TGFβ-activated PDHB promotes mitochondrial pyruvate metabolism and contributes to human endoderm differentiation via ATP-dependent BRG1

Domain: chemistry
Published: 2026-02-17
Editorial outcome: major_revision

Abstract
Cell fate determination is closely linked to metabolic state, yet how metabolic remodeling influences human pluripotent stem cells differentiation into three germ layers remains in completely understood. Here, we reveal that definitive endoderm differentiation from human pluripotent stem cells requires a TGFβ-driven metabolic switch characterized by reduced lactate production and enhanced TCA cycle activity and oxidative phosphorylation, mediated by PDHB. Disruption of glucose utilization or pyruvate entry into the TCA cycle markedly impairs endoderm differentiation, whereas inhibition of lactate production enhances differentiation efficiency. Mechanistically, blockade of glucose metabolism or the TCA cycle reduces intracellular ATP levels, compromising the activity of BAF complex, an ATP-dependent chromatin remodeling complex centered on BRG1. This complex promotes chromatin accessibility and activates endodermal gene programs during differentiation. Together, these findings highlight metabolic reprogramming as a key regulator of human endoderm fate through ATP-dependent control of chromatin remodeling.

Introduction
Cell fate determination is accompanied by significant metabolic changes. Glucose metabolism provides a crucial source of cellular energy and metabolic intermediates, which primarily includes two major pathways: glycolysis to produce lactate, and the pyruvate entry into mitochondria, followed by the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation. Metabolic switches not only affect energy production but also influence the concentrations of various metabolic intermediates, such as acetyl coenzyme A (CoA) and S-adenosylmethionine (SAM). These metabolic intermediates could further regulate the post-translational modifications of proteins, including histone, by serving as substrates of acetylation and methylation modifications, and finally affecting cell fate determination1,2. For instance, the mitochondrial pyruvate carrier (MPC) regulates the downstream TCA cycle during the differentiation of memory T cells, and thereby influences global H3K27ac modification levels. This modulation ultimately governs T cell differentiation and anti-tumor function3. During early embryo development, the mouse two-cell embryo prefers methionine, polyamine and glutathione metabolism, whereas the blastocyst exhibits increased TCA cycle metabolites and presents a more oxidative state4. Interestingly, glucose is necessary for the transition from morula to blastocyst mainly via supporting anabolic processes but not energy production. Moreover, during this process, both the differentiation of the inner cell mass (ICM) into the three germ layers and the specification of the trophectoderm (TE) are highly dependent on glucose metabolism. Notably, glucose deprivation causes a severe developmental arrest and a significant loss of CDX2-positive TE cells, highlighting the essential role of glucose metabolism in early embryogenesis5. In addition, the high level of phospholipid unsaturation at the blastocyst stage is conserved and necessary for early mammalian embryo development. The lipid desaturases, including SCD1, are needed during the blastocyst development and implantation6. Overall, increasing evidence indicates a metabolic shift occurs and plays important roles during early embryo development.

Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) derived from the ICM of human blastocysts, and induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells, are defined by the abilities of self-renewal and to differentiate into three germ layers7. Recent studies have revealed that human PSCs exhibit a metabolic profile similar to the cancer cells, which relies predominantly on glycolysis as their primary glucose metabolism pathway even under aerobic conditions. This phenomenon is commonly referred to as aerobic glycolysis or the Warburg effect8. Several reports indicate that human PSCs undergo metabolic remodeling, transition from a state heavily reliant on glycolysis to a state more dependent on the TCA cycle and oxidative phosphorylation, when they exit pluripotent state9–11. Consistent with the high aerobic glycolysis observed in PSCs, Moussaieff and colleagues found that inhibition of glycolysis can promote spontaneous differentiation and pluripotent state exit1. During the iPSC reprogramming, the investigators also found the upregulation of glycolysis and dynamics of mitochondrial networks, from mature somatic mitochondria with tubular and cristae-rich structures transformed into immature, spherical forms with sparse cristae in iPSCs12.

Germ layer differentiation (i.e., ectoderm, mesoderm and endoderm), which correlates to gastrulation, is a critical event in early embryo development, thus representing a suitable model to investigate the molecular regulation during early embryo development. During lineage specification, different germ layers show different characteristics and requirements for metabolic patterns. Cliff and colleagues have discovered that a significant metabolic switch, from the high glycolysis in ESCs to a lower glycolysis and higher oxidative phosphorylation in differentiated cells, occur during the mesoderm and definitive endoderm (DE) differentiation13. The following studies identified that glutamine metabolism has similar differences during germ layer differentiation. Glutamine is a major metabolic precursor of the TCA cycle, which can reflect the levels of TCA cycle and oxidative phosphorylation to a certain extent. The investigators found that exogenous glutamine is necessary for mesoderm and endoderm differentiation, whereas the ectoderm could survive in glutamine-free media14,15. In addition, metabolism changes also occur during extra-embryonic endoderm (XEN) cells differentiation from mouse ESCs. The XENs exhibit high glycolysis and maintain elevated lactate levels by increasing LDHA expression, and exogenous lactate supplement could promote the XEN differentiation16. Consistent with this study, another group found that the lactylation of ESRRB protein by increased lactate levels mediates the elevated differentiation efficiency17. Despite the metabolic switch appears during germ layer differentiation, how such a metabolic switch happens and how these precise metabolic reprogramming contributes to lineage differentiation are still largely undocumented.

Our group previously revealed the mitochondrial homeostasis plays a critical role in mesoderm and definitive endoderm differentiation from human ESCs18,19. Consistent with the crucial role of mitochondrial homeostasis, we recently found elevated fatty acid oxidation and reduced synthesis would result in the increased acetyl-CoA, then led to the SMAD3 acetylation to promote the expression of endoderm-related genes20. Inspired from previous studies, here we begin with a CRISPR-based screen targeting metabolic regulators during DE differentiation and then focus on glucose metabolism. We then characterize the glucose metabolism switch between lactate production and the TCA cycle, and demonstrate that both glucose utilization and biased pyruvate-TCA cycle flux promote DE differentiation. Disrupting the glucose metabolic switch using chemical inhibitors or genetic depletion significantly affects DE differentiation. Importantly, the inhibition of lactate production to promote pyruvate entry into the TCA cycle enhances DE differentiation efficiency. Furthermore, based on the rescue experiments using different metabolites, we figure out that the ATP production and the function of the ATP-dependent chromatin remodeling complex BAF facilitate DE differentiation.

Results
Glucose metabolism is essential for DE differentiation
First, we performed a CRISPR-based screening in our DE differentiation model, using a metabolism-related sgRNA library21. In CRISPR screening, we used CXCR4 expression as a quantitative readout to evaluate DE differentiation efficiency. As a well-established surface marker of definitive endoderm, CXCR4 (also named CD184) has been extensively utilized in previous studies to assess DE differentiation efficiency from pluripotent stem cells20,22. Interestingly, we obtained many genes associated with glycolysis and the TCA cycle in primary hints. And the key rate-limiting enzyme of fatty acid oxidation CPT2 also appears in these hints, which we reported recently20, indicating the reliability of this screening (Supplementary Fig. 1a). We then performed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, which significantly enriched in pathways related to glucose metabolic process, glycolytic process, and electron transfer activity as well as the TCA cycle (Fig. 1a and Supplementary Fig. 1b). Moreover, the distribution of different sgRNAs targeting the individual genes shown in Supplementary Fig. 1a also confirmed our finding (Supplementary Fig. 1c). Therefore, to further investigate the impact of glucose metabolism on DE differentiation, we assessed whether the alterations in overall glucose utilization affect differentiation. We differentiated human ESCs into endoderm with different glucose concentrations (from 2.8 mM to 25 mM) and determined the differentiation efficiency. The intracellular flow cytometric analyses of key DE markers CXCR4 and SOX17 showed that higher glucose concentrations significantly resulted in higher differentiation efficiency (Fig. 1b). Moreover, RNA levels of DE-related genes FOXA2, SOX17, and CXCR4 were upregulated along with increased glucose concentrations. In contrast, RNA expression levels of ESC-specific genes OCT4, SOX2, and NANOG were decreased at high glucose conditions (Fig. 1c). These results indicate that insufficient glucose supply impairs DE differentiation, and high glucose facilitates DE differentiation.

To further validate the promotive role of glucose metabolism, we utilized the hexokinase inhibitors 3-Bromopyruvic acid (BrPA) and 2-Deoxy-D-glucose (2DG)23 to block glucose utilization during differentiation (Fig. 1d). Apoptosis levels showed no significant toxicity under the concentrations of BrPA and 2DG used in our experiments (Supplementary Fig. 1d). Importantly, both inhibitors remarkably reduced the proportion of CXCR4 and SOX17 double-positive cells in a dose-dependent manner (Fig. 1e). And the suppression of glucose metabolism was further confirmed by the decreased level of acetyl-CoA, a key intermediate of this pathway (Supplementary Fig. 1e). Consistent with the flow cytometric result, we found endoderm marker genes (CXCR4, SOX17 and FOXA2) were down-regulated and pluripotent genes (OCT4, SOX2 and NANOG) were up-regulated along with the increased concentration of inhibitors (Fig. 1f and Supplementary Fig. 1f). Together, these results highlight the crucial role of glucose metabolism in DE differentiation, which is positively correlated with the differentiation efficiency.

Glucose-pyruvate pathway promotes DE differentiation
To further validate the downstream of glucose metabolism, we used phosphofructokinase (PFK) inhibitor Aurintricarboxylic acid (ATA)24, which blocks phosphofructokinase and possibly facilitates the PPP pathway to generate reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate25, crucial for regulating cellular oxidative stress and producing metabolic intermediates needed for cell proliferation (Fig. 1d). Interestingly, addition of ATA significantly decreased the protein levels of DE markers SOX17 and CXCR4 (Fig. 1g). Consistently, quantitative real-time PCR (qRT-PCR) results confirmed that DE differentiation was markedly inhibited upon ATA treatment (Supplementary Fig. 1g), indicating that the fructose-1,6-bisphosphate (FBP)-pyruvate metabolism, rather than PPP, functions in DE differentiation.

In addition, we supplemented the metabolite, pyruvate, the downstream product of glucose utilization, followed by FBP and evaluated whether pyruvate is involved in DE differentiation. The data showed that pyruvate can effectively promote differentiation. Most importantly, supplementation with pyruvate can rescue the impaired DE differentiation caused by BrPA treatment (Fig. 1h). Taken together, we concluded that the glucose-pyruvate pathway is required for human DE differentiation.

