Lactate promotes H3K18 lactylation in human neuroectoderm differentiation

Abstract

In mammals, early embryonic gastrulation process is high energy demanding. Previous studies showed that, unlike endoderm and mesoderm cells, neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process, which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/β-catenin signaling during neuroectoderm differentiation. Notably, histone lactylation, especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments, we further identified that four genes, including PAX6, were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-024-05510-x.

Introduction

Mammalian early embryo gastrulation is a complex and dynamic process characterized by rapid cell proliferation and morphogenesis to establish the three primary germ layers: ectoderm, mesoderm, and endoderm [1, 2]. This process is high energy demanding and thus energy metabolism plays a fundamental role in supporting embryonic development and influencing their fate decisions [3–5]. Recent studies have highlighted the intricate relationship between metabolic regulation and cell differentiation. Both nutrient availability and metabolite concentrations, can influence cellular signaling pathways, gene expressions, and epigenetic modifications, thereby impacting cell fate decisions [6–9].

Therefore, it is important to study how energy metabolism influence early embryonic development process. However, it is very difficult to investigate early embryonic development process in vivo; while in vitro human embryonic stem cells (hESCs) differentiation models provided very useful information. HESCs are derived from the inner cell mass of early blastocysts with the full potential to differentiate into all embryonic cell lineages [10]. They can differentiate into all three germ layers in vitro and energy metabolism exhibits dramatic changes during in vitro differentiation process. HESCs use glycolysis as the major energy metabolic process, then endoderm and mesoderm undergo a metabolic transition to oxidative phosphorylation [11–13]. Surprisingly, neuroectoderm retains their reliance on aerobic glycolysis [14]. Glycolysis generates ATP and pyruvate under aerobic conditions or lactate under anaerobic conditions [15]. Notably, both hESCs and neuroectoderm cells, even with abundant oxygen, generate high level of lactate as the final product [16].

In recent years, lactate has been reported as a key metabolite not only involved in central carbon metabolism for biomass synthesis and energy production [17, 18], but also in tumorigenesis [19, 20], neurodevelopment [18, 21], and immunological responses [22, 23]. Moreover, lactate could also act as a precursor for histone lysine lactylation [24], and such epigenetic modification can regulate gene transcription in various biological processes [25–28]. Therefore, we hypothesized that lactate might act as an important regulator in early embryonic development, especially in neuroectoderm cells, which maintained high level of glycolysis.

In this study, by using two hESCs differentiation models, we found that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Notably, histone lactylation, especially H3K18la was significant upregulated during this process. Genome-wide analysis revealed that H3K18la is highly concentrated at gene promoters, and we further identified four genes upregulated by lactate during neuroectoderm differentiation. We verified a H3K18la modification site at PAX6 promoter and exogenous lactate could also rescue the level of PAX6 mRNA after shPAX6 inhibition.

Method and materials

Human stem cell culture

H9 (WA09) human embryonic stem cells were cultured in mTESR1 media (cat. # 85850, StemCell Technologies) on six-well plates pre-coated with diluted Matrigel (cat. # 354277, Corning). Medium was replenished daily. hESCs were passaged every 4–6 days at a split ratio of 1:4 to 1:8 using EDTA (cat. # CA3001500, Cellapy). H9-derived neuroectoderm were generated following neural progenitor induction protocol and cultured in STEMdiff Neural Induction Medium (cat. # 05835, StemCell Technologies) with different supplements including 25 mM sodium L-lactate (cat. # L7022, Sigma-Aldrich), 10 µM GSK2837808A (cat. # HY-100681, MCE), and 10 µM FX-11 (cat. # HY-16214, MCE). Medium was replenished daily. All cells were cultured at 37 °C in 20% O₂ and 5% CO₂.

Spontaneous and induced hESCs differentiation

To set up differentiation models, hESCs were dissociated with Accutase (cat. # A1110501, Gibco) when reached a growth confluence of ~ 80%, and seeded as single cells at a density of 200,000 cells/cm² in 12-well plates pre-coated with diluted Matrigel (cat. # 354277, Corning) in different medium. For spontaneous differentiation model, unconditioned DMEM/F12 medium (composed of 80% DMEM/F12, 20% KOSR, 1% GlutaMAX and 1%NEAA) were used. For induced differentiation model, STEMdiff Neural Induction Medium were used. On the day cells were set up for differentiation, it was designated as ‘Day 0’. Rock inhibitor Y-27,632 (cat. # HY-10071, MCE) was added at Day 0 in both models and changed to fresh medium without Y-27,632 after one day. Medium was replaced daily in both models to support cell differentiation.

qRT-PCR

Total RNA was extracted from cells with TRIzol reagent (cat. # 15596026, Invitrogen) and chloroform, precipitated with isopropanol, washed by 70% RNase-free ethanol and reverse transcribed into cDNA. 1 µg RNA were used to reverse to cDNA with the HiScript II Q RT SuperMix (cat. # R222, Vazyme) according to the manufacture’s protocol. Then the cDNAs were used for qRT-PCR experiments with ChamQ Universal SYBR qPCR Master Mix (cat. # Q711, Vazyme) and performed on The LightCycler^® 480 II Real-Time PCR System (Roche). Relative expression of each target was calculated with human GAPDH as benchmark based on the delta Ct method. All qRT-PCR experiments were carried out at least three replicates. Comparison between samples was performed using Student’s t test. The primers used in the qRT-PCR assays are listed in Supplementary Table S1.

