of Zscan4 in Hdac1 KO ESCs, KQ Hdac1 is most potent in reducing Zscan4 expression, while KR Hdac1 is least efficient (Fig. 6A). IF and qRT-PCR assays further confirmed that KR mutation, but not KQ mutation, fails to rescue the enhanced fraction of Zscan4+ cells and the elevated expression of 2C genes in Hdac1 KO ESCs (Fig. 6B–D). These data implicate that lactylation of Hdac1 is required for silencing 2C genes.

Fig. 6
figure 6

Lactylation of Hdac1 promotes its function of silencing 2C genes. A Hdac1 KO-1 ESC lines stably expressing FLAG-tagged WT, KQ and KR Hdac1, were established and named as WT, KQ, and KR ESCs. Western blot was performed to detect the expression levels of Hdac1 and Zscan4 in these cell lines. Two clones of WT, KQ, and KR ESCs were included. B IF detecting Zscan4 expression in E14, Hdac1 KO, WT, KQ, and KR ESCs. Scale bar: 50 μm. C The fraction of Zscan4+ cells quantified from the IF data (B). D qRT-PCR analysis of 2C genes in E14, Hdac1 KO, WT, KQ, and KR ESCs. E Experimental procedures of the 8-cell embryo injection. F Brightfield and fluorescence overlayed images of chimeric embryos. Tat-PFBD nanodot labeled E14, Hdac1 KO-1, WT, KQ, and KR ESCs were microinjected into 8-cell embryos, and images were taken at the blastocyst stage. Scale bar: 20 μm. G Summary of the incorporation of E14, Hdac1 KO-1, WT, KQ, and KR ESCs into chimeric embryos. The bar charts show the percentage of chimeras with ICM and TE contributions from Tat-PFBD nanodot labeled E14, Hdac1 KO-1, WT, KQ, and KR ESCs. The number of chimeric embryos is shown. For qRT-PCR and IF, n = 3. Data are presented as average ± SD. Statistical analysis is performed with two-way ANOVA. **, p < 0.01; ***, p < 0.001

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The activation of 2C genes indicates the entry to the 2C-like state with both embryonic and extraembryonic developmental potency. To further verify the effect of Hdac1 lactylation on the developmental potential of ESCs, E14, Hdac1 KO, WT, KQ, and KR ESCs fluorescently labeled with Trans-Activator of the Transcription-Poly(9,9-dihexylfluorene-alt-2,1,3-benzoxadiazole) (Tat-PFBD) nanodots [36], were microinjected into 8-cell embryos (Fig. 6E). E14, WT, and KQ ESCs are restricted to the ICM, whereas Hdac1 KO and KR ESCs contribute to both ICM and trophectoderm (TE) (Fig. 6F, G), suggesting that Hdac1 KO and KR ESCs are indeed 2CLCs with expanded developmental potential.

Hdac1 lactylation enhances its binding to 2C genes and deacetylation activity

Given that Hdac1 is a histone deacetylase and that H3K27ac is involved in the regulation of 2C genes [41, 42], we first examined how Hdac1 lactylation affects the level of global H3K27ac. Upon Hdac1 KO, H3K27ac level is increased, consistent with the role of Hdac1 as a histone deacetylase. Interestingly, WT and KQ Hdac1, but not KR Hdac1, efficiently suppress the enhanced H3K27ac level in Hdac1 KO ESCs (Fig. 7A), suggesting that Hdac1 lactylation facilitates the removal of H3K27ac. It has been demonstrated that HDAC1 is not only a deacetylase, but also a delactylase, catalyzing histone delactylation [43]. Moreover, histone lactylation H3K18la facilitates the expression of major ZGA genes [44]. Thus, we tested whether Hdac1 lactylation regulates 2C gene expression through delactylating H3K18la. Western blot experiments showed that the global H3K18la level is not changed by Hdac1 KO, and that global H3K18la levels in WT, KQ, and KR ESCs are almost the same (Fig. S6A). Thus, it is more likely that Hdac1 lactylation regulates the expression of 2C genes through modulating its deacetylase activity, rather than its delactylase activity.