To further investigate the effects of BrPA treatment on differentiation potential toward other germ layers, we performed embryoid body (EB) formation assays. We found that BrPA treatment did not significantly affect EB size (Supplementary Fig. 2a). qRT-PCR analysis revealed that BrPA treatment slightly impaired expressions of mesendodermal markers (EOMES, MIXL1, T) and terminal endodermal markers (SOX17, FOXA2, CXCR4). In addition, ectodermal differentiation was also disrupted (Supplementary Fig. 2b). In addition, we performed directed differentiation toward ectoderm and mesoderm lineages under BrPA treatment. For ectoderm differentiation, neuroectodermal markers (NES, PAX6, SOX1) were examined on days 3 and 7. These markers remained largely unchanged at day 3 but were significantly reduced by day 7 upon BrPA treatment (Supplementary Fig. 2c). For mesoderm differentiation, we used a previously reported cardiomyocyte differentiation protocol26. The expression of cardiac-specific markers (TNNT2, GATA4, MYH6) was impaired in BrPA-treated cells (Supplementary Fig. 2d), indicating impaired differentiation toward the mesodermal cardiac lineage. Interestingly, similar to what we observed in DE differentiation, the reduced ectodermal differentiation in EBs could be also rescued by pyruvate or glutamine supplement (Supplementary Fig. 2e). These results together suggest that the glucose-pyruvate pathway is crucial for proper lineage differentiation of human ESCs.

Pyruvate entering the TCA cycle supports DE differentiation
The direction of glucose metabolic flux is largely determined by whether pyruvate is converted into lactate by the lactate dehydrogenase (LDH) complex, or into acetyl-CoA by the pyruvate dehydrogenase (PDH) complex (Fig. 2a)27,28. In our RNA-seq data, we found glycolysis-related genes were predominantly downregulated, whereas the TCA cycle-related genes were largely upregulated during DE differentiation (Supplementary Fig. 3a, b). To determine which direction is functional and required for DE differentiation, we attempted to interrupt the lactate production and the TCA cycle, respectively.

We first used LDH inhibitors oxamic acid sodium (OXA)29 and galloflavin (GF)30 to block lactate production and thus promote pyruvate entry into the TCA cycle. We found that OXA and GF treatment increased the proportion of CXCR4 and SOX17 double-positive cells (Fig. 2b), and the analysis of DE-related gene expressions under both treatments also showed that OXA and GF promoted cell differentiation. (Supplementary Fig. 3c). As expected, OXA treatment indeed inhibited lactate production while promoting pyruvate entry into the TCA cycle (Fig. 2d, e), and GF also increased the acetyl-CoA levels (Supplementary Fig. 3d). Therefore, we concluded that lactate production is harmful for DE differentiation.

Meanwhile, we also used the mitochondrial pyruvate carrier (MPC) inhibitor UK5099 to impede pyruvate entry into mitochondria, and the pyruvate dehydrogenase kinase (PDK) inhibitor sodium dichloroacetate (DCA) to enhance PDH activity31,32. The results showed that UK5099 significantly decreased the differentiation efficiency (Fig. 2c). Consistent with this finding, RNA expression analysis also indicated that UK5099 inhibited the DE-related gene expression (Supplementary Fig. 3c). And the levels of lactate and acetyl-CoA also indicated that UK5099 suppressed pyruvate conversion to acetyl-CoA, thus promoting lactate production and decreasing the acetyl-CoA content (Fig. 2d, e). Importantly, treatment with PDK inhibitor DCA, which facilitates the pyruvate utilization, also led to the anticipated differentiation-promoting effects (Supplementary Fig. 3e). Notably, acetyl-CoA levels also were elevated under DCA treatments, indicating an increase in TCA cycle flux (Supplementary Fig. 3d). We also evaluated the effect of glutamine, a TCA cycle metabolic precursor. The data indicate that glutamine significantly promotes differentiation (Fig. 2f). More importantly, supplementation with glutamine effectively rescues DE differentiation following treatment with BrPA or 2DG (Fig. 2g). Taken together, these results indicated that the metabolism switch from the glycolysis to TCA cycle could effectively facilitate the DE differentiation.

TGFβ promotes pyruvate metabolism during DE differentiation
Next, we wanted to investigate the exact regulation mechanism of the metabolism switches. The LDH and PDH complexes are major regulators of pyruvate metabolism flux. We first examined the gene expression changes of these complexes during DE differentiation. qPCR results revealed that PDH-related genes were obviously upregulated during differentiation (Fig. 3a). We also assessed the genes encoding mitochondrial pyruvate transporters, MPC1 and MPC2, exhibiting notable increases (Supplementary Fig. 4a). Therefore, we hypothesized that a specific signal during differentiation controlled the upregulation of PDH-related genes, thereby mediating the metabolic switching. Following this hypothesis, we focused on the TGFβ and WNT signaling pathways, which are the major regulatory pathways of DE differentiation33. We examined the chromatin immunoprecipitation followed by sequencing (ChIP-seq) data of SMAD2/3 and β-catenin34, the downstream transcription factor of the TGFβ pathway and WNT pathway, respectively. Interestingly, SMAD2/3 significantly bound to the PDHB with two distinct peaks, while β-catenin showed no bindings (Fig. 3b). We also confirmed this conclusion by ChIP-qPCR (Fig. 3c). In contrast, SMAD2/3 does not show significant binding peaks on LDHA or LDHB, correlating with the expression changes of LDH-related genes (Supplementary Fig. 4b).

To further confirm the regulatory role of the TGFβ signaling pathway on PDHB expression, we activated the TGFβ signal pathway by activator Activin A or overexpressing SMAD3. Western blot analysis showed that TGFβ activation significantly increased PDHB expression (Fig. 3d, e). Similar results were observed in HEK293T cells, and both RNA and protein levels of PDHB were significantly upregulated following TGFβ pathway activation (Fig. 3f, g). Overall, these data indicate that the TGFβ signaling pathway can activate PDHB expression during DE differentiation, thereby mediating the metabolic model switch.

PDHB is critical for DE differentiation
The PDH complex is essential for converting pyruvate to acetyl-CoA, which is a critical step for sustaining TCA cycle flux35. To further validate that pyruvate entry into the TCA cycle is important for DE differentiation, we employed CRISPR-Cas9 technology to target PDHB, a critical component of PDH complex, based on the HUES8 cell line (Supplementary Fig. 5a). A previous study demonstrated that PDHB depletion markedly reduces PDH activity and disrupts metabolic patterns36. Although we generated ten heterozygous clones out of 53 genotyped clones, we did not obtain any homozygous PDHB-knockout human ESCs (Supplementary Fig. 5b), suggesting PDHB might be essential for ESC survival.

Nevertheless, heterozygous cell lines displayed a significant decrease of PDHB expression (Supplementary Fig. 5c) and metabolic alterations, including reduced acetyl-CoA levels and increased lactate levels (Fig. 4a). The pluripotency of PDHB+/- cells was not influenced by decreased PDHB and acetyl-CoA (Supplementary Fig. 5d). Therefore, we proceeded with the heterozygous lines for further experiments. We first assessed whether this metabolic switch affected differentiation efficiency using flow cytometry and an immunofluorescence assay. Results indicated that PDHB+/- cells had a reduced proportion of CXCR4 and SOX17 double-positive cells and fewer FOXA2-positive cells than wild-type cells (Fig. 4b and Supplementary Fig. 5e), suggesting that the TCA cycle inhibition impairs endoderm differentiation. Then we performed RNA-seq using differentiated PDHB+/- cells. The results revealed profound changes in gene expression (Supplementary Fig. 5f). We also applied single-cell flux estimation analysis (scFEA) to investigate the metabolic flux changes in PDHB+/- cells37. Consistently, metabolites related to TCA cycle intermediates (e.g., acetyl-CoA, oxaloacetate, Fumarate) were enriched in wild-type cells. And metabolites related to glycolysis (e.g., lactate) were remarkably enriched in PDHB+/- cells, suggesting PDHB heterozygous knockout blocked TCA cycle metabolism fluxes and upregulated glycolysis (Fig. 4c). Furthermore, the gene-set enrichment analysis (GSEA) highlighted the enrichment of DE-related genes in wild-type cells and ESC-related genes in PDHB+/- cells (Fig. 4d), reinforcing that differentiation efficiency was impaired in PDHB+/- cells. Overall, these findings demonstrate that the PDHB-mediated pyruvate utilization in the TCA cycle supports endodermal differentiation, and its inhibition adversely impairs the differentiation efficiency.

We also performed EB differentiation assays on the PDHB+/- cell line. The results showed that PDHB heterozygous knockout attenuated the downregulation of pluripotency markers (OCT4, SOX2, NANOG) and impaired endodermal and ectodermal differentiation. Specifically, it led to decreased expression of endodermal markers (SOX17, FOXA2, CXCR4) and ectodermal genes (NES, PAX6). Interestingly, we found that the expression of early mesendodermal stage markers (EOMES, MIXL1, T) are increased (Fig. 4e). Given that pluripotent stem cells first transition through a mesendodermal stage, marked by the expression of EOMES, MIXL1, and T (Brachyury), before further differentiating into either mesoderm or DE38, we analyzed gene expression dynamics at three time points (Day 3, 5, and 9) during EB differentiation. We observed that PDHB+/− cells exhibited a delayed downregulation of pluripotency genes and a postponed peak in mesendodermal gene expression: mesendodermal markers peaked around Day 5 in wild-type cells, while the PDHB+/− cells reached this stage around Day 9 or later (Supplementary Fig. 5g). This data suggests that PDHB depletion impairs both the exit from pluripotency and the initial mesendodermal differentiation. Notably, the upregulation of DE markers was continually delayed in PDHB+/− cells.

The DE differentiation represented an early stage of pancreatic lineage commitment. We further examined whether impaired DE differentiation affected subsequent pancreatic development based on our published pancreatic differentiation protocol39. At both the early pancreatic progenitor (PP1) and late pancreatic progenitor (PP2) stages, BrPA treated and PDHB+/- cells exhibited markedly reduced RNA expression of key pancreatic genes, including FOXA2, NKX6-1, NKX2-2, and PDX1 (Supplementary Fig. 5h), which was further confirmed by the immunofluorescence analysis (Supplementary Fig. 5i). Overall, these results suggest that metabolic alterations caused by BrPA treatment or PDHB deficiency not only impair three germ layers differentiation but also disrupt the progression of pancreatic differentiation.

We next established PDHB-overexpressing ESC lines and confirmed efficient overexpression at both the RNA and protein levels (Fig. 4f). PDHB upregulation enhanced the conversion of pyruvate to acetyl-CoA, thereby stimulating the TCA cycle activity and ATP production (Fig. 4g). To further assess the metabolic consequences, [U-13C]-glucose tracing revealed increased glucose-derived labeling in key TCA intermediates, including citrate, aconitate, succinate, and succinyl-CoA (Supplementary Fig. 6a, b and Supplementary Data 1), consistent with an overall metabolic shift toward enhanced oxidative metabolism. More importantly, PDHB overexpression significantly improved DE differentiation efficiency (Fig. 4h). Moreover, under reduced Activin A concentrations, PDHB overexpression further promoted DE differentiation (Fig. 4i), suggesting that PDHB activity becomes rate-limiting when signaling input is weakened. Collectively, these findings support that PDHB drives a metabolic shift toward increased TCA cycle flux, thereby enhancing DE differentiation.