Histone extraction

Histones from cells were extracted using a standard acid-extraction protocol [29]. Cells were grown in 6-well plates pre-coated with diluted Matrigel, and harvested on ice using hypotonic lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl₂, and 1 mM DTT) containing Protease Inhibitor Cocktail (cat. # GK10014, GLPBIO) for nuclei extraction. The nuclei were resuspended in 0.2 M HCl with no clumps left in solution, and incubated on rotator at 4 °C for at least 30 min or overnight, then followed by centrifugation at 16,000 g for 10 min at 4 °C to collect histones in supernatants. After adjusting pH to 7.5 by 1 M Tris-HCl pH 8.0, the histone pellet was prepared for western blotting analysis.

Western blotting

Protein lysates were collected and mixed with 5 x SDS-PAGE loading buffer and denatured at 95 °C for 5 min. Proteins were analyzed with 12% SDS–PAGE and then transferred to a polyvinyl difluoride membrane (Millipore). The blots were probed with primary antibodies at 4 °C overnight and then labeled by appropriate secondary antibodies. Blotting images were acquired with a Tanon-5200 imaging system. Densitometric quantification of protein bands was calculated by ImageJ software and the relative expression of proteins or modifications was compared to the loading control. At least three biological replicates were performed. Detailed information on the primary and secondary antibodies used for Immunoblotting is summarized in Table S2.

Immunofluorescence

Human stem cells were seeded on removable 9 mm round coverslips (cat. # BS-09-RC, biosharp) pre-coated with diluted Matrigel in 48-well plates. Cells were fixed in 4% (w/v) paraformaldehyde at RT for 10 min, and then gently rinsed 1–3 times with phosphate-buffered saline (PBS). After fixed, cells were incubated with primary antibodies diluted in blocking buffer (5% (v/v) goat serum and 0.3% (v/v) Triton X-100 in PBS) at 4℃ overnight and then labeled by appropriate secondary antibodies and fluorescent dyes diluted in blocking buffer at RT for 2 h. All images were acquired using Zeiss LSM700 confocal microscopes. ImageJ was used to perform quantitative analysis of the ratio of indicated positive cells. At least three biological replicates were performed. Detailed information on the primary and secondary antibodies used for immunostaining analysis is summarized in Table S2.

Measurement of lactate concentration

Cultured cells were harvested by cell scraping and washed with cold PBS, following by homogenization with Lactate Assay Buffer, and then centrifugated at 13,000 rpm for 10 min at 4 °C. The supernatants were collected for further test. The culture medium was also collected, centrifuged (13,000 rpm at 4 °C for 10 min) to remove cellular debris and diluted 10x in Lactate Assay Buffer. Then the intracellular or extracellular lactate concentration was measured using a L-Lactate Assay Kit (cat. # ab65330, Abcam) according to the manufacturer’s instructions. At least three biological replicates were performed.

Cell viability assay

Cells were grown in 96-well plate pre-coated with diluted Matrigel and treated with different exogenous supplementary. Cell viability was analyzed by CCK-8 cell counting kits (cat. # A311-01, Vazyme) according to the manufacturer’s instructions. Cells were incubated with 10 µl CCK-8 reagents for 2 h in cell incubator, and detected at 450 nm absorbance by a Multimode Plate Reader (cat. #HH34000000, PerkinElmer). At least three biological replicates were performed. Cell viability was represented as a percentage of the control.

CUT&Tag-seq analysis

CUT&Tag was performed with CUT&Tag-IT Assay Kit (cat. # 53160, ActiveMotif) according to the manufacturer’s instructions. Cultured cells were collected and bounded to Concanavalin A–coated beads at RT for 10 min. Subsequently, cells were resuspended in antibody buffer and incubated with primary antibodies against H3K18la at 4℃ overnight and Guinea pig-anti rabbit IgG in Dig-wash buffer for 1 h at RT in order. Samples were then mixed with pA-Tn5-adapter transposases (pA-Tn5) following by transposon activation and DNA fragmentation. After that, DNA was isolated, amplified, and purified to construct the CUT&Tag libraries for sequencing. The libraries were sequenced on an Illumina NovaSeq 6000 platform. The reads were then aligned to the human genome (hg38) using the BWA-MEM program.

RNA-seq analysis

For RNA sequencing, total RNA samples were collected by TRIzol reagent as described in the “qRT-PCR” section. RNA quality was confirmed using a Qubit 4.0 fluorometer to determine concentration and total yield, an Agilent 2100 Bioanalyzer to evaluate RNA integrity, and a NanoDrop spectrophotometer to measure RNA purity. After that, mRNA was enriched by Oligo(dT) beads, reversed, and purified to construct the cDNA libraries for sequencing. The libraries were sequenced on a DNBSEQ-T7 platform. RNA-seq reads were aligned to the human genome (hg19) with STAR [30]. Differential gene analysis was performed using DESeq2 (fold change ≥ 2 and p_adjust < 0.05) [31].

