GCN5 drives MASLD progression through LXRα/SREBP1c signaling pathway–mediated de novo lipogenesis

Domain: neuroscience
Published: 2026-02-16
Editorial outcome: minor_revision

Abstract
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern that affects nearly one-quarter of the world’s population. General control non-repressed protein 5 (GCN5), a histone acetyltransferase (HAT), has been implicated in the progression of several diseases, but its role in MASLD remains unclear. Here, we provide the experimental evidence that progressive human and male murine MASLD is driven by GCN5, but not by p300/CREB binding protein associated factor (PCAF) activation. Hepatocyte-specific GCN5 overexpression accelerates MASLD progression, whereas its ablation alleviates disease severity. Moreover, pharmacological inhibition of GCN5 with CPTH2 protects against MASLD. Metabolomics and RNA-seq analyses demonstrate that GCN5 promotes de novo lipogenesis (DNL) by upregulating SREBP1c-mediated transcription of lipogenic genes. Mechanistically, GCN5 acetylates histone H3 at the SREBP1c promoter, enhancing transcription through its intrinsic acetyltransferase activity. Our findings further identify GCN5 as a key regulator of LXRα-induced SREBP1c expression, suggesting that targeting GCN5 may selectively inhibit SREBP1c-driven DNL without impairing LXRα-mediated reverse cholesterol transport (RCT). Notably, combined treatment with the Liver X Receptor (LXR) agonist T0901317 and CPTH2 synergistically reduced lipid accumulation in vitro and in vivo, highlighting a promising therapeutic strategy for MASLD.

Introduction
Non-alcoholic fatty liver disease (NAFLD), now known as metabolic dysfunction-associated steatotic liver disease (MASLD), is a global health concern, that affects nearly one-quarter of the world’s population1. MASLD encompasses a spectrum of conditions, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), which markedly increases the risk of cirrhosis, hepatocellular carcinoma (HCC), and systemic metabolic disorders2. In 2024, the U.S. Food and Drug Administration (FDA) approved Resmetirom, an oral selective agonist of thyroid hormone receptor (THR)-β, for the treatment of MASH with liver fibrosis. This approval marked the introduction of the first targeted drug therapy for MASH3. However, further validation of its clinical efficacy is warranted. Thus, there is an urgent need to elucidate the mechanisms underlying the onset and progression of MASLD to enable the development of effective therapeutic interventions4.

MASLD arises from a complex interplay of various environmental and genetic factors. According to the double-hit theory of pathogenesis, the first hit is the accumulation of triglycerides in hepatocytes, followed by a second hit characterized by the activation of inflammatory mediators leading to hepatocellular damage, inflammation, and fibrosis5,6. More recent models propose that multiple concurrent factors contribute to MASLD development. Fatty acids and their metabolites have been implicated in the transition from simple steatosis to MASH. Insulin resistance (IR) promotes hepatic accumulation of free fatty acids, resulting in endoplasmic reticulum (ER) stress, oxidative stress, and ultimately hepatocyte apoptosis. In turn, fatty acids exacerbate IR in the liver by promoting lipid storage7. Despite these insights, the precise mechanisms driving MASLD pathogenesis remain unclear.

Epigenetics provides a compelling framework for understanding how lifestyle, environmental exposures, and other risk factors influence MASLD development8. Histone acetylation, a post-translational modification that occurs mainly at specific lysine residues in the N-terminal tails of histones H3 and H4, regulates chromatin accessibility and gene expression9. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain histone acetylation in a dynamic equilibrium. Evidence indicates that histone acetylation patterns are altered in MASLD. For example, studies in Japanese macaques have demonstrated increased acetylation at H3K14 in fetal hepatic tissue, as well as elevated acetylation at H3K9 and H3K18. In addition, a maternal high-fat diet (HFD) reduced HDAC1 protein levels in the fetal liver. These findings suggest that maternal obesity induced by a HFD alters fetal chromatin structure through histone modifications10–13.