ATP and BAF complex play crucial roles in DE differentiation
Next, we wanted to investigate how such a metabolism switch regulated DE differentiation. Various metabolic intermediates could affect protein post-translational modifications or chromatin structure by acting as substrates or cofactors of epigenetic enzymes8. Thus, we supplemented SAM, α-KG, acetate, citrate, or ATP to determine which metabolite could rescue DE differentiation upon BrPA treatment and PDHB heterozygous knockout. Interestingly, we found that SAM, α-KG, acetate and citrate showed no apparent rescue effect (Supplementary Fig. 7a), whereas ATP significantly restores endoderm differentiation efficiency under both conditions (Fig. 5a). Consistently, we observed a significant ATP levels reduction in both BrPA-treated and PDHB+/- cells (Fig. 5b). Moreover, ATP content correlated with differentiation efficiency across different inhibitor treatments: 2DG, ATA and UK5099 (inhibit DE differentiation) decreased ATP levels, while OXA, GF, and DCA (accelerate DE differentiation) increased ATP levels (Supplementary Fig. 7b). Moreover, upon treated with another PDH inhibitor CPI61340, DE differentiation was blocked along with decreased ATP content, while the exogenous supplement of ATP could rescue the differentiation defect (Supplementary Fig. 7c), consistent with the observations from PDHB+/- cells. Overall, these findings suggest that the impact of metabolic switch on differentiation efficiency is mainly mediated by the changes in ATP content.

Then we analyzed the difference of gene expression pattern between PDHB+/- and wild-type cells to further investigate the interface between ATP level and DE differentiation. GO enrichment analysis showed that metabolic-related gene sets, such as acetyl-CoA metabolism and differentiation-associated gene sets like the WNT signaling pathway, cell fate determination, and endoderm formation were enriched in differentially expressed genes, as expected. Interestingly, we found that chromatin accessibility associated terms were also enriched, such as DNA-binding transcription activator activity and regulation of DNA binding (Supplementary Fig. 7d). Literature further highlights the crucial roles of ATP-dependent chromatin remodeling complexes during the exit from pluripotency, such as Brahma-associated factor (BAF) complexes and the NURD complex41. Therefore, we hypothesized that metabolic switching and ATP content might influence the function of ATP-dependent chromatin remodeling complexes, thereby affecting differentiation efficiency.

To test this hypothesis, we first examined whether the ATP-dependent chromatin remodeling complex and which complex regulated endoderm differentiation. We used specific inhibitors of the BAF complex (BRM014 targeting core subunits BRM/BRG1, and BI7273 targeting BRD7/9) and NURD complex (BPK-25) during DE differentiation. The results showed that the inhibition of BAF complex activity significantly impaired differentiation efficiency, whereas inhibition of the NURD complex did not have any obvious impact (Fig. 5c), suggesting that the differentiation inhibition was more likely mediated by the BAF complex. The BAF complex consists of two mutually exclusive core subunits, BRM and BRG142. We further examined the expression of these core subunits during differentiation and found that BRG1 is predominantly expressed and progressively upregulated during differentiation, while BRM is scarcely expressed (Fig. 5d and Supplementary Fig. 7e). Next, we attempted to generate BRG1 knockout ESCs. Given that BRG1 knockdown disrupts pluripotency maintenance in human43,44, we employed the dTAG-induced degradation system for cell line construction45. Western blotting confirmed complete BRG1 degradation within 8 hours of dTAG induction in these two HUES8-derived ESC clones (Fig. 5e). Consistent with the inhibitor treatment, BRG1 loss during DE differentiation significantly impaired endoderm formation (Fig. 5f). Taken together, these data indicate that the BAF complex, especially the BRG1-centered BAF complex, is necessary for endoderm differentiation.

To further establish that ATP functions upstream of BRG1, we inhibited BAF activity during ATP supplementation. Inhibition of the BAF complex effectively abolished the rescuing effect of ATP on DE differentiation (Fig. 5g). Similarly, the decreased RNA levels of DE marker genes FOXA2, SOX17, CXCR4 due to blocked glucose utilization and mitochondrial pyruvate metabolism could be also rescued by ATP supplement, then inhibited by BAF inhibitors (Supplementary Fig. 8a). Moreover, the result that ATP supplementation failed to restore DE differentiation defect in BRG1-depletion cells (Fig. 5h) further confirmed that BRG1 is required for ATP-mediated enhancement of differentiation. In addition, we employed BRM014, a specific inhibitor of the ATPase activity of BRM/BRG146. The results showed that exogenous ATP supplementation failed to promote DE differentiation when BRG1’s ATPase activity was inhibited (Supplementary Fig. 8b, c), indicating ATP exerted its effect on differentiation through the ATPase function of BRG1. More importantly, overexpression of wild-type or ATPase-dead (K798R47,48) BRG1 in dTAG-induced BRG1-knockout cells revealed that only wild-type BRG1 restored DE differentiation, whereas the ATPase-dead mutant failed to rescue (Fig. 5i). Together, these results demonstrate that BRG1 regulates DE differentiation through its ATPase activity.

Metabolic remodeling during human embryonic development
To further investigate whether similar mechanisms including metabolic reprogramming and the ATP-BAF regulatory axis operate in vivo, we analyzed a publicly available single-cell RNA-seq dataset of 3D-cultured human pre-gastrulation embryos49, encompassing multiple cell types present during the gastrulation stage, including the embryonic disc, amnion, yolk sac and the primitive streak anlage (PSA)50 (Supplementary Fig. 9a). Based on the data, we examined the expression patterns of genes involved in glycolysis, the TCA cycle, oxidative phosphorylation, and the BAF chromatin remodeling complex across different embryonic tissues. Our analysis revealed that during the developmental transition from the inner cell mass (ICM) to epiblast (EPI) and subsequently to PSA-EPI, glycolytic gene expression (including canonical glycolytic genes such as GAPDH, GPI, HK2, and LDHA) was markedly decreased, whereas genes related to the TCA cycle and oxidative phosphorylation were significantly upregulated (Supplementary Fig. 9a, b). These results indicate a clear metabolic shift toward mitochondrial oxidative metabolism during this stage. Furthermore, genes associated with the BAF complex, such as BRG, BRD7, BRD9 and ARID1A, also exhibited a moderate increase in expression throughout the ICM-EPI-PSA-EPI differentiation process (Supplementary Fig. 9c). Together, these findings suggest that the metabolic and epigenetic changes described in our study also occur during normal human embryonic development. Notably, such a metabolic transition was not observed in lineages differentiating toward extraembryonic fates (Supplementary Fig. 9a, b).

PDHB promotes chromatin accessibility in DE genes by BAF
To confirm whether the function of the BAF complex is affected upon inhibiting mitochondrial pyruvate metabolism in the PDHB+/- cells, we performed the ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing) to assess changes of chromatin accessibility. We first confirmed the data quality evidenced by the nice reproducibility among different samples (Fig. 6a and Supplementary Fig. 10a). Differential peaks analysis revealed that PDHB depletion significantly impacted the overall chromatin accessibility. Interestingly, most of the differential peaks showed decreased accessibility following metabolic suppression (Fig. 6a). The finding further indicated that the ATP-dependent chromatin remodeling complex BAF was functionally impaired in PDHB+/- cells. We next performed GSEA of the top 500 upregulated and downregulated genes identified by ATAC-seq, and the results confirmed that changes in chromatin accessibility were correlated with RNA-seq data (Supplementary Fig. 10b). Moreover, we conducted GO enrichment analysis on genes with reduced chromatin accessibility in PDHB+/−. The results also showed significant enrichment in gene sets associated with pancreas development, endoderm development, and the WNT signaling pathway (Fig. 6b), consistent with the inhibited DE differentiation we observed previously.

Subsequently, to investigate the relationship between the chromatin accessibility changes and BAF complex function, we analyzed BRG1 ChIP-seq data51 and overlapped the genes with downregulated ATAC-seq signal and BRG1-binding signal (Fig. 6c). The Venn diagram showed that the majority of genes with decreased chromatin accessibility also bind to the BRG1 protein. Notably, most of these genes were upregulated during DE differentiation and were enriched in DE cells compared with ESCs (Fig. 6d, e). And the up-regulated genes in day 5 also enriched the terms related to the endoderm differentiation, including the stem cell differentiation and canonical WNT signal pathway (Fig. 6f). Moreover, DE-related transcription factors, including TCF4 and FOXI1, showed marked reductions in chromatin accessibility and gene expression following PDHB depletion (Fig. 6h). Taken together, the functional impairment of BAF complex lead to more closed chromatin accessibility of BAF binding DE-related genes, consequently disrupting the cell differentiation.

Several studies have demonstrated that the BAF complex primarily remodels the chromatin environment of enhancers52–54. To investigate whether BRG1 exerts its function through the regulation of enhancer accessibility in DE differentiation, we further analyzed the distribution of BRG1-binding peaks across different genomic elements. The results revealed that BRG1 preferentially binds to intergenic and intronic regions, accounting for approximately 74.9% of the total binding sites (Supplementary Fig. 10c), while it also bound to promoter regions for about 10.76%. Then we compared chromatin accessibility of enhancer and active transcription start site (TSS) regions between wild-type and PDHB-depleted cells. The data indicated that only the chromatin accessibility at enhancers was significantly reduced in the PDHB-deficient cells, while there were no notable changes in TSS regions (Fig. 6g). As examples, BRG1 primarily binds the non-promoter regions of TCF4 and FOXI1, which were typically marked by two enhancer-associated histone modifications, H3K27ac and H3K4me1 (Fig. 6h). To further confirm the BRG1 binding was dependent on the metabolic pattern, we performed ChIP-qPCR on these non-promoter regions of DE-related genes in wild-type and PDHB-deficient cells. The results revealed that PDHB depletion led to an obvious decrease in BRG1 enrichment at DE-related gene loci, such as FOXI1, TCF4, SMAD3 and SOX17 (Fig. 6i and Supplementary Fig. 10d), concomitant with the reduced chromatin accessibility observed in ATAC-seq. These findings indicated that PDHB and ATP production facilitated the BRG1/BAF complex to bind with enhancers and carries out its function of facilitating chromatin opening.

Methods
Cell culture
Human embryonic stem cell line HUES8 and induced pluripotent stem cell line PGP1 were cultured on Matrigel-coated plates (Corning, Cat#354277) with mTeSR1 medium (STEMCELL Technologies, Cat#85850) at 37°C with 5% CO2. Subculturing was performed every 4 days using Accutase (STEMCELL Technologies, Cat#07922), and the culture medium was changed daily. Human embryonic kidney cell line 293T (HEK293T) was grown on tissue culture-treated plates to ensure cell adherence, in DMEM mixed with 10% fetal bovine serum (FBS, Gibco, Cat#10100147) and 1x penicillin/streptomycin (Gibco, Cat#10378016). Passaging was carried out using 0.05% trypsin (Gibco, Cat#25300120) when cells reached 90–100% confluence.