Native qChIP

Native ChIP was performed in two biological replicates following a previous described protocol with minor changes [32]. Cells were grown in 6-well plates pre-coated with diluted Matrigel. Isolated nuclei in Native ChIP were extracted by NIB 400 buffer (10 mM Tris-HCl pH 7.5, 60 mM KCl, 1.5 mM MgCl₂, 1 mM CaCl₂, 0.25 M Sucrose, 10% (v/v) Glycerol, 400 mM NaCl, 1mM DTT) with Protease Inhibitor Cocktail, and incubated with 3 µl of MNase (0.2 U/µl) (cat. # N3755, Sigma-Aldrich) at 28 °C for 15 min to digest chromatin. Then, the sample was immunoprecipitated with appropriate antibody conjugated Protein A/G agaroses at 4℃ overnight, to enrich mono- and di-nucleosomes size DNA. Subsequently, the sample was followed by series washing procedures in NIB 500 solution (10 mM Tris-HCl pH 7.5, 60 mM KCl, 1.5 mM MgCl₂, 1 mM CaCl₂, 0.25 M Sucrose, 10% (v/ v) Glycerol, 500 mM NaCl), Lithium solution (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.25 M LiCl, 0.5% (v/v) Na-deoxycholate, 10% (v/ v) NP-40), and TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0), and then eluted in freshly prepared ChIP direct elution buffer (10 mM Tris-HCl pH 7.5, 5mM EDTA pH 8.0, 300 mM NaCl, 0.5% (v/ v) SDS), with RNase A digestion at 37 °C for 30 min. DNA extraction was followed phenol/chloroform/isoamyl alcohol (PCI) protocol after proteinase K digestion, and DNA was precipitated by TE buffer. qChIP was performed for PAX6 promoter.

Human reference genome and other external data used for comparative analysis

CUT&Tag data was mapped to hg38 and RNAseq data was mapped to hg 19. Public RNAseq-DEGs were generated by intersection analysis of published data GSE161531 [33] (GSM4909467, GSM4909468, GSM4909469, GSM4909470, GSM4909471, GSM4909472) and GSE137129 [34] (GSM4067415, GSM4067416, GSM4067421, GSM4067422). ATAC-seq data used for comparative analysis were analyzed from published data GSE174727 (GSM5324891, GSM5324892, GSM5324893, GSM5324894, GSM5324897, GSM5324898).

Quantification and statistical analysis

All experiments were repeated at least three times, and the statistical significance was evaluated. All data are expressed as mean ± standard error of the mean (SEM) unless otherwise described. Statistical analyses were performed using GraphPad Prism software (version 8.0). The significance of differences was assessed by two-tailed unpaired Student’s t test for two datasets and one-way ANOVA followed by Bonferroni post-hoc test for multiple datasets. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, # < 0.0001, and ns for not significant. A Kolmogorov-Smirnov test was performed to assess the compare the distribution of H3K18la peaks between the Day 0 cand Day 5 cells.

Results

Intracellular lactate was increased during hESCs spontaneous differentiation

HESCs are maintained in mTESR1 media and they can spontaneously differentiate into three germ layer cells in vitro in DMEM/F12 media [35] (Fig. 1A). Therefore, we firstly investigate the function of lactate in this spontaneous differentiation model. After 48 h of differentiation, the intracellular lactate levels in those cells were measured by colorimetric assays and the results showed that the level of lactate was increased approximately two-fold compared to that in the undifferentiated cells (Fig. 1B). In comparison, both the intercellular and extracellular lactate levels were not changed in hESCs culture when those cells were maintained as stem cells (Figure S1A). qRT-PCR analysis on the differentiated cells confirmed that the expression of pluripotency markers OCT4 (also known as POU5F1) and NANOG were significantly decreased after 48 h (Figure S1B), while the expressions of neuroectoderm markers PAX6 and SOX1 (Figure S1C) and mesoderm markers TBXT and HAND1 (Figure S1D) were significantly increased. No significant change was observed in endoderm markers SOX17 during differentiation (Figure S1E).

Fig. 1.

Exogenous lactate promotes hESCs differentiation towards neuroectoderm in the spontaneous differentiation model. (A) Illustration for the hESCs spontaneous differentiation procedure. (B) Relative intracellular lactate levels of Day 0 cells and Day 2 cells in the spontaneous differentiation model (n = 3). (C) 25 mM Sodium lactate or 10 µM LDHA inhibitors were added at Day 0 as illustrated. (D) Relative intracellular lactate levels of Day 2 cells with either sodium lactate or LDHA inhibitors treatments (n = 3). (E-H) qRT-PCR analysis of OCT4, NANOG, PAX6, SOX1, TBXT, HAND1, and SOX17 mRNA expression in Day 2 cells with sodium lactate or Gska treatment (n = 4)