General control non-repressed protein 5 (GCN5) was the first HAT to be cloned and identified in yeast and belongs to the N-acetyltransferase superfamily14. GCN5 is widely conserved across eukaryotes and contains a C-terminal bromodomain (BRD) that recognizes acetylated histones, a catalytically active and relatively conserved HAT domain, and a PCAF homology domain that recognizes the N-terminus of nucleosomes15. GCN5 is primarily expressed in the liver, brain, thyroid, kidney, and spleen, and is mainly localized in the nucleus and associated with the microtubule cytoskeleton. In mammals, GCN5 shares 73% sequence homology with p300/CREB binding protein–associated factor (PCAF); both possess histone acetyltransferase and lysine acetyltransferase activity16. GCN5 acetylates histone H3 at lysine residues K9 and K14, and it also acetylates non-histone proteins. Functionally, GCN5 regulates cell proliferation, metabolism, and inflammation, and its dysregulation has been linked to the progression of cancer, metabolic, autoimmune, and neurological disorders17. Recently, GCN5 has emerged as a promising therapeutic target, and high-throughput screening approaches have been established to identify selective inhibitors. To date, more than 20 small-molecule inhibitors with distinct structural scaffolds have been reported18,19.

Studies indicate that GCN5 regulates hepatic gluconeogenesis and is closely associated with diabetes20–23, but its role in MASLD has not been elucidated. Based on clinical and experimental evidence, we investigated the involvement of hepatocyte GCN5 in MASLD progression. We demonstrate that GCN5 promotes hepatic de novo lipogenesis (DNL) by regulating the transcriptional activity of LXRα/RXRα-mediated SREBP1c.

Results
GCN5 expression is triggered in patients and mice with MASLD
We first examined the dynamics of GCN5 (KAT2A) and PCAF (KAT2B) expression in liver tissue from individuals with healthy livers (control group, n=11) and patients with progressive MASLD (mild to severe steatosis, n=117) (Supplementary Data 1). Western blotting and immunohistochemistry analyses showed that GCN5, but not PCAF protein levels, were markedly higher in liver tissue from patients with MASLD than in healthy controls (Fig. 1A–D). Consistently, qRT-PCR analysis revealed significantly higher KAT2A, but not KAT2B, mRNA expression in liver tissue from patients with MASLD (Fig. 1E). Both protein and mRNA levels of hepatic GCN5, but not PCAF, positively correlated with MASLD progression (Fig. 1A–E). Supporting the role of GCN5 in disease acceleration, we observed a positive correlation of intrahepatic GCN5 expression with MASLD progression determined by the MASLD activity score (MAS), and the levels of AST, ALT, TG, TC, LDL-c, and bile acid in blood samples from patients with MASLD (Fig. 1F–L). The blood levels of HDL-c, insulin, and glucose did not correlate with the protein levels of GCN5 or PCAF (Supplementary Fig. S1A–C).

Based on these findings, we further evaluated GCN5 and PCAF expression during MASLD progression and investigated GCN5 induction and nuclear localization in a murine model. Mice were fed an HFD for 8, 16, or 32 weeks (Fig. 1M). This model is well established for studying sequential stages of MASLD progression. Consistent with earlier studies, HFD-fed mice developed hepatic steatosis with ballooned hepatocytes and varying degrees of lobular inflammation24. Moreover, in line with our findings in human MASLD, western blotting and immunohistochemistry confirmed strong induction and nuclear accumulation of GCN5, but not PCAF, in hepatocytes during MASLD progression (Fig. 1N–Q). Hepatic Kat2a, but not Kat2b, mRNA levels were also elevated with disease progression (Fig. 1R). We next examined GCN5 expression across different liver cell types. GCN5 protein and mRNA levels were significantly increased in HL-7702 and HepG2 cells, as well as in mouse primary hepatocytes treated with PAOA (Supplementary Fig. S1D–L).