The chemical small molecules used in this study were as follows: 3-Bromopyruvic acid (BrPA), at concentrations of 30-40 µM (APExBio, Cat#B7922); 2-Deoxy-D-glucose (2DG), at concentrations of 1-2 mM (APExBio, Cat#B1027); ATA, at concentrations of 100–200 µM (Sigma, Cat#A1896); Oxamate (OXA), at a concentration of 5 mM (MCE, Cat#HY-W013032A); Galloflavin (GF), at a concentration of 50 µM (APExBio, Cat#B5716); UK5099, at a concentration of 4 µM (MCE, Cat#HY-15475); Dichloroacetate (DCA), at a concentration of 10 mM (APExBio, Cat#B7174); BRM/BRG1 Inhibitor-1, at a concentration of 25 nM (MCE, Cat#HY-119374); BI-7273, at a concentration of 10 µM (Selleck, Cat#S8179); and BPK-25, at a concentration of 5 µM (MCE, Cat#HY-141550). Notably, the concentrations of these chemicals used in this study had a minimal impact on cell survival.

Definitive endoderm (DE) differentiation
For the induction of DE, experiments were conducted using 24-well cell culture plates coated with growth factor-reduced Matrigel (Corning, Cat#354230). The initial cell seeding density was 100,000 cells per well. After cultivating the cells in mTeSR1 for one day, the DE differentiation medium was switched for the next three days. The DE differentiation medium was based on DMEM (Gibco, Cat#C11995500BT) and supplemented with 100 ng/mL Activin A (PeproTech, Cat#120-14P), 1x penicillin/streptomycin (Gibco, Cat#10378016), and 0.2% bovine serum albumin (Yeasen Biotechnology, Cat#36104ES25).

CRISPR-screen
Human ESCs were infected by metabolism-related lentiviral library which contains Cas9 cassette. Then the cells were selected by 2 μg/ml puromycin for 2 days. After the selection, we passaged the cells to DE differentiation medium, which contained 100 ng/mL Activin A, 1x penicillin/streptomycin, and 0.2% fatty acid-free bovine serum albumin. Then we tested the differentiation efficiency with APC-conjugated mouse anti-human CXCR4 antibody. Genomic DNA was isolated from the top 30% positive and bottom 30% negative cells, and the sgRNA sequences were amplified by PCR and then sequenced.

Gene overexpression constructs
The coding sequence (CDS) of PDHB was subcloned into the doxycycline-inducible pCW lentiviral vector. Lentiviral particles were generated by co-transfecting HEK293T cells with the pCW-PDHB construct together with the packaging plasmids psPAX2 and pMD2.G. The viral supernatant was collected and transfected into HUES8 cells. Stable PDHB-overexpressing cells were obtained following selection with 2 μg/mL puromycin for about 7 days. Wild-type and ATPase-mutant BRG1 were overexpressed in HUES8 cells using the PiggyBac system.

[U-13C]-glucose Metabolic flux analysis
Cells were cultured in glucose-free medium supplemented with 17.5 mM [U-13C]-glucose (MCE, HY-B0389A). After incubation, metabolites were extracted with 80% methanol at −80°C and analyzed in the National Protein Science Technology Center at Tsinghua University using a Dionex Ultimate 3000 UPLC coupled to a TSQ Quantiva Ultra triple-quadrupole mass spectrometer (Thermo Fisher). Data were acquired in selected reaction monitoring (SRM) under positive/negative ion switching mode. TCA cycle intermediates (citrate, α-ketoglutarate, succinate, etc.) were detected for 13C isotopologue analysis, and quantified using TraceFinder 3.2 (Thermo, USA).

RNA preparation and qRT-PCR
Cells were first washed with Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Cat#C14190500BT) and then harvested using lysis buffer. Total RNA was extracted using the HiPure Total RNA Mini Kit (Magen, Cat#R4111-03) according to the manufacturer’s protocol. Qualified total RNA, assessed with NanoDrop 2000 spectrophotometer (Thermo Fisher), was reverse-transcribed into complementary DNA (cDNA) using the ABScript II RT Master Mix (ABclonal, Cat#RK20402) following the manufacturer's guidelines. qRT-PCR was conducted using a C1000 Touch Thermal Cycler (Bio-Rad) with a 384-well plate format. The reaction included 2x SYBR Green qPCR Master Mix (Bimake, Cat#B21203).

Flow cytometry
Cultured cells were detached using TrypLE (Gibco, Cat#12604013) at 37°C for 2 minutes. After detachment, cells were washed twice with DPBS containing 2% FBS and collected by centrifugation at 300 g for 3 minutes at 4°C. Cells were then resuspended in DPBS with 2% FBS and incubated with APC-conjugated mouse anti-human CD184 antibody (1:200, BD, Cat#555976) for 30 minutes at 4°C in the dark. Following incubation, the cells were washed twice with cold DPBS containing 2% FBS. The cells were fixed and permeabilized using Fix/Perm Buffer (BD Biosciences, Cat#562574) for 1 hour at 4°C. After fixation, cells were washed twice with Perm Buffer (BD Biosciences, Cat#562574). Cells were then incubated with Alexa Fluor 488-conjugated mouse anti-human SOX17 antibody (1: 200, BD Biosciences, Cat#562205) for 45 minutes at 4°C. After staining, cells were washed and resuspended in 200 µL of DPBS. The stained cells were analyzed using the CytoFLEX flow cytometer (Beckman).

Immunofluorescence staining
Cells were initially fixed with 4% paraformaldehyde for 15 minutes at room temperature. After fixation, they were washed three times with DPBS and subsequently blocked and permeabilized with a solution containing 10% donkey serum and 0.3% Triton X-100 in DPBS for 1 hour at room temperature. Primary antibodies were then applied in a solution of DPBS with 0.3% Triton X-100 and 10% donkey serum and incubated overnight at 4°C. The following day, the cells were washed three times with DPBS and then incubated with secondary antibodies in the same blocking solution for 1-2 hours at room temperature. After washing with DPBS, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Sigma, Cat#10236276001) for 10 minutes at room temperature. The primary antibodies used included: rabbit anti-FOXA2 (1: 200, HUABIO, China, Cat#ET1703-76), anti-PDX1 (1: 200, R&D, Cat#AF2419), anti-NKX6-1 (1: 200, ABclonal, Cat#A20419), and anti-NKX6-1 (1: 200, Cell Signaling Technology, Cat#54551).

Western blot
Cells were washed with DPBS, dissociated using TrypLE, and collected by centrifugation at 300 × g for 3 minutes at 4°C. Whole cell extracts were prepared using RIPA buffer (Beyotime, Cat#P0013C) supplemented with a protease inhibitor cocktail (Roche, Cat#4693116001). The protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Cat#A53225). Approximately 20 µg of protein was separated by SDS-PAGE (10% gel) and transferred to a nitrocellulose membrane (Millipore, Cat#Z746010). The membrane was then blocked with TBST, which contained 5% BSA, incubated overnight at 4°C with primary antibodies, and washed with TBST for 30 minutes. HRP-conjugated secondary antibodies were applied for 1 hour at room temperature, followed by washing and treatment with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Cat#WBKLS0500) for visualization. The antibodies for western blot used in the present work include: α-Tubulin (1: 3000, Proteintech, Cat#11224-1-AP), PDHB Polyclonal antibody (1: 2000, Proteintech, Cat#14744-1-AP), and SMARCA4/BRG1 Polyclonal antibody (1: 5000, Proteintech, Cat#21634-1-AP). The original western blots were shown in the Supplementary material.

Generation of knockout cell lines
To generate PDHB-knockout ESCs, CRISPR/Cas9 genome editing technology was employed. The guide RNA sequence of PDHB (5’-CTAGAGAATGAATTGATGTA-3’) was designed by the online CRISPR design tool (http://crispr.mit.edu/). Then the sgRNA oligos were annealed and ligated into the pX459 expression vector, and then the construct was co-electroporated with a GFP vector (approximately 1/10 of the pX459 amount) into ESCs using the P3 Primary Cell 4D-Nucleofector X Kit. The transfected cells were sorted based on GFP expression via flow cytometry and subsequently plated into a 96-well plate. Genotyping of the resulting colonies was performed by Sanger sequencing, and PDHB expression levels were verified by Western blot analysis.

For BRG1-dTAG cells, we used CRISPR/Cas9-mediated homology-directed recombination HDR to knock in a sequence encoding 3×Flag-FKBP-T2A-Puromycin at the C-terminus of BRG1. The pX459 vector carrying the guide RNA (5’-GCTGCTACCCGTTACTGCTA-3’) and the HDR template was transfected into HUES8 cells using Lipofectamine (Thermo Fisher, Cat#STEM00001). After puromycin selection, surviving cells were single-cell sorted, and successful knock-in clones were verified by PCR.

Measurement of acetyl-CoA, L-lactate and ATP levels
Cells were washed with DPBS, dissociated with TrypLE, incubated at 37°C for 2 minutes, resuspended in DMEM, and collected by centrifugation at 300 × g for 3 minutes at 4°C. The quantification of acetyl-CoA was performed by the Acetyl CoA Assay Kit (sigma, Cat#MAK039), the quantification of L-Lactate was performed by the L-Lactate Assay Kit (abcam, Cat#ab65330), and the quantification of ATP was performed by the ATP Assay Kit (Beyotime, Cat#S0026B). For normalization of cell mass, protein concentration was measured with the Pierce BCA Protein Assay Kit (Thermo Fisher, Cat#23225).

RNA-Seq and Data analysis
The RNA-seq data were processed based on a previous pipeline76: reads were aligned to the human genome (hg38) by bowtie2 (2.3.4.3). Secondary alignments were performed to ensure that each read was uniquely mapped to a single genomic location. Subsequently, read counts were obtained with featureCounts (1.6.3). RNA expression levels were normalized as TPM (transcripts per million). Differential expression analysis was conducted with DESeq2, and GSEA was performed on the GSEA software to identify specifically enriched pathways.

The sequencing data in this study have been uploaded to the Gene Expression Omnibus (GEO) database under accession codes GSE285132, GSE285133, GSE285135. The public data used in this study was list as follow: BRG1 ChIP-seq data from GSE18284251, H3K27ac ChIP-seq data and H3K4me ChIP-seq data from GSE5447177, β-catenin ChIP-seq data from GSE9920234, the DE RNA-seq data from GSE16862518.

Single-cell flux estimation analysis (scFEA)
The scFEA was used to predict the metabolite changes based on the single-cell or bulk RNA-seq37. The detailed protocol of scFEA can be found on GitHub (https://github.com/changwn/scFEA). In brief, TPM from RNA-seq was the input of the scFEA package. And analysis was performed in default parameters. Finally, the results were visualized using the pheatmap package.

ChIP-seq and ATAC-seq
For each sample, 1×10⁷ cells were crosslinked with 1% formaldehyde for 10 minutes and quenched with 0.125 M glycine for 5 minutes. Cells were washed with cold DPBS, collected by scraping, and resuspended in Cell Lysis Buffer (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 0.2% NP-40) with 1x protease inhibitor. The lysis was sonicated to generate 200–500 bp fragments. Chromatin was immunoprecipitated overnight with 5 μg SMAD2/3 antibody (R&D, Cat#AF3797) or Protein A/G Dynabeads. After extensive washing, DNA was eluted and reverse crosslinked at 67°C for 5 h. The purified DNA was sent for ChIP-seq library construction.