As the intercellular lactate level was increased during hESCs spontaneous differentiation, we used sodium lactate or LDHA inhibitors (Gska or Fx11) to change the intracellular lactate during hESCs spontaneous differentiation (Fig. 1C). Again, the intracellular lactate level was examined after 48 h and the results confirmed that sodium lactate treatment significantly elevated the intracellular lactate level, with an increase of approximately eight-fold; whereas Gska or FX11 treatment significantly suppressed the intracellular lactate level to about 15% of that in the control cells (Fig. 1D). We further analyzed the expression of multiple pluripotency markers and lineage-specific genes under these conditions. Exogenous lactate significantly upregulated the expression of neuroectoderm markers PAX6 and SOX1 with a nearly three-fold increase (Fig. 1F) and downregulated the expression of mesoderm marker HAND1 by 84% (Fig. 1G). No significant changes were observed in the expression of TBXT and SOX17 (Figure S1G&H). At the same time, exogenous lactate partially attenuated the loss of pluripotency markers OCT4 and NANOG during spontaneous differentiation as they were higher in lactated treated cells and untreated cells (Fig. 1E). On the other hand, Gska treatment had no significant effect on most markers except a slight decrease in NANOG expression (Fig. 1E-H).

Collectively, these results showed that exogenous lactate could boost the intracellular lactate level and facilitate the spontaneous differentiation of hESCs towards the neuroectoderm.

Exogenous lactate promoted neuroectoderm differentiation in the induced differentiation model

We observed that exogenous lactate could facilitate the spontaneous differentiation of hESCs towards the neuroectoderm. However, there are mixed cell populations in this spontaneous model. HESCs can be specifically induced to neuroectoderm cells by SMAD inhibition [14, 36] (Fig. 2A). Again, we first confirmed that the induced differentiation was successful in our hands. The decrease of pluripotent marker OCT4 and the increase of neuroectoderm marker PAX6 in this induced model were confirmed by qRT-PCR, western blotting, and immunofluorescent experiments (Figure S2A-C). We also checked the intracellular lactate level at Day 5, at which most cells have differentiated into neuroectoderm cells. The results showed that the intercellular lactate at Day 5 was approximately four-fold of that in Day 0 undifferentiated cells (Fig. 2B). This result was in consistent with what we had observed in the spontaneous differentiation model. We examined the expression of PDHA1, LDHA, and LDHB in this induced differentiation model and the results showed that the expression of LDHA was significantly increased at Day 5, while the levels of PDHA1 and LDHB were significantly decreased at Day 5 (Fig. 2C). Such expression pattern suggested that more pyruvate might be catalyzed to lactate in those neuroectoderm cells.

Fig. 2.

Exogenous lactate promotes neuroectoderm differentiation in the induced differentiation model. (A) Illustration for the induced neuroectoderm differentiation model from hESCs. (B) Relative intracellular lactate levels of Day 0 cells and Day 5 cells in the induced differentiation model (n = 3). (C) qRT-PCR analysis of LDHA, LDHB, and PDHA1 mRNA expression in Day 0, Day 3, and Day 5 cells during neuroectoderm differentiation (n = 4). (D) 25 mM Sodium lactate or 10 µM LDHA inhibitors were added at Day 0 as illustrated. (E) Relative intracellular lactate levels of Day 5 cells with sodium lactate or Gska treatment (n = 3). (F-G) qRT-PCR analysis of OCT4, NANOG, PAX6, and SOX1 mRNA expression in Day 5 cells treated with sodium lactate or Gska (n > 5)

Again, we used sodium lactate and Gska to change the intercellular lactate level in this model (Fig. 2D). The intracellular lactate level was significantly upregulated about nine-fold by sodium lactate and reduced to nearly 15% upon Gska treatment (Fig. 2E). Before we examined the effect of intercellular lactate on differentiation, we tested whether changes on lactate levels affect cell viability, proliferation, and apoptosis. Neither sodium lactate nor LDHA inhibitors (Gska or Fx11) had any significant influence on the viability of cells in this model (Figure S3A). No obvious effect on cell numbers was observed under different treatments (Figure S3B). Immunofluorescent experiments with antibodies against cleaved caspase 3 (apoptosis marker) or Ki67 (proliferation marker) on Day 5 cells also revealed no significant changes upon those treatments (Figure S3C-D). Therefore, neither cell proliferation nor apoptosis was affected by those treatments.

Then, we examined the effect of sodium lactate or Gska on neuroectoderm differentiation in this induced differentiation model. The expression of neuroectoderm markers PAX6 and SOX1 were both significantly increased by sodium lactate treatment in Day 5 cells (Fig. 2G); while the expression of pluripotency markers OCT4 and NAONG were decreased to about 50% (Fig. 2F). We did not observe any significant changes upon Gska treatment compared to the control cells (Fig. 2F&G). Immunostaining experiments for OCT4 and PAX6 on Day 5 cells also confirmed these results (Figure S4A&B). As the expression of OCT4 was no longer detectable in Day 5 cells, we tested OCT4 expression in Day 3 cells, and found that sodium lactate treatment indeed downregulated the ratio of OCT4 positive cells, while Gska treatment had no significant effects (data not shown). Together, our results suggested that exogenous lactate further promotes neuroectoderm differentiation without affecting cell viability, proliferation or apoptosis.