Fig. 7
figure 7

Lactylation of Hdac1 enhances its binding to 2C genes and deacetylase activity, hence facilitating H3K27ac removal. A Western blot analysis for H3K27ac levels in E14, Hdac1 KO-1, WT, KQ, and KR ESCs. B–D H3K27ac ChIP-seq assay was performed in WT, KQ, and KR ESCs. B Heatmap plots (up panel) and averaged profiles (bottom panel) of the H3K27ac ChIP-seq signal at transcription start sites (TSSs) in WT, KQ, and KR ESCs. C Heatmap plots (up panel) and averaged profiles (bottom panel) of the H3K27ac ChIP-seq signal at 2C gene TSSs in WT, KQ, and KR ESCs. D H3K27ac ChIP-seq tracks at 2C gene loci, Pramel7 and Nfam1, in WT, KQ, and KR ESCs. E ChIP-qPCR quantification of Hdac1 binding at Pramel7 and Nfam1 loci in WT, KQ, and KR ESCs. F The deacetylase activities of WT, KQ, and KR Hdac1 proteins were measured by in vitro deacetylation assay. G A working model for Hdac1 lactylation to silence 2C genes in ESCs. For ChIP-qPCR, n = 3. Data are presented as average ± SD. Statistical analysis is performed with one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001

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To depict the H3K27ac profiles regulated by Hdac1 lactylation, chromatin immunoprecipitation coupled with sequencing (ChIP-seq) for H3K27 was performed in Hdac1 KO ESCs stably expressing FLAG-tagged WT, KQ, and KR Hdac1 (WT, KQ, and KR cells). With similar number of clean reads in the three ChIP-seq data, more peaks are detected in KR ESCs, than WT and KQ ESCs expressing WT and KQ Hdac1, indicating the highest level of H3K27ac in KR ESCs (Fig. S6B, C). Heatmap, pileup and peak signal analyses further demonstrate that the enrichment of H3K27ac at overall and 2C gene promoter regions is higher in KR ESCs, than that in ESCs expressing WT and KQ Hdac1 (Fig. 7B, C, and S6D, E). Consistently, elevated H3K27ac at individual 2C genes in KR ESCs, such as Pramel7, Nfam1, Dppa2 and Zfp668 was observed (Fig. 7D and S6F). Due to the repetitive sequences and limited mappability of Zscan4, direct visualization of the ChIP-seq data at the Zscan4 locus was not feasible [45]. Thus, H3K27ac ChIP-qPCR was performed to demonstrate that the enrichment of H3K27ac at the Zscan4 locus in KR ESCs is the highest (Fig. S6G). These data suggest that lactylation of Hdac1 increases its activity to remove H3K27ac modification, hence promoting the silence of 2C genes.

Hdac1 lactylation might facilitate deacetylation of H3K27 by enhancing the binding of Hdac1 at target genes or by elevating its deacetylase activity. We first tested whether Hdac1 binding at target genes is regulated by lactylation. ChIP-qPCR detected stronger binding of KQ Hdac1 at the Pramel7 and Nfam1 loci than that of WT and KR Hdac1 (Fig. 7E), suggesting that Hdac1 lactylation promotes its binding to target genes. Next, we addressed whether lactylation of Hdac1 increases its deacetylase activity. FLAG-tagged WT, KQ and KR Hdac1 proteins, expressed in ESCs, were purified for in vitro deacetylation assay. WT and KQ Hdac1 proteins have higher deacetylase activity than KR Hdac1, indicated by more reduced H3K27ac levels (Fig. 7F). Taken together, these results suggest that lactylated Hdac1 binds stronger to 2C genes and exhibits a higher deacetylase activity, resulting in more efficient deacetylation of H3K27 and suppression of 2C genes.

Discussion

It has been widely accepted that cellular metabolism plays an important role in cell fate determination, including the regulation of pluripotency [3, 6,7,8, 17]. Yet, the mechanism for cellular metabolism to regulate cell fate remains largely unknown. In this study, we revealed that lactate derived from glycolysis contributes to the pluripotent-to-2C state conversion, through the lactylation of Hdac1. In ESCs with high glycolytic activity and producing more lactate, lactylated Hdac1 removes H3K27ac from 2C genes to maintain the silence of 2C genes. Conversely, glycolysis is reduced in 2CLCs, resulting in less lactate and more unlactylated Hdac1. Unlactylation of Hdac1 not only suppresses its binding at target genes, but also decreases its deacetylase activity, leading to the activation of 2C genes (Fig. 7G).