Cells were prepared for ATAC-seq library construction using the Hyperactive ATAC-Seq Library Prep Kit for Illumina (Vazyme, Cat#TD711-01) and the TruePrep Index Kit V2 for Illumina (Vazyme, Cat#TD202). The ATAC-seq data were processed as follows: all reads were aligned using Bowtie2 (2.3.4.3), and duplicates were removed. Subsequent data visualization followed a similar approach to ChIP-seq (reported in our previous works)76, where deeptools (3.3.0) was used to generate bigWig (bw) files, which were then utilized to produce heatmaps and average profile plots78–80.

Statistics and reproducibility
All experiments were conducted independently with at least three biological replicates if not specifically mentioned. Data were analyzed using GraphPad Prism 8 and were presented as means ± standard deviation (SD). For figures with three or more groups, statistics were calculated using one-way ANOVA, followed by Dunnett’s multiple comparisons test. Two-way ANOVA was used when two variables were involved, followed by Tukey’s multiple comparisons test. For comparisons between two groups, two-tailed paired t tests were applied directly. Statistical significance is indicated in each figure. No statistical method was used to predetermine sample size; no data were excluded from the analyses; the experiments were not randomized; and investigators were not blinded to allocation during experiments and outcome assessment.

Ethics
The human ESCs and iPSCs reported in this study have been approved by the Biomedical Ethics Committee of Wuhan University (approval number: WHU-LFMD-IRB2024026). The established human ESC line HUES8, acquired from the Harvard Stem Cell Institute, was used in this study75.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Meng et al. provides compelling evidence that glucose metabolism, specifically, mitochondrial pyruvate flux governed by TGFβ-induced PDHB expression, plays a pivotal role in definitive endoderm (DE) differentiation from human pluripotent stem cells (hPSCs). The authors demonstrate that ATP generated through this metabolic reprogramming enhances the activity of the BRG1/BAF chromatin remodeling complex, thereby promoting chromatin accessibility at DE-related gene loci. Through a comprehensive combination of CRISPR screening, small molecule perturbations, metabolite supplementation, RNA-seq, ATAC-seq, and ChIP-seq, the study establishes a mechanistic link between extracellular signaling, cellular metabolism, epigenetic remodeling, and cell fate determination. The study employs innovative approaches to uncover a previously underappreciated role of TGFβ-PDHB signaling in directing glucose metabolism toward mitochondrial pyruvate oxidation. However, several concerns should be addressed prior to publication:\n1. While the study implicates BRG1 as a downstream effector of ATP-mediated chromatin remodeling, direct evidence of its ATPase activity modulation is lacking. Incorporating experiments with a dominant-negative or ATPase-dead BRG1 mutant would strengthen the causal relationship.\n2. It remains unclear whether PDHB overexpression or activation is sufficient to enhance DE differentiation. Addressing this would clarify whether PDHB acts as a limiting factor in this process.\n3. Some figures, particularly Fig. 6 depicting ATAC-seq and BRG1 ChIP-seq overlap, are densely annotated. Simplifying these visuals and including schematic models would improve accessibility and reader comprehension.\n4. Terminology such as “mainstream glucose metabolism” versus “PPP” should be clearly defined earlier in the manuscript to avoid ambiguity and improve clarity.", "reviewer_2": "The authors revealed a metabolic switch with decreased lactate production and increased mitochondrial pyruvate metabolism, which is controlled by TGFβ-activated PDHB, is necessary for the definitive endoderm differentiation from human pluripotent stem cells. The experiments data, especially the pyruvate rescue and ATP rescue, are convincing and impressive, and the following are the additional points that can be addressed.\nIn line 55-56 in the introduction, the glucose metabolism is also required for the cell type specification of TE during blastocyst development.\nFor figure 1, the authors can add one or two sentence to explain the rationale of using CXCR4 as the readout of DE differentiation in their CRISPR screening.\nFor figure 1, whether glucose metabolism inhibition leads to compensation of other lineage differentiation, such as ectoderm and mesoderm, or it generally inhibits differentiation and pluripotency exit in this 2D differentiation system. It was discussed in the embryoid body section but not here. Also, the toxicity effect data upon BrPA/2DG treatment was mentioned but not shown.\nFor figure 2, the pyruvate and glutamine rescue experiments are interesting and convincing in elucidating the role of glucose metabolism in DE differentiation. Can they also rescue the slight changes of ectoderm differentiation in Fig 2I?\nIn line 276-279, the authors should better explain the difference of mesoendoderm and definitive endoderm to make it coherent through the article\nCan the authors use a PDH inhibitor to show ATP decrease and reduced DE differentiation, which can be rescued by ATP, in addition to the general glycolysis inhibitor?\nFor fig 5, the ATP rescue experiments are impressive. Can supplemented ATP be permeable to the cell membrane to change the intracellular ATP level, and is there other reports or this is the first time to show ATP can participate in the ATP-dependent chromatin remodeling processes. These points can be at least discussed in the discussion section.\nSome of the grammar should be polished such as :\nLine 139 To further validate the promotional role\nLine 144 evident by the decreased acetyl-CoA\nLine 169 Pyruvate entering TCA cycle facilitates DE differentiation\nTCA cycle should be the TCA cycle.", "reviewer_3": "Summary\nMeng et al. investigates how glucose metabolism regulates endoderm differentiation from human iPS cells. The authors demonstrate that mitochondrial pyruvate metabolism is required for ATP production, which in turn supports the chromatin remodeling activity of the BRG1-containing BAF complex. They further link this metabolic switch to TGFβ signaling through transcriptional regulation of PDHB by SMAD2/3. Loss of PDHB impairs DE differentiation, which can be partially rescued by exogenous ATP, and this rescue is blocked by BAF complex inhibition. These findings are supported by extensive molecular analyses including CRISPR screens, metabolite measurements, ATAC-seq, ChIP-seq, and RNA-seq.\nWhile the study makes a compelling case for the involvement of mitochondrial metabolism in endoderm fate commitment, several major conceptual, technical, and interpretational limitations lessen its current suitability for publication in Nature Communications.\nConceptual Impact and Novelty / Overall Big Picture Issues\nThe manuscript brings forward a conceptually appealing model that connects growth factor signaling (TGFβ), metabolic state (pyruvate flux → ATP), and chromatin remodeling (BAF complex) to orchestrate cell fate decisions in human-iPS cells. The novelty resides in linking ATP-dependent chromatin remodeling to a specific metabolic switch regulated by PDHB, as a direct target of SMAD2/3. This integration of metabolism and epigenetics during germ layer specification is timely and of interest to a broad audience.\nHowever, several gaps undermine the model’s completeness and generalizability:\n- The central mechanism (ATP → BAF) is not fully validated, and alternative ATP-dependent processes are not addressed.\n- The metabolic reprogramming is inferred, not directly measured via flux analysis.\n- Findings are derived from a single cell line, limiting generalizability.\n- The in vivo relevance is not addressed at all.\nTogether, these issues limit the physiological and mechanistic depth required for publication in a high-impact journal.\nMajor Concerns\n1. Ambiguity in Cell Line Usage\nAlthough the Methods section states the use of both HUES8 and PGP1 cells, the figures, legends, and Results fail to specify which cell line was used in each experiment. This raises concerns about reproducibility and generalizability.\n➤ Authors must clarify which experiments were performed in which cell lines, and replicate key findings (e.g., ATP rescue, PDHB KD) in the second line.\n2. Mechanistic Inference of ATP → BAF is Not Fully Supported\nFigure 5A shows that ATP rescues DE differentiation in PDHB+/- or BrPA-treated cells, and that this rescue is blocked by BAF inhibitors. While this is consistent with the proposed model, it does not exclude other ATP-dependent regulators of chromatin or differentiation.\n➤ Authors should directly assess BAF complex activity in response to ATP changes (e.g., BRG1 occupancy at DE loci, ATPase activity, or nucleosome repositioning assays).\n➤ Alternatively, loss-of-functon(e.g., BRG1 KO) combined with ATP rescue would strengthen causal inference.\n3. Lack of Direct Metabolic Flux Measurements\nThe metabolic switch is deduced from static metabolite levels and inhibitor use. These are suggestive but not conclusive.\n➤ Authors should include stable isotope tracing (e.g., 13C-glucose or 13C-pyruvate) to directly confirm increased flux through the TCA cycle during differentiation and after TGFβ/PDHB activation.\n4. No Exploration of In Vivo or Cross-Lineage Relevance\nAll findings are based on in vitro differentiation of human iPSCs to endoderm. It remains unknown whether this TGFβ–PDHB–ATP–BRG1 axis operates in in vivo contexts, or other lineages (mesoderm, ectoderm), where PDHB expression and mitochondrial flux are also relevant.\n➤ Analysis of publicly available embryo scRNA-seq datasets could help address in vivo relevance.\n➤ Directed differentiation to mesoderm or ectoderm in PDHB+/- or BrPA-treated cells would assess lineage specificity.\n5. Statistical Concerns\nThe manuscript reports the use of t-tests throughout, including in experiments with three or more groups, where ANOVA with appropriate post-hoc correction would be more statistically appropriate.\n➤ Authors should re-analyze all multi-group comparisons using ANOVA or other appropriate statistical frameworks.\nMinor Concerns\n1. Figure legends should specify which cell line was used in each experiment.\n2. The Western blot data are not accompanied by quantification or normalization to loading controls.\n3. Clarify terminology: In some sections, PDH and PDHB are used interchangeably. Clearly distinguish PDHB as a subunit and gene target.\n4. Figures 5–6 could benefit from improved resolution and annotations."}, "author_response": "Response to reviewers’ comments\nWe sincerely thank the reviewers for their insightful comments and constructive\nsuggestions, which have greatly helped us improve the quality of our manuscript. In\nresponse to the reviewers’ concerns, we have conducted additional experiments,\nperformed further data analyses, and revised the manuscript to clarify the statements in\nthe previous version. The new data and results mainly include: (1) establishing BRG1-\ninducible degradation and ATPase activity mutant systems to investigate the\nrequirement of its ATPase activity in ATP/BRG1-mediated DE differentiation; (2)\ngenerating a stable PDHB-overexpressing cell line and performing ¹³C-glucose tracing\nto confirm enhanced mitochondrial pyruvate utilization; (3) reproducing key findings\nin another hPSC line (PGP1); (4) performing ChIP-qPCR to assess BRG1 occupancy\nchanges at DE-associated loci; (5) analyzing single-cell RNA-seq data from human pre-\ngastrulation embryos to support the in vivo relevance of our model.