Intracellular lactate did not affect BMP or WNT/β-catenin signaling

Differentiation of hESCs to neuroectoderm requires inhibition of BMP signaling and low WNT/β-catenin signaling [36, 37]. It has been reported that lactate can act as a signaling molecule to activate WNT/β-catenin signaling [38, 39], we wonder whether the intracellular lactate level could affect WNT/β-catenin and BMP signaling during neuroectoderm differentiation in our models. Therefore, we examined the levels of phosphorated-SMAD1/5 and active β-catenin by western blot to analyze the activity of BMP and WNT/β-catenin signaling. Neither sodium lactate nor Gska treatment had any effect on the levels of phosphorated-SMAD1/5 or active β-catenin in Day 5 cells (Figure S5A&B) or Day 2 cells (Figure S5C&D) of the induced differentiation model. Moreover, we also analyzed the activity of BMP and WNT/β-catenin signaling on the spontaneous differentiation model. Again, neither sodium lactate nor Gska treatment had any effect on the expression of phosphorylated SMAD1/5 and active β-catenin compared to the control cells (Figure S5E&F). Collectively, our results suggested that changes on the intracellular lactate level do not affect the BMP or WNT/β-catenin signaling during neuroectoderm differentiation.

Histone lactylation was increased during differentiation

It has been reported that lactate is the precursor for lysine lactylation [24, 40], and especially exposed lysine of histone can be lactylatized [24]. Therefore, it is possible that the intracellular lactate might affect the level of protein lactylation, including histone lactylation. We first analyzed the Pan-lysine lactylation levels during induced neuroectoderm differentiation. Besides a band at approximately 70 kDa was decreased in differentiated cells compared to undifferentiated hESCs, there were no significant changes in overall Pan-lysine lactylation during this process (Fig. 3A). On the other hand, the Pan-histone lysine lactylation level (Pan HKla) was significantly increased in neuroectoderm compared to that in the hESCs (Fig. 3B&C). It has been reported that H3K18 and H4K12 are important sites for histone lactylation [25–27], therefore we further analyzed the lacylation levels of H3K18 (H3K18la) and H4K12 (H4K12la) by western blot. The results showed that the level of H3K18la were significantly increased in neuroectoderm compared to that in hESCs; while no significant change was detected in the levels of H4K12la (Fig. 3B&C). We also examined the levels of histone lactylation in induced differentiation model upon sodium lactate or Gska treatment. At Day 5, the level of Pan HKla was not significantly affected by sodium lactate treatment; while the levels of H3K18la and H4K12la were upregulated about 1.7 and 1.2 folds, respectively (Fig. 3D&E). Gska treatment can approximately decrease the Pan HKla level to 56%, H3K18la level to 69%, and H4K12la level to 64%, respectively (Fig. 3D&E). These results indicated that changes on intracellular lactate level could affect histone lactylation, especially H3K18la levels during neuroectoderm differentiation.

Fig. 3.

Intracellular lactate modulates histone lactylation during neuroectoderm differentiation. (A) Western blotting analysis of Pan lysine lactylation in Day 0, Day 3, and Day 5 cells from the induced neuroectoderm differentiation model (representative graphs of four independent experiments). (B) Representative graphs of Pan HKla, H3K18la, and H4K12la in Day 0, Day 3, and Day 5 cells during the induced neuroectoderm differentiation by western blotting analysis. Quantification of their protein levels were shown in (C) (n = 4). (D) Representative graphs of Pan HKla, H3K18la and H4K12la in Day 5 cells with sodium lactate or Gska treatments in the induced differentiation model by western blotting analysis. Quantification of their protein levels were shown in (E) (n = 3)

Genome-wide analysis of H3K18la during neuroectoderm differentiation showed it was enriched at promoter regions and engaged in gene transcription

Since exogenous lactate could promote histone lactylation, especially H3K18la level during neuroectoderm differentiation, we performed CUT&Tag experiment on cells from induced differentiation model by using H3K18la antibodies to analyze genome-wide chromatin locations of H3K18la. H3K18la peaks were found in over 15,000 genes in both Day 0 and Day 5 cells (Figure S6A), suggesting that the H3K18la modifications were ubiquitous in genome during neuroectoderm differentiation. Further analysis revealed a predominantly enrichment of H3K18la peaks at transcriptional start sites (TSSs), with over 40% of the peaks located within the 3 kb promoter regions (Fig. 4A and S6B). Analysis with deeptools [41] also showed that the enrichment peaks at TSS in Day 5 cells was higher than that in Day 0 cells (Fig. 4B). To elucidate the potential epigenetic impacts of H3K18la in this process, we further analyzed the CUT&Tag data and identified 2,172 genes exhibiting significant changes in H3K18la modification levels between Day 0 and Day 5 cells (Pvalue < 0.05, abs (log2FoldChange) > 0.8). Gene ontology (GO) analysis showed that those genes were enriched in neural development related pathways, including axon development, axonogenesis, and modulation of chemical synaptic transmission (Fig. 4C).

Fig. 4.