Exogenous lactate activates 2C genes in ESCs (Fig. S7A, B) [46]. It seems to be conflicted with our data with SO treatment and Ldha KO. However, lactate has a diverse range of functions other than Hdac1 lactylation. For example, lactate supplementation enhances the global Kla level, including the lactylation of histones and Hdac1. It has been shown that H3K18la facilitates ZGA, including 2C gene expression [44]. Thus, lactate may activate 2C genes through H3K18la, which overwrites the suppressive effect of Hdac1 lactylation. In addition, other data are indeed in support the role of Hdac1 lactylation in suppressing 2C genes. First, inhibiting glycolysis, the primary lactate production pathway [3], induces 2C genes in mouse ESCs. Second, sodium lactate (NALA), which also increases global Kla level, suppresses 2C gene expression (Fig. S7C, D).

It has been shown that Hdac1 is essential for proper ZGA and preimplantation development. Inhibition of Hdac activity by Trichostatin A (TSA) results in elevated levels of H3K27ac and minor ZGA genes in 2C embryos and developmental arrest primarily at the 4-cell stage [41, 42]. Nonetheless, we cannot exclude the possibility that other acetylation modifications, such as H3K9ac, play a role in regulating 2C genes expression [47, 48]. In addition, knockdown of Hdac1 enhances the expression of a subset of genes at the late 2C stage, while the global transcription rate remains unaffected [49]. The activity of Hdac1 is primarily regulated by its expression level. During preimplantation development, Hdac1 mRNA is expressed at low levels before the 2C stage, and upregulated in late 2C embryos (Fig. S8) [50]. Meanwhile, Hdac1 protein enters the nucleus at the 2C stage [49, 51]. To ensure the proper regulation of 2C genes, fine tune of Hdac1 activity through lactylation might be necessary in 2C embryos. In early 2C embryos, lack of Hdac1 lactylation might further suppress Hdac1 activity to facilitate the ZGA. In contrast, during the transition from 2-cell to 4-cell embryos where 2C genes need to be silenced, lactylation of Hdac1 might enhance its deacetylase activity and the binding at target genes, allowing rapid silencing of 2C genes. In supporting with this view, treatment of zygotes with GNE-140, which is a dual inhibitor of Ldha/b and prevents the conversion from pyruvate to lactate, leads to developmental arrest from the 8- to 16-cell stages [42]. Nevertheless, the function of Hdac1 lactylation in preimplantation development requires additional experimental validation.

Pyruvate is the sole substrate required for the first division of mouse zygote and ZGA, while either pyruvate or lactate is able to support development from the 2C stage [52, 53]. Interestingly, a subset of mitochondrial TCA cycle enzymes, including pyruvate dehydrogenase (Pdh), pyruvate carboxylase (Pcb), Citrate synthase (Cs), mitochondrial enzyme aconitase 2 (Aco2), and mitochondrial isocitrate dehydrogenase (Idh3), are transiently localized to the nucleus of 2C embryos and pluripotent stem cells (PSCs) [53, 54]. This translocation of TCA cycle enzymes is regulated by pyruvate and essential for ZGA [53]. Interestingly, our lactylome analysis of ESCs identified 27 lactylated mitochondrial proteins, such as CS, succinate dehydrogenase complex flavoprotein subunit A (Sdha) and Malate dehydrogenase 2 (Mdh2) (Table S1). Among these latctylated proteins, Cs, Sdha, and Mdh2 are translocated into the nucleus of 2C embryos and/or PSCs [53, 54]. Whether lactylation regulates the nuclear translocation of TCA cycle enzymes is worth investigating.

So far, it has been demonstrated that histone acetyltransferase, p300 and CBP, and histone deacetylase, HDAC1-3 and SIRT1-3, catalyze lactylation and delactylation, respectively [18, 32, 55,56,57,58]. It is very likely these histone acetyltransferase and histone deacetylase are also responsible for the lactylation and delactylation of Hdac1. However, further experiments are required to identify the exact lactylation writer and eraser of Hdac1 K412 and K438.