\nWe hope that our revisions can address the concerns satisfactorily. With regard to\nthe specific comments, our point-by-point responses are listed below:\nReviewer #1:\nThis manuscript by Meng et al. provides compelling evidence that glucose metabolism,\nspecifically, mitochondrial pyruvate flux governed by TGFβ-induced PDHB expression,\nplays a pivotal role in definitive endoderm (DE) differentiation from human pluripotent\nstem cells (hPSCs). The authors demonstrate that ATP generated through this metabolic\nreprogramming enhances the activity of the BRG1/BAF chromatin remodeling\ncomplex, thereby promoting chromatin accessibility at DE-related gene loci. Through\na comprehensive combination of CRISPR screening, small molecule perturbations,\nmetabolite supplementation, RNA-seq, ATAC-seq, and ChIP-seq, the study establishes\na mechanistic link between extracellular signaling, cellular metabolism, epigenetic\nremodeling, and cell fate determination. The study employs innovative approaches to\nuncover a previously underappreciated role of TGFβ-PDHB signaling in directing\nglucose metabolism toward mitochondrial pyruvate oxidation. However, several\nconcerns should be addressed prior to publication:\nResponse: We sincerely appreciate the reviewer’s positive and encouraging comments,\nparticularly the recognition that “The study employs innovative approaches to uncover\na previously underappreciated role of TGFβ-PDHB signaling in directing glucose\nmetabolism toward mitochondrial pyruvate oxidation”. We have accordingly\nperformed additional experiments and analyses and believe the revised version should\nbe significantly strengthened.\n1. While the study implicates BRG1 as a downstream effector of ATP-mediated\nchromatin remodeling, direct evidence of its ATPase activity modulation is lacking.\nIncorporating experiments with a dominant-negative or ATPase-dead BRG1 mutant\nwould strengthen the causal relationship.\nResponse: We thank the reviewer for this insightful suggestion. We agree that direct\nperturbation of BRG1’s ATPase activity is critical to establish the causal link between\nATP levels and BRG1 function. Given that BRG1 knockdown disrupts pluripotency\nmaintenance in human1, 2, we employed the dTAG-induced degradation system for\nsubsequent experiments3. First, we constructed dTAG-inducible BRG1 degradation\nESC lines to circumvent the potential effects of constitutive BRG1 loss on pluripotency\nmaintenance. Then we rescued with wild-type or ATPase-dead (K798R4, 5) BRG1 to\ninvestigate whether the ATP influenced DE differentiation by ATPase domain of BRG1.\nWestern blotting confirmed complete BRG1 degradation within 8 hours of dTAG\ninduction in these two HUES8-derived clones (Fig. 5E). Consistent with the\nBRM/BRG1 inhibitor experiments, BRG1 loss during DE differentiation impaired\nendoderm formation (Fig. 5F). Bright-field imaging further revealed that, while control\ncells lost their colony morphology during differentiation, dTAG-treated BRG1-depleted\ncells retained compact and colony-like morphology, indicating that they had not exited\nthe pluripotent state.\nThen we further overexpressed wild-type and ATPase-dead (K798R) BRG1 in\ndTAG-induced BRG1 knockout hESCs. The results showed that only wild-type BRG1\nwas able to rescue DE differentiation, whereas the ATPase-dead mutant failed (Fig. 5I).\nThis result indicates that BRG1 regulates the differentiation process via its ATPase\nactivity.\nAfter BRG1 depletion, we supplemented ATP to examine whether it could rescue\nthe DE differentiation defect caused by BRG1 knockout. The results showed that ATP\nsupplementation failed to restore DE differentiation, further supporting that BRG1 is\nessential for ATP-mediated enhancement of DE differentiation efficiency (Fig. 5H).\nAdditionally, we employed BRM014, a specific inhibitor of the ATPase activity of\nBRM/BRG16, to confirm the requirement of BRG1’s ATPase function in ATP-mediated\ndifferentiation. Consistent with previous results, exogenous ATP supplementation\nfailed to promote DE differentiation when BRG1’s ATPase activity was inhibited,\nindicating that ATP exerts its effect on differentiation through the ATPase function of\nBRG1 (Fig. S8A).\nTogether, these complementary approaches—pharmacological inhibition,\ninducible degradation, and ATPase-dead mutant analysis—provide strong evidence that\nBRG1 functions as an ATP downstream effector through its ATPase activity, thereby\nregulating the DE differentiation process.\n2. It remains unclear whether PDHB overexpression or activation is sufficient to\nenhance DE differentiation. Addressing this would clarify whether PDHB acts as a\nlimiting factor in this process.\nResponse: We appreciate the reviewer’s constructive suggestion. To determine whether\nPDHB activation or overexpression is promotive for DE differentiation, we employed\ntwo complementary approaches.\nFirst, we established PDHB-overexpressing ESC lines (Fig. 4F) and performed\nDE differentiation. PDHB overexpression markedly enhanced the conversion of\npyruvate to acetyl-CoA, thereby facilitating its entry into the TCA cycle and promoting\nATP production (Fig. 4G).\nDuring DE differentiation, PDHB overexpression significantly increased\ndifferentiation efficiency (Fig. 4H). Notably, when the Activin A concentration was\nreduced from the standard 100 ng/mL to lower levels, PDHB overexpression further\nenhanced DE differentiation, suggesting that PDHB activity serves as a limiting factor\nunder reduced signaling conditions (Fig. 4I).\nNext, we also used sodium dichloroacetate (DCA; APExBio, Cat# B7174), a\nspecific inhibitor of pyruvate dehydrogenase kinase (PDK)7, which negatively regulates\npyruvate dehydrogenase (PDH) activity through phosphorylation8, 9, and acts as a\npharmacological activator of PDH activity10, 11 to confirm the results of PDHB\noverexpression. The results showed that DCA treatment elevated acetyl-CoA levels and\nsignificantly enhanced DE differentiation efficiency (Fig. S2B-C).\nTogether, PDHB overexpression (genetic manipulation) and DCA treatment\n(pharmacological activation) demonstrate that PDHB overexpression or activation is\nable to enhance DE differentiation, suggesting that PDHB acts as a limiting factor in\nthis process.\n3. Some figures, particularly Fig. 6 depicting ATAC-seq and BRG1 ChIP-seq overlap,\nare densely annotated. Simplifying these visuals and including schematic models would\nimprove accessibility and reader comprehension.\nResponse: We thank the reviewer for this valuable suggestion. In response, we have\nsimplified Figure 6 by reorganizing the ATAC-seq and BRG1 ChIP-seq overlap panels\nand removing redundant annotations to enhance visual clarity.\n4. Terminology such as “mainstream glucose metabolism” versus “PPP” should be\nclearly defined earlier in the manuscript to avoid ambiguity and improve clarity.\nResponse: We appreciate the reviewer’s thoughtful comment. To improve clarity and\nensure terminological consistency, we have revised the manuscript to explicitly define\nthe terms “mainstream glucose metabolism” and “PPP pathway” (in line 165-168).\nSpecifically, “mainstream glucose metabolism” is now defined as the glycolytic flux\nleading to pyruvate generation and subsequent mitochondrial oxidation via the PDH–\nTCA axis, whereas “PPP” refers to the parallel branch responsible for NADPH and\nribose-5-phosphate production for biosynthetic processes.\nReviewer #2 (Remarks to the Author):\nThe authors revealed a metabolic switch with decreased lactate production and\nincreased mitochondrial pyruvate metabolism, which is controlled by TGFβ-activated\nPDHB, is necessary for the definitive endoderm differentiation from human pluripotent\nstem cells. The experiments data, especially the pyruvate rescue and ATP rescue, are\nconvincing and impressive, and the following are the additional points that can be\naddressed.\nResponse: We sincerely appreciate the reviewer’s comment that “The experiments data,\nespecially the pyruvate rescue and ATP rescue, are convincing and impressive”. We\nhave accordingly performed additional experiments and analyses and believe the\nrevised version should be significantly strengthened.\nIn line 55-56 in the introduction, the glucose metabolism is also required for the cell\ntype specification of TE during blastocyst development.\nResponse: We thank the reviewer for this insightful comment. We have now revised\nthe Introduction to include this important point. In the revised manuscript, we have\nadded the following sentences to the Introduction: “Moreover, during this process, both\nthe differentiation of the inner cell mass (ICM) into the three germ layers and the\nspecification of the trophectoderm (TE) are highly dependent on glucose metabolism.\nNotably, glucose deprivation causes a severe developmental arrest and a significant\nloss of CDX2-positive TE cells, highlighting the essential role of glucose metabolism in\nearly embryogenesis12.”\nFor figure 1, the authors can add one or two sentence to explain the rationale of using\nCXCR4 as the readout of DE differentiation in their CRISPR screening.\nResponse: We appreciate this helpful suggestion. In the revised manuscript, we have\nadded that: “In CRISPR screening, we used CXCR4 expression as a quantitative readout\nto evaluate DE differentiation efficiency. As a well-established surface marker of\ndefinitive endoderm, CXCR4 has been extensively utilized in previous studies to assess\nDE differentiation efficiency from pluripotent stem cells13, 14.”\nFor figure 1, whether glucose metabolism inhibition leads to compensation of other\nlineage differentiation, such as ectoderm and mesoderm, or it generally inhibits\ndifferentiation and pluripotency exit in this 2D differentiation system. It was discussed\nin the embryoid body section but not here. Also, the toxicity effect data upon BrPA/2DG\ntreatment was mentioned but not shown.\nResponse: We appreciate the reviewer’s valuable suggestion. To evaluate the impact of\nglucose metabolism inhibition on the differentiation of other germ layers, we performed\nembryoid body (EB) differentiation to assess the effects of BrPA treatment across all\nthree germ layers. The results showed that BrPA treatment slightly impaired the\nexpression of mesendodermal and ectodermal markers (Fig. S2B, in revised\nmanuscript). In Figure 1, we specifically focused on the effect of glucose metabolism\ninhibition on DE differentiation; therefore, data on mesoderm and ectoderm\ndifferentiation were presented somewhere else.\nIn addition, we performed directed differentiation toward ectoderm and mesoderm\nlineages under BrPA treatment. For ectoderm differentiation15, ectoderm markers\n(NESTIN, PAX6, SOX1) were examined on day 3 and 7. Under BrPA treatment,\nectodermal markers remained largely unchanged at day 3 but were markedly reduced\nby day 7 (Fig. S2C). Mesoderm differentiation was induced using a previously\nestablished cardiomyocyte differentiation protocol16. The expression of cardiac-\nspecific markers (TNNT2, GATA4, MYH6) was impaired in BrPA-treated cells,\nindicating impaired differentiation toward the mesodermal cardiac lineage (Fig. S2D).\nCollectively, these results suggest that inhibition of glucose metabolism generally\nsuppresses both ectodermal and mesodermal differentiation.\nFor inhibitor usage, we applied concentrations within the non-cytotoxic range.\nCliff and colleagues selected 2.2 mM as the final concentration of 2-DG17. In our study,\nwe used 1 mM and 2 mM as the working concentration for 2-DG. To assess potential\ncytotoxicity, we measured the apoptosis rate in BrPA or 2-DG-treated, and PDHB+/-\ncells using the Annexin V-FITC/PI Apoptosis Detection Kit. Our results showed no\nsignificant increase in apoptosis compared to the control, indicating that the inhibitors\nwe used did not affect cell survival (Fig. S1D).