Genome-wide analysis of H3K18la during neuroectoderm differentiation. (A) The density heatmap of H3K18la binding peaks in Day 0 and Day 5 cells during neurectoderm differentiation which was visualized by the deeptools tool and ordered by signal intensity. (B) H3K18la peaks distribution of Day 0 and Day 5 cells; KS-test & T-test < 2.2e-16. (C) GO analysis (biological process) of 2,172 genes with the significantly different H3K18la binding peaks between Day 0 and Day5 cells. (D) Overlap of our RNAseq-DEGs and H3K18la genes were analyzed and showed in Scatterplot (left) and bar plot (right). 275 genes were divided into 4 groups (H3K18la-up & activated; H3K18la-up & repressed; H3K18la-down & activated; H3K18la-down & repressed) by their H3K18la levels (log2[Day 5/ Day 3]) and transcriptional levels (log2[Day 5/ Day 0]). (E) Similar analysis was performed on published RNA-seq datasets. (F) Intersection analysis for “H3K18la-up & activated” genes between two datasets: 93 genes from (D) and 46 genes from (E). (G) The heatmap showed the qRT-PCR analysis of the expression of PLK3, PAX6, TMEM169, PCDH9, RUSC2, and C1QTNF6 in Day 0, Day 3, and Day 5 cells from the induced differentiation model (n = 4). (H) qRT-PCR analysis of mRNA expressions of PAX6, TMEM169, RUSC2, C1QTNF6 and PCDH9 in Day 5 cells treated with sodium lactate or Gska from the induced differentiation model (n = 4)

To gain insights into the transcriptional landscape during hESCs differentiation towards neuroectoderm, we also conducted RNA sequencing (RNA-seq) analysis of Day 0 and Day 5 cells from induced differentiation model. 3,874 genes were identified as differentially expressed genes (DEGs) under the threshold of P value < 0.05 and a fold-change cutoff of absolute value of abs (log2FoldChange) > 1 (Figure S6C). In order to identify genes potentially regulated by H3K18la modification in neuroectoderm differentiation, we performed an intersection analysis of H3K18la genes (2172 genes) and RNAseq-DEGs (3,874 genes). There are 275 genes exhibiting both robust changes in transcript abundance and H3K18la modification (Figure S6F). Based on their expression patterns and H3K18la levels, these 275 genes were further classified into four groups: H3K18la-up & activated (93 genes), H3K18la-up & repressed (49 genes), H3K18la-down & activated (34 genes), and H3K18la-down & repressed (99 genes) (Fig. 4D). We also downloaded two published transcriptome datasets of hESCs induced differentiation (GSE161531 [33] and GSE137129 [34]) and performed similar analysis. 2,739 DEGs from intersection of GSE161531 and GSE137129 were identified after applying a significance threshold of P value < 0.05 and a fold-change cutoff of absolute value of abs (log2FoldChange) > 1 (Figure S6D&E). After intersection analysis with 2172 H3K18la genes, there are 183 genes exhibiting both robust changes in transcript abundance and H3K18la modification (Figure S6G). They also can be further divided into four groups: H3K18la-up & activated (46 genes), H3K18la-up & repressed (37 genes), H3K18la-down & activated (33 genes), and H3K18la-down & repressed (67 genes) (Fig. 4E). Since we observed that the level of H3K18la was elevated during differentiation, and H3K18la was previously reported as an activator to gene transcription [24, 25], we focused on the “H3K18la-up & activated” genes. After intersection analysis of the 93 H3K18la-up & activated genes from our RNA-seq data and the 46 H3K18la-up & activated genes from public RNA-seq data, there are 19 genes shared by both groups (Fig. 4F). As there was enriched H3K18la signals at the promoter regions, we looked at the H2K18la sites on those 19 genes. Within these 19 genes, there are 6 genes have H3K18la at the promoter regions, which are PAX6, PLK3, TMEM169, RUSC2, PCDH9 and C1QTNF6 (Fig. 4F).

Next, we verified the mRNA expression pattern of those 6 genes during neuroectoderm differentiation by qRT-PCR. Except PLK3, the other 5 genes were confirmed to be upregulated in induced neuroectoderm differentiation (Fig. 4G). As sodium lactate treatment can promote the level of H3K18la, we further tested the expression levels of these 5 genes after sodium lactate treatment. The results showed that the transcription levels of 4 genes, PAX6, TMEM169, RUSC2, and C1QTNF6, could be significantly upregulated by sodium lactate (Fig. 4H).

Moreover, an open chromatin conformation is essential for gene transcriptional process [42]. Therefore, we analyzed chromatin accessibility from published ATAC-seq data (GSE174727), which was induced neuroectoderm differentiation from human induced pluripotent stem cells (iPSCs) [33]. We found that H3K18la modifications of all these 4 genes (PAX6, TMEM169, RUSC2, and C1QTNF6) are located within accessible chromatin regions of their respective promoters in neural stem cells (NSCs) or neural progenitor cells (NPCs), but not in iPSCs (Fig. 5). These findings suggested that the accessibility of chromatin at these gene promoters may facilitate H3K18la-mediated transcriptional activation during neuroectoderm differentiation.

Fig. 5.