In summary, our results reveal that glycolysis-derived lactate promotes Hdac1 lactylation, silences 2C genes and prevents the pluripotent-to-2C transition, providing a novel mechanistic link between glycolysis and cell fate regulation.

Conclusions

In conclusion, this study 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.

Data and code availability

Sequencing data generated in this study have been deposited into the Gene Expression Omnibus database under accession numbers GSE247959 (RNA-seq, the access token gtgliomgnjgllqx) and GSE247958 (ChIP-seq, the access token crshimykpjuplkj).

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Acknowledgements

This study was supported by the National Key R&D Program of China (Grant No. 2021YFA1101002), and the 111 Project Grant (B08011).

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Authors and Affiliations

  1. 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

    Qiman Dong, Xiaoqiong Yang, Lingling Wang, Qingye Zhang, Nannan Zhao, Shanshan Nai, Xiaoling Du & Lingyi Chen

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  1. Qiman Dong
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Contributions

Q.D., X.Y., L.W., N.Z., Q.Z., S.N. and X.D. performed experiments, Q.D. analyzed the data and contributed to the paper writing, L.C. designed the experiments and wrote the paper.

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Correspondence to Lingyi Chen.

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All procedures and protocols have been reviewed and approved by the Nankai University Animal Care and Use Committee. Ethics has been reviewed under the title “Application Form for Ethical Review of the Use of Experimental Animals” on December 20,2021 (Approval Number: 2021-SYDWLL-000469).