\nFor figure 2, the pyruvate and glutamine rescue experiments are interesting and\nconvincing in elucidating the role of glucose metabolism in DE differentiation. Can\nthey also rescue the slight changes of ectoderm differentiation in Fig 2I?\nResponse: We sincerely thank the reviewer for this insightful comment. As shown in\nFig. S2B (in revised manuscript), inhibition of glucose metabolism by BrPA treatment\nslightly suppressed ectoderm differentiation. Following the reviewer’s suggestion, we\nperformed rescue experiments by supplementing exogenous pyruvate, glutamine, and\nATP during embryoid body (EB) formation. Consistent with the results observed in DE\ndifferentiation, supplementation with these metabolites effectively restored ectoderm\ndifferentiation efficiency impaired by glucose metabolic inhibition (Fig. S2E).\nIn line 276-279, the authors should better explain the difference of mesoendoderm and\ndefinitive endoderm to make it coherent through the article.\nResponse: We appreciate the reviewer’s suggestion. During pluripotent stem cell\ndifferentiation, cells can directly commit to the ectodermal lineage. However, for\nmesoderm and endoderm lineages, cells first transition through a mesendodermal stage,\nmarked by the expression of EOMES, MIXL1, and T (Brachyury), before further\ndifferentiating into either mesoderm or definitive endoderm (DE)18.\nIn Figure 5E (in revised manuscript), we observed that PDHB depletion (PDHB+/-)\nresulted in an increase in mesendodermal markers. To further investigate this process,\nwe analyzed gene expression dynamics at three time points (Day 3, 5, and 9) during\nembryoid body (EB) differentiation. PDHB+/− cells exhibited a delayed downregulation\nof pluripotency genes and a postponed peak in mesendodermal gene expression:\nmesendoderm markers peaked around Day 5 in wild-type cells, the PDHB+/− cells\nreached this stage around Day 9 or later (Fig. S5G). This suggests that PDHB+/− impairs\nboth the exit from pluripotency and the initial mesendodermal differentiation. Notably,\nthe upregulation of DE markers was also significantly delayed in PDHB+/− cells.\nIn revised manuscript, we also provided a further explanation for this phenotype.\nCan the authors use a PDH inhibitor to show ATP decrease and reduced DE\ndifferentiation, which can be rescued by ATP, in addition to the general glycolysis\ninhibitor?\nResponse: We thank the reviewer for this excellent suggestion. To specifically inhibit\nPDH activity, we used CPI-613 (Selleck S2776), a mitochondrial enzyme inhibitor that\nblocks pyruvate dehydrogenase activity19. Similar to the results observed in PDHB+/-\ncells, CPI-613 treatment led to a dose-dependent reduction in DE differentiation\nefficiency, accompanied by a significant decrease in intracellular ATP levels.\nImportantly, both ATP content and differentiation efficiency were successfully rescued\nby exogenous ATP supplementation (Fig. S7C), further confirming that PDH-regulated\nmetabolic changes influence DE differentiation via ATP availability.\nFor fig 5, the ATP rescue experiments are impressive. Can supplemented ATP be\npermeable to the cell membrane to change the intracellular ATP level, and is there other\nreports or this is the first time to show ATP can participate in the ATP-dependent\nchromatin remodeling processes. These points can be at least discussed in the\ndiscussion section.\nResponse: We sincerely thank the reviewer for this constructive comment. ATP is a\ncharged and hydrophilic molecule that is difficult to freely cross the plasma membrane.\nMoreover, no plasma membrane-associated ATP transporter has been identified.\nTherefore, whether exogenously supplied ATP can enter the cell and regulate\nintracellular energy levels is a critical question.\nSeveral earlier studies have demonstrated that ATP can be both released from and\ntaken up by cells20, 21, 22. However, the exact mechanisms underlying this process\nremained unclear due to technical limitations. Recent work using nonhydrolyzable\nfluorescent ATP (NHF-ATP) tracing methods has shown that multiple cell types,\nincluding human lung cancer cell A549 and human non-small cell lung cancer (NSCLC)\ncells, can internalize extracellular ATP via macropinocytosis, leading to an increase in\nintracellular ATP levels23, 24, 25, 26. These findings provide strong evidence for the\ninternalization of extracellular ATP and suggest that cellular energy status can be\nmodulated through exogenous ATP supplementation.\nIn our experiments, we directly measured intracellular\nATP levels and confirmed that ATP supplementation\nsuccessfully restored ATP concentrations in DE cells (Fig.\nS8A). This restoration occurred both under normal conditions\nand when BRG1 ATPase activity was inhibited, further\nsupporting the idea that extracellular ATP can influence\nintracellular ATP levels and, consequently, impact cellular\nprocesses dependent on ATP availability.\nRegarding the role of ATP in ATP-dependent chromatin\nremodeling, we acknowledge that our study is not the first to report such an effect.\nPrevious structural and biochemical studies of the BAF complex have demonstrated\nthat ATP binding and hydrolysis at the ATPase domain are essential for chromatin\nremodeling activity. This has been validated through ATPase activity assays and\nrestriction enzyme accessibility assays (REAA) (shown below) 27. Together, these\nfindings provide mechanistic support for our results, which show that ATP-mediated\nactivation of BRG1 facilitates chromatin accessibility and contributes to cell\ndifferentiation.\n[FIGURE REDACTED]\nSome of the grammar should be polished such as:\nLine 139 To further validate the promotional role\nLine 144 evident by the decreased acetyl-CoA\nLine 169 Pyruvate entering TCA cycle facilitates DE differentiation\nTCA cycle should be the TCA cycle.\nResponse: We appreciate the reviewer’s careful review. In response, we have made the\nfollowing revisions to improve clarity and readability:\nLine 139: \"To further validate the promotional role\" has been revised to \"To further\nvalidate the promotive role\" for clarity.\nLine 144: \"Evident by the decreased acetyl-CoA\" has been revised to \"Evident from\nthe decreased acetyl-CoA\" to improve the phrasing.\nLine 169: \"Pyruvate entering TCA cycle facilitates DE differentiation\" has been revised\nto \"Pyruvate entering the TCA cycle facilitates DE differentiation\" to ensure correct\narticle usage.\nReviewer #3 (Remarks to the Author):\nSummary\nMeng et al. investigates how glucose metabolism regulates endoderm differentiation\nfrom human iPS cells. The authors demonstrate that mitochondrial pyruvate\nmetabolism is required for ATP production, which in turn supports the chromatin\nremodeling activity of the BRG1-containing BAF complex. They further link this\nmetabolic switch to TGFβ signaling through transcriptional regulation of PDHB by\nSMAD2/3. Loss of PDHB impairs DE differentiation, which can be partially rescued\nby exogenous ATP, and this rescue is blocked by BAF complex inhibition. These\nfindings are supported by extensive molecular analyses including CRISPR screens,\nmetabolite measurements, ATAC-seq, ChIP-seq, and RNA-seq.\nWhile the study makes a compelling case for the involvement of mitochondrial\nmetabolism in endoderm fate commitment, several major conceptual, technical, and\ninterpretational limitations lessen its current suitability for publication in Nature\nCommunications.\nConceptual Impact and Novelty / Overall Big Picture Issues\nThe manuscript brings forward a conceptually appealing model that connects growth\nfactor signaling (TGFβ), metabolic state (pyruvate flux → ATP), and chromatin\nremodeling (BAF complex) to orchestrate cell fate decisions in human-iPS cells. The\nnovelty resides in linking ATP-dependent chromatin remodeling to a specific metabolic\nswitch regulated by PDHB, as a direct target of SMAD2/3. This integration of\nmetabolism and epigenetics during germ layer specification is timely and of interest to\na broad audience.\nHowever, several gaps undermine the model’s completeness and generalizability:\n- The central mechanism (ATP → BAF) is not fully validated, and alternative ATP-\ndependent processes are not addressed.\n- The metabolic reprogramming is inferred, not directly measured via flux analysis.\n- Findings are derived from a single cell line, limiting generalizability.\n- The in vivo relevance is not addressed at all.\nTogether, these issues limit the physiological and mechanistic depth required for\npublication in a high-impact journal.\nResponse: We sincerely appreciate the reviewer’s evaluation of our work, particularly\nthe recognition that our study “brings forward a conceptually appealing model” and\nhighlights the novelty of linking TGFβ signaling, mitochondrial metabolism, and\nchromatin remodeling in endoderm fate commitment. In response to the reviewer’s\ninsightful comments, we have performed additional experiments and analyses to\naddress the raised concerns, and we believe that these revisions have substantially\nstrengthened the mechanistic and conceptual depth of the study.\nMajor Concerns\n1. Ambiguity in Cell Line Usage\nAlthough the Methods section states the use of both HUES8 and PGP1 cells, the figures,\nlegends, and Results fail to specify which cell line was used in each experiment. This\nraises concerns about reproducibility and generalizability.\n➤ Authors must clarify which experiments were performed in which cell lines, and\nreplicate key findings (e.g., ATP rescue, PDHB KD) in the second line.\nResponse: We thank the reviewer for this important comment. In our study, the majority\nof experiments were performed using the human embryonic stem cell line HUES8,\nwhile selected key experiments were independently validated in another cell line PGP1,\nsuch as the Fig. 1E (in revised manuscript). We have now clearly indicated the specific\ncell line used in each figure and corresponding legend in the revised manuscript.\nIn the revised manuscript, we also replicated more critical experiments in PGP1\ncells. Specifically, we used BrPA to inhibit global glycolytic activity and CPI-613\n(Selleck S2776) as a selective PDH inhibitor19. Consistent with the results obtained in\nHUES8 cells (in answers for reviewer#2, Fig. S7C), both treatments led to decreased\nintracellular ATP levels and impaired definitive endoderm differentiation, while\nexogenous ATP supplementation successfully restored ATP levels and differentiation\nefficiency. Furthermore, inhibition of BRG1 ATPase activity (by BRM/BRG1 inhibitor,\nBRM014) abolished this ATP-mediated rescue effect (Fig. S8C), supporting that the\nobserved phenomena are conserved across different stem cell lines.\n2. Mechanistic Inference of ATP → BAF is Not Fully Supported\nFigure 5A shows that ATP rescues DE differentiation in PDHB+/- or BrPA-treated cells,\nand that this rescue is blocked by BAF inhibitors. While this is consistent with the\nproposed model, it does not exclude other ATP-dependent regulators of chromatin or\ndifferentiation.\n➤ Authors should directly assess BAF complex activity in response to ATP changes\n(e.g., BRG1 occupancy at DE loci, ATPase activity, or nucleosome repositioning\nassays).\nResponse: We thank the reviewer for the insightful suggestion. Previous structural and\nbiochemical studies of the BAF complex have demonstrated that ATP binding and\nhydrolysis at the ATPase domain are essential for chromatin remodeling activity, being\nvalidated through ATPase activity assays and restriction enzyme accessibility assays\n(REAA) 27. To investigate whether ATP influences DE differentiation by modulating\nBRG1 occupancy at DE-associated loci in our experiment, we performed ChIP-qPCR\non several key regulators of DE differentiation. The results revealed that PDHB\nheterozygous deletion led to a marked decrease in BRG1 enrichment at these loci\n(including FOXI1, TCF4, SMAD3 and SOX17) (Fig.6I), concomitant with the reduced\nchromatin accessibility observed in ATAC-seq. These findings provide mechanistic\ninsight into how reduced chromatin accessibility caused by BRG1 dysfunction is linked\nto impaired DE differentiation, highlighting the role of ATP and PDHB in regulating\nBRG1 activity at these loci.