H3K18la modifications were located within accessible chromatin regions. Genome browser tracks of H3K18la CUT&Tag signal (H3K18la antibody) loci at the promotors of 4 genes (PAX6, TMEM169, RUSC2, and C1QTNF6) were presented here. Day 0 and Day 5 cells are from the induced differentiation model. Open chromatin changes between iPSCs, in vitro-differentiated NPCs, and commercially available NSCs were defined by reported ATAC-seq; ATAC. Yellow box highlights regions where H3K18la overlaps open chromatin on those gene promoters. Scale bar, 1 kb

H3K18la positively regulates PAX6 transcription

PAX6 exhibited a particular higher elevation the mRNA level during induced differentiation (Fig. 4G) and upon lactate treatment compared to other genes (Fig. 4H). Therefore, we focused on PAX6 in the following study. PAX6 a is a key transcriptional factor to induce neurogenesis [43, 44]. The loss of PAX6 could also impair human neuroectoderm differentiation in vitro [45]. We further explored how intracellular lactate promoting PAX6 transcription. To confirm the H3K18la level at PAX6 promoter, we also performed a quantitative chromatin immunoprecipitation (qChIP) analysis on PAX6 promoter by using H3K18la antibody. The results showed that there is H3K18la enrichment at PAX6 promoter and it was significantly elevated during neuroectoderm differentiation (Fig. 6A). Moreover, sodium lactate treatment could significantly enhance the H3K18la level at PAX6 promoter, while Gska treatment would slightly attenuate the H3K18la level at PAX6 promoter in Day 5 cells (Fig. 6B). Collectively, these results demonstrated that lactate promoted H3K18la modification at PAX6 promoter during neuroectoderm differentiation.

Fig. 6.

Exogeneous lactate upregulates the H3K18 lactyaltion level and transcription of PAX6. (A) qChIP analysis of the PAX6 promoter was performed using antibodies against H3K18la and control IgG in Day 0 and Day 5 cells from the induced neuroectoderm differentiation model (n = 4). (B) qChIP analysis of the PAX6 promoter was performed using antibodies against H3K18la and control IgG in Day 5 cells with sodium lactate or Gska treatments in the induced neuroectoderm differentiation model (n = 4). (C) Illustration of shPAX6 and sodium lactate treatment in the induced differentiation model. shPAX6 was introduced to human neuroectoderm differentiation in vitro at Day 1 with or without sodium lactate; and cells were harvested at Day 3. (D) qRT-PCR analysis of PAX6, OCT4, SOX2, SOX1, OTX2, and NES mRNA expression in Day 3 cells treated with shPAX6 or scramble shRNA (shScr) with or without sodium lactate

Since H3K18la modifications on the PAX6 promoter can be upregulated by lactate, we hypothesized that lactate treatment could rescue the level of PAX6 mRNA after shRNA treatment. To test this hypothesis, we introduced shPAX6 at day 1 of the induced differentiation model to degrade PAX6 mRNA; then cells were divided into two groups either with or without lactate treatment and all cells were harvested at Day 3 (Fig. 6C). The levels of PAX6 mRNA were quantified by qRT-PCR and the results showed that the expression of PAX6 was significantly suppressed by shPAX6, and it could be rescued by sodium lactate treatment to the level similar to control cells (Fig. 6D). We also examined the level of other markers, including pluripotency marker OCT4, neuroectoderm markers SOX1, SOX2, OTX2, and NES. The transcriptional level of SOX1 was significantly downregulated by shPAX6, while no obvious difference was observed on the expression levels of OCT4, SOX2, OTX2 and NES upon shPAX6 treatment (Fig. 6D). Sodium lactate treatment not only rescued the expression of PAX6 but also that of SOX1 (Fig. 6D), suggesting that PAX6 is upstream of SOX1. These results demonstrated that exogenous lactate could promote PAX6 transcription during neuroectoderm differentiation.

Discussion

Lactate is previously regarded as a metabolic waste, but recent studies have revealed its unique functions in different biological processes [46]. In our study, we discovered that lactate can promote human neuroectoderm differentiation in vitro (Fig. 7). We found that increased intracellular lactate could upregulate the level of H3K18la modification during neuroectoderm differentiation, and H3K18la modification predominantly located at gene promoters. Moreover, the transcription levels of PAX6, TMEM169, RUSC2, and C1QTNF6 were upregulated with increased H3K18la modifications at their promoters during neuroectoderm differentiation. Further studies verified the H3K18la modification at the PAX6 promoter and exogenous lactate could also rescue the level of PAX6 mRNA after shPAX6 inhibition.

Fig. 7.

Schematic illustration for lactate promoting neuroectoderm differentiation. Increased intracellular lactate can upregulate H3K18la level and promote hESCs differentiation towards neuroectoderm

HESCs differentiation has been extensively dissected from the angles of transcription factors, signaling pathways, and epigenetics [47–49]. During hESCs differentiation towards neuroectoderm, it has been well-established that BMP inhibition and low WNT/β-catenin signaling are essential for hESCs directly differentiation into neuroectoderm, while activation of BMP signaling and high WNT/β-catenin signaling would lead hESCs to mesendoderm [50–53]. SMAD1/5 and β-catenin serve as key signaling transduction molecules in the BMP and WNT/β-catenin pathways, respectively. Phosphorylation of SMAD1/5 and non-phosphorylation of β-catenin (active β-catenin) could translocate to the nucleus, and function as transcriptional co-activators, promoting the expression of target genes. In our study, we found that changes in intracellular lactate levels did not affect the levels of phosphorylated SMAD1/5, nor active β-catenin, suggesting that lactate is unlikely to regulate these two signaling pathways during neuroectoderm differentiation.