To investigate the mechanism underlying lipotoxicity-induced GCN5 expression, we used JASPAR analysis to predict transcription factors (TFs) potentially binding to the KAT2A promoter. This analysis identified four candidates: KLF5, KLF15, KLF16, and ZNF384 (Supplementary Fig. S1M). Functional validation was performed using luciferase reporter constructs containing either the full-length (–2000 to +200bp) or truncated versions of the KAT2A promoter. Dual-luciferase assays showed that only KLF15 significantly activated the full-length promoter (Supplementary Fig. S1N). This result was corroborated in PAOA-treated HL-7702 hepatocytes, where KLF15 overexpression specifically increased KAT2A transcription (Supplementary Fig. S1O). To further characterize this interaction, we performed chromatin immunoprecipitation (ChIP) assays with primers targeting the predicted KLF15 binding sites identified by JASPAR. The results confirmed direct binding of KLF15 to the KAT2A promoter region spanning –1330 to –1180bp (Supplementary Fig. S1P). Importantly, site-directed mutagenesis of the core binding motif within this region abolished KLF15-mediated promoter activation (Supplementary Fig. S1Q), providing mechanistic evidence that KLF15 regulates KAT2A expression through this cis-element. Together, these experiments in progressive human and murine MASLD establish the first experimental evidence that GCN5 activation contributes to disease acceleration.

Hepatocellular GCN5 overexpression exacerbates MASLD progression in vivo and increases lipid accumulation in hepatocytes in vitro
To confirm the association between hepatic GCN5 expression and MASLD progression, we generated a wild-type (WT) KAT2A expression vector using adeno-associated virus serotype 8 (AAV-Kat2a WT) (Fig. 2A). Western blotting confirmed that tail vein injection of AAV-Kat2a WT selectively increased GCN5 expression in the liver, but not in other tissues (Fig. 2B, and Supplementary Fig. S2A, B). Compared with AAV-LacZ controls, Kat2a WT overexpression in the liver did not alter body weight or food intake (Supplementary Fig. S2C, D). However, Kat2a WT overexpression increased liver weight (LW) and the liver-to-body weight ratio (LW/BW) in high-fat diet (HFD)-fed mice (Fig. 2C). Hematoxylin and eosin (H&E) and Oil-Red O staining further showed that Kat2a WT markedly promoted hepatic lipid accumulation in HFD-fed mice (Fig. 2D, and Supplementary Fig. S2E), consistent with elevated liver and serum TG, TC, HDL-c, and LDL-c levels (Fig. 2E, F). In addition, hepatocyte ballooning was more pronounced in HFD-fed mice overexpressing Kat2a WT (Fig. 2D). Serum AST and ALT levels were also significantly elevated in Kat2a WT-overexpressing HFD mice compared with control-HFD mice (Fig. 2G). As a negative control, we expressed an acetyltransferase-defective mutant (GCN5-E575Q, AAV-Kat2a MUT) (Fig. 2H). Tail vein injection of AAV-Kat2a MUT successfully increased hepatic GCN5 expression without affecting other tissues (Fig. 2I and Supplementary Fig. S2F, G). Like AAV-LacZ controls, Kat2a MUT overexpression did not affect body weight or food intake (Supplementary Fig. S2H, I). Unlike Kat2a WT, however, Kat2a MUT did not promote abnormal lipid accumulation in the liver or blood of HFD-fed mice (Fig. 2J–N, and Supplementary Fig. S2J).

To further assess the effects of GCN5 on hepatocellular lipid metabolism, WT and acetyltransferase-defective (E575Q) KAT2A constructs were transfected into HepG2, HL-7702, and primary mouse hepatocytes (Supplementary Fig. S2K–M). Overexpression of KAT2A WT, but not KAT2A MUT, significantly increased cellular TG and TC levels (Fig. 2O, Q, S). Consistently, staining with the neutral lipid dyes Nile Red and BODIPY 493/503 (Bodipy) revealed that lipid droplet size and number were significantly higher in hepatocytes overexpressing KAT2A WT, but not KAT2A MUT (Fig. 2P, R, T). Together, these results demonstrate that hepatocellular GCN5 overexpression exacerbates