\n➤ Alternatively, loss-of-function (e.g., BRG1 KO) combined with ATP rescue would\nstrengthen causal inference.\nResponse: We thank the reviewer for this insightful suggestion. We agree that direct\nperturbation of BRG1’s ATPase activity is critical to establish the causal link between\nATP levels and BRG1 function. Given that BRG1 knockdown disrupts pluripotency\nmaintenance in human, we employed the dTAG-induced degradation system for\nsubsequent experiments1, 2. First, we constructed dTAG-inducible BRG1 degradation\ncell lines to circumvent the potential effects of constitutive BRG1 loss on pluripotency\nmaintenance. Then we rescued with wild-type or ATPase-dead (K798R4, 5) BRG1 to\ninvestigate whether the ATP influenced DE differentiation by ATPase domain of BRG1.\nWestern blotting confirmed complete BRG1 degradation within 8 hours of dTAG\ninduction (Fig. 5E). Consistent with the BRM/BRG1 inhibitor experiments, BRG1 loss\nduring DE differentiation impaired endoderm formation (Fig. 5F). Then we further\noverexpressed wild-type and ATPase-dead (K798R) BRG1 in dTAG-induced BRG1\nknockout hESCs. The results showed that only wild-type BRG1 was able to rescue DE\ndifferentiation, whereas the ATPase-dead mutant failed (Fig. 5I). These results indicate\nthat BRG1 regulates the differentiation process via its ATPase activity.\nAfter BRG1 deletion, we supplemented ATP to examine whether it could rescue\nthe DE differentiation defect caused by BRG1 knockout. The results showed that ATP\nsupplementation failed to restore DE differentiation, further supporting that the ATPase\nactivity of BRG1 is essential for ATP-mediated enhancement of DE differentiation\nefficiency (Fig. 5H). Additionally, we employed BRM014, a specific inhibitor of the\nATPase activity of BRM/BRG16, to confirm the requirement of BRG1’s ATPase\nfunction in ATP-mediated differentiation. Consistent with previous results, exogenous\nATP supplementation failed to promote DE differentiation when BRG1’s ATPase\nactivity was inhibited (Fig. S8A), indicating that ATP exerts its effect on differentiation\nthrough the ATPase function of BRG1.\nTogether, these complementary approaches—pharmacological inhibition,\ninducible degradation, and ATPase-dead mutant analysis—provide strong evidence that\nBRG1 functions as an ATP downstream effector through its ATPase activity, thereby\nregulating the DE differentiation process.\n3. Lack of Direct Metabolic Flux Measurements\nThe metabolic switch is deduced from static metabolite levels and inhibitor use. These\nare suggestive but not conclusive.\n➤ Authors should include stable isotope tracing (e.g., 13C-glucose or 13C-pyruvate)\nto directly confirm increased flux through the TCA cycle during differentiation and after\nTGFβ/PDHB activation.\nResponse: We thank the reviewer for the good advice. In response, we performed [U-\n¹³C]-glucose tracing experiments to analyze metabolism alterations during\ndifferentiation and PDHB activation. During DE differentiation, we indeed observed a\ndecrease in the ¹³C labeling ratio of several glycolytic intermediates, including fructose-\n1,6-bisphosphate (FBP), 1,3-bisphosphoglycerate (1,3-BPG), 3- phosphoglycerate /2-\nphosphoglycerate (3PG/2PG), phosphoenolpyruvate (PEP), as well as the end product\nlactate. These results further confirm the reduced glycolytic activity following DE\ndifferentiation.\nNext, we established a PDHB-overexpressing hESC line (Fig. 4F) to examine\nchanges in TCA cycle intermediates upon PDHB overexpression. In terms of metabolic\nchanges, PDHB overexpression increased glucose-derived labeling in key TCA cycle\nmetabolites, including citrate,\naconitate, succinate, and\nsuccinyl-CoA (Fig. S6A-B).\nCollectively, our ¹³C metabolic\nflux analysis demonstrates that\nPDHB overexpression promotes\na metabolic shift toward elevated\nTCA cycle activity, providing a\nmechanistic explanation for the\nenhanced DE differentiation.\n4. No Exploration of In Vivo or Cross-Lineage Relevance\nAll findings are based on in vitro differentiation of human iPSCs to endoderm. It\nremains unknown whether this TGFβ–PDHB–ATP–BRG1 axis operates in in vivo\ncontexts, or other lineages (mesoderm, ectoderm), where PDHB expression and\nmitochondrial flux are also relevant.\n➤ Analysis of publicly available embryo scRNA-seq datasets could help address in\nvivo relevance.\nResponse: We thank the reviewer for this constructive suggestion. To further address\nthis issue, we analyzed the single-cell RNA-seq dataset of 3D-cultured human pre-\ngastrulation embryos 28, which includes multiple cell types at the gastrulation stage,\nsuch as the embryonic disc, amnion, basement membrane, primary and primate-specific\nsecondary yolk sac, anterior–posterior polarity formation, and primitive streak anlage\n(PSA)29. Based on these data, we examined the expression patterns of genes involved\nin glycolysis, the TCA cycle, oxidative phosphorylation, and the BAF chromatin\nremodeling complex across different embryonic tissues.\nOur analysis revealed that during the developmental transition from the inner cell\nmass (ICM) to epiblast (EPI) and subsequently to PSA-EPI, glycolytic gene\nexpression—including canonical glycolytic genes such as GAPDH, GPI, HK2, and\nPDHB—was markedly decreased, whereas genes related to the TCA cycle and\noxidative phosphorylation were significantly upregulated. These results indicate a clear\nmetabolic shift toward mitochondrial oxidative metabolism during this stage.\nFurthermore, genes associated with the BAF complex, such as SMARCA4 (BRG1),\nBRD7, BRD9, and ARID1A, also exhibited a moderate increase in expression\nthroughout the ICM-EPI-PSA-EPI differentiation process. Together, these findings\nsuggest that the metabolic and epigenetic changes described in our study also occur\nduring normal human embryonic development. Notably, such a metabolic transition\nwas not observed in lineages differentiating toward extraembryonic fates. (CTBs:\ncytotrophoblasts; STBs: syncytiotrophoblasts; EVTs: extravillous cytotrophoblasts;\nPrE: primitive endoderm/hypoblast)\n[FIGURE REDACTED]\n➤ Directed differentiation to mesoderm or ectoderm in PDHB+/- or BrPA-treated cells\nwould assess lineage specificity.\nResponse: We appreciate the reviewer’s valuable suggestion. To assess lineage\nspecificity, we performed ectoderm differentiation using the protocol described in our\nprevious publication15. On day 3 and 7 of differentiation, we examined the expression\nof pluripotency genes and ectoderm-associated markers, including NESTIN, PAX6, and\nSOX1. Under BrPA treatment, ectodermal gene expression showed no significant\nreduction at day 3 but was markedly decreased at day 7. Consistently, the PDHB+/-\ncell\nline exhibited suppression of ectodermal gene expression at both time points. Regarding\npluripotency genes, we observed a slightly increase at day 3, reflecting delayed\ndifferentiation. However, this increase diminished at day 7, indicating that despite\nimpaired ectoderm differentiation, the cells still exited the pluripotent state.\nTo further clarify the effect on mesoderm differentiation, we performed directed\ncardiomyocyte differentiation16. We observed that the expression of cardiac-specific\nmarkers (TNNT2, GATA4, MYH6, MEF2C) was impaired in BrPA treated and PDHB+/-\ncell line, indicating an impaired differentiation phenotype toward the mesodermal\ncardiac lineage.\n5. Statistical Concerns\nThe manuscript reports the use of t-tests throughout, including in experiments with\nthree or more groups, where ANOVA with appropriate post-hoc correction would be\nmore statistically appropriate.\n➤ Authors should re-analyze all multi-group comparisons using ANOVA or other\nappropriate statistical frameworks.\nResponse: We appreciate the reviewer’s insightful comment regarding the statistical\nanalysis. In response, we have re-analyzed all multi-group comparisons. For figures\nwith three or more groups, statistics were calculated using one-way ANOVA, followed\nby Dunnett’s multiple comparisons test. Two-way ANOVA was used when two\nvariables were involved, followed by Tukey's multiple comparisons test. For\ncomparisons between two groups, paired t-tests were applied directly. The revised\nstatistical analyses and updated results are now included in the manuscript and\ncorresponding figure legends.\nMinor Concerns\n1. Figure legends should specify which cell line was used in each experiment.\nResponse: We thank the reviewer for pointing this out. In the revised manuscript, we\nhave updated the figure legends to clearly specify which cell line was used in each\nexperiment, ensuring better clarity for readers.\n2. The Western blot data are not accompanied by quantification or normalization to\nloading controls.\nResponse: We appreciate the reviewer’s comment regarding the Western blot data. In\nresponse, we have now included quantification of the Western blot signals and have\nnormalized the data to appropriate loading controls (e.g., α-tubulin or H3) for all\nrelevant experiments. These updates are described in the figure legends.\n3. Clarify terminology: In some sections, PDH and PDHB are used interchangeably.\nClearly distinguish PDHB as a subunit and gene target.\nResponse: We thank the reviewer for the careful review. We have revised the\nmanuscript to clarify the terminology. Specifically, we now distinguish PDHB as the\nsubunit of pyruvate dehydrogenase (PDH) and the gene encoding this subunit. The\nrevised manuscript clearly differentiates these terms to avoid any confusion.\n4. Figures 5–6 could benefit from improved resolution and annotations.\nResponse: We appreciate the reviewer’s suggestion to improve the resolution and\nannotations of Figures 5 and 6. In response, we have enhanced the resolution of these\nfigures and added additional annotations to improve clarity and accessibility.\nReference\n1. Ho L, et al. An embryonic stem cell chromatin remodeling complex, esBAF, is essential for\nembryonic stem cell self-renewal and pluripotency. Proc Natl Acad Sci U S A 106, 5181-\n5186 (2009).\n2. 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Extracellular ATP is internalized by macropinocytosis and induces intracellular\nATP increase and drug resistance in cancer cells. Cancer Lett 351, 242-251 (2014).\n26. Qian Y, Wang X, Li Y, Cao Y, Chen X. Extracellular ATP a New Player in Cancer Metabolism:\nNSCLC Cells Internalize ATP In Vitro and In Vivo Using Multiple Endocytic Mechanisms.\nMol Cancer Res 14, 1087-1096 (2016).\n27. Mashtalir N, et al. A Structural Model of the Endogenous Human BAF Complex Informs\nDisease Mechanisms. Cell 183, 802-817 e824 (2020).\n28. Xiang L, et al. A developmental landscape of 3D-cultured human pre-gastrulation\nembryos. Nature 577, 537-542 (2020).\n29. Jiang L, et al. Cell size regulates human endoderm specification through actomyosin-\ndependent AMOT-YAP signaling. Stem Cell Reports 19, 1137-1155 (2024)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All previous concerns have been addressed.", "reviewer_2": "The authors have addressed all my questions, and the manuscript is qualified for the publication.", "reviewer_3": "You have done a great job and attempting to address all of my outlined concerns. I have no further comments. Congrats on a wonderful study."}, "author_response": ""}]

Decision letter