Recent years, cellular metabolites are reported to play important roles in epigenetic modifications. For example, acetyl-CoA, α-ketoglutarate, succinate, and fumarate could all participate in chromatin modifications to regulate gene transcriptions [54]. Histone lactylation has been recently reported as a novel epigenetic modification regulated by cellular lactate levels and appearing to directly promote gene transcription from chromatin [24–26, 55]. Our study found the elevated level of H3K18la during neuroectoderm differentiation, which was regulated by intracellular lactate levels. Genome-wide CUT&Tag analysis revealed widespread H3K18la on genome and a high enrichment at the promoter regions. We further identified 4 genes (PAX6, TMEM169, RUSC2, and C1QTNF6) were upregulated by exogenous lactate during neuroectoderm differentiation, with H3K18la modification specifically located at the opening phases of their respective promoter regions. PAX6, a paired box transcription factor, is uniformly expressed in hESC-derived neuroectoderm and has been reported to be essential for both hESCs differentiation towards neuroectoderm as well as the following neurogenesis [43, 45]. Here we verified that H3K18la modifications at the PAX6 promoter were upregulated during neurectoderm differentiation and exogenous lactate could also rescue the transcription level of PAX6 after shPAX6 inhibition. Therefore, it is possible that lactate could upregulate PAX6 transcription via H3K18la modification during neuroectoderm differentiation. Besides PAX6, we also observed other genes have similar H3K18la modification site correlate with open chromatin at the promoter regions. However, very little is known for the other three genes during neuroectoderm differentiation. Therefore, it would be interesting to investigate the functions of those genes during neuroectoderm differentiation in the future.

While we showed that H3K18la is related to neuroectoderm differentiation, other histone modifications could also be involved in this process. Histone acetylation modifications, such as H3K9ac [56, 57] and H3K27ac [58], are known regulators of neuroectoderm differentiation, which are also enriched at gene promoters. Unfortunately, both histone lactylation and acetylation are acylated by acetyltransferases (such as CBP/P300), and deacylated by HDAC1-3 or SIRT1-3 [59], making it difficult to dissect the sole function of H3K18la, especially it is very difficult to investigate whether erase H3K18la modification alone on genome could block neuroectoderm differentiation. It has been reported that HDAC inhibition could promote human pluripotent stem cells toward neural progenitors [60], suggesting that increased histone acylation could promote neuroectoderm differentiation. However, which type of acylation is regulated by HDAC during this process will need further investigation in the future.

In our study, we used LDHA inhibitor Gska to downregulate the level of H3K18la. We noticed that although the intracellular lactate levels can be dramatically downregulated (85%) by Gska (Fig. 2E), the decrease on H3K18la levels was only about 30% (Fig. 3E). In addition, there were only about 23% decrease on H3K18la signal at PAX6 promoter after Gska treatment compared to untreated cells (Fig. 6B). This limited decrease on H3K18la level upon Gska treatment may not enough to cause changes on gene expression levels and may partially explain why we did not observe the inhibition effect of Gska on differentiation. In addition, although we observed highly enriched signal of H3K18la at the promoter regions, H3K18la modifications are ubiquitously distributed in the whole genome. What are the functions of H3K18la at other sites will need further investigation in the future.

In summary, we explored the role of lactate during hESCs differentiation to neuroectoderm and our results showed that increased intracellular lactate could increase H3K18la level and promote neuroectoderm differentiation. Genome-wide analysis of H3K18la indicates it highly enriched at gene promoters and we identified 4 genes can be upregulated by lactate during neuroectoderm differentiation, including PAX6. These results suggested that lactate is sufficient but not necessary to promote neuroectoderm differentiation under our conditions and H3K18la is an important epigenetic factor during neuroectoderm differentiation.

Electronic supplementary material

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Abbreviations

hESC

Human embryonic stem cells

Gska

GSK2837808A

Pan HKla

Pan-histone lysine lactylation level

TSS

Transcriptional start sites

Author contributions

Y.W. and Y.Z. designed the study. Y.W. performed the experiments with technical support from Y.-M.W. Y.W. and Y.H. performed CU&Tag analysis. Y.W. wrote the draft of the manuscript. Y.Z. supervised the study and edited the manuscript. All authors discussed the results and approved the final manuscript.

Funding

This study was supported by grants from the National Natural Science Foundation of China (32370861) and the National Key R&D Program of China 2018YFA0800303 to Y-F. Z.

Data availability

CUT&Tag data generated in this study have been deposited to NCBI BioProject: PRJNA1125585; RNAseq data generated in this study have been deposited to NCBI BioProject: SUB14764048. Both data will be publicly available as of the date of publication. The data can be obtained from the corresponding author upon reasonable request.

Declarations

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Competing interests

The authors declare no competing interests.