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Supplementary Information

13287_2024_4027_MOESM1_ESM.docx

Additional file 1: Figure S1. The extracellular lactate level decreases upon SO treatment, related to Figure 1. (A) Bar graph of extracellular lactate levels in control or SO-treated ESCs. (B) Bar graph of intracellular lactate levels in control or SO-treated ESCs. n=3. Data are presented as average ± SD. Statistical analysis was performed with unpaired two-tailed Student’s t test. **, p < 0.01. Figure S2. Construction of Ldha and Ldhb knockout ESC lines, related to Figure 2. (A) The expression levels of Ldha, Ldhb, Ldhc and Ldhd in ESCs analyzed from ¬¬¬¬RNA-seq data. (B) A schematic illustration of the experimental d-esign to knock out Ldha. Blue rectangles represent the exons of Ldha. The sgRNA targeting sequence is shown, and the protospacer-adjacent motif (PAM) is marked in red. (C) Sequencing chromatograph of the Ldha gene around the Cas9 targeting site in Ldha KO-1 and KO-2 ESCs. Red triangles mark the indel mutations. (D) Sequence alignment of the Ldha gene around the Cas9 targeting site in E14 and Ldha KO ESCs. (E) A schematic illustration of the experimental design to knock out Ldhb. (F) Sequencing chromatograph of the Ldhb gene around the Cas9 targeting site in Ldhb KO-1 and KO-2 ESCs. (G) Sequence alignment of the Ldhb gene around the Cas9 targeting site in E14 and Ldhb KO ESCs. (H) The expression of Kla and Ldhb in E14 and Ldhb KO ESCs, detected by Western blot. Figure S3. Deletion of Ldhb does not affect the expression of 2C genes, related to Figure 2. E14 and Ldha KO ESCs were subjected to RNA-seq (A-C) and qRT-PCR (D). (A) Scatterplot of gene expression in E14 and Ldhb KO ESCs. (B) Scatterplot of retroelement expression in E14 and Ldhb KO ESCs. Parallel diagonal lines indicate the two-fold threshold in expression difference. Red dots mark genes with more than 2-fold upregulation upon Ldhb KO, while green indicates genes with a greater than 2-fold reduction in expression. (C) GSEA shows the activation of 2C gene set in Ldhb KO ESCs. (D) qRT-PCR analysis of 2C genes in E14 and Ldhb KO ESCs. For qRT-qPCR, n=3. Data are presented as average ± SD. Statistical analysis was performed with one-way ANOVA. Figure S4. SO treatment or Ldha KO activates 2C genes and decreases global lactylation in J1 ESCs, related to Figure 2. (A) The expression of Kla in J1 and 20 mM SO-treated ESCs, detected by Western blot. (B) qRT-PCR analysis of 2C genes in J1 and 20 mM SO-treated ESCs. Statistical analysis was performed with unpaired two-tailed Student’s t test. (C) A schematic illustration of the experimental design to knock out Ldha. Blue rectangles represent the exons of Ldha. The sgRNA targeting sequence is shown, and the PAM is marked in red. (D) Sequencing chromatograph of the Ldha gene around the Cas9 targeting site in Ldha KO-1 and KO-2 ESCs. Red triangles mark the indel mutations. (E) Sequence alignment of the Ldha gene around the Cas9 targeting site in J1 and Ldha KO ESCs. (F) The expression of Kla and Ldha in J1 and Ldha KO ESCs, detected by Western blot. Quantification of tracks is shown in the right. Regions with reduced Kla signal are highlighted with vertical lines. (G) qRT-PCR analysis of 2C genes in J1 and Ldha KO ESCs. Statistical analysis was performed with one-way ANOVA. For qRT-PCR, n=3. Data are presented as average ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Figure S5. Validation of Hdac1 KO ESCs, related to Figure 5. (A) Sequencing chromatograph of the Hdac1 gene around the Cas9 targeting site in Hdac1 KO-1 and KO-2 ESCs. (B) Sequence alignment of the Hdac1 gene around the Cas9 targeting site in E14, and Hdac1 KO ESCs. Figure S6. Analyzing H3K18la and H3K27ac in WT, KQ and KR ESCs, related to Figure 7. (A) Western blot analysis for H3K18la levels in E14, Hdac1 KO-1, WT, KQ, and KR ESCs. The specific band of H3K18la is marked by a red asterisk. (B) The number of clean reads from WT, KQ and KR ESC samples. (C) The count of peaks in WT, KQ and KR ESCs. (D) Scatterplot depicting RPKM normalized ChIP-seq peaks at TSSs in WT, KQ, and KR ESCs. Centre lines show mean values. (E) Scatterplot depicting RPKM normalized ChIP-seq peaks at 2C gene TSSs in WT, KQ, and KR ESCs. Centre lines show mean values. (F) H3K27ac ChIP-seq tracks at 2C gene loci, Dppa2 and Zfp668, in WT, KQ, and KR ESCs. (G) ChIP-qPCR quantification of Hdac1 binding at Zscan4 loci in WT, KQ, and KR ESCs. For ChIP-qPCR, n=3. Data are presented as average ± SD. Statistical analysis was performed with one-way ANOVA. *, p < 0.05; ***, p < 0.001. Figure S7. Exogenous lactate might activate other signaling pathway(s) to increase the expression of 2C genes, related to discussion. (A) qRT-PCR analysis of 2C genes in E14 ESCs treated with or without 20 mM L-lactate. (B) The expression of Kla in E14 ESCs treated with or without 20 mM L-lactate, detected by Western blot. (C) qRT-PCR analysis of 2C genes in E14 ESCs treated with or without 50 mM NALA. (D) The expression of Kla in E14 ESCs treated with or without 50 mM NALA, detected by Western blot. For qRT-qPCR, n=3. Data are presented as average ± SD. Statistical analysis was performed with unpaired two-tailed Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Figure S8. The expression level of Hdac1 in different pre-implantation stages and mESCs, related to discussion. The expression level of Hdac1 in different pre-implantation stages and mESCs (accession number GSE165782). FGO, full-grown oocytes; LP1, late prometaphase I oocytes; MII, metaphase II oocytes; PN3, early one-cell stage; PN5, late one-cell stage; E2C, early two-cell stage; L2C, late two-cell stage; 4C, four-cell stage; 8C, eight-cell stage; ICM, inner cell mass.

Additional file 2.

13287_2024_4027_MOESM3_ESM.xlsx

Additional file 3: Table S1. Lactylated sites and proteins identified by lactylome analysis (Table S1.xlsx). Table S2. Primers used in this study.

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Dong, Q., Yang, X., Wang, L. et al. Lactylation of Hdac1 regulated by Ldh prevents the pluripotent-to-2C state conversion. Stem Cell Res Ther 15, 415 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-024-04027-1

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Stem Cell Research & Therapy

ISSN: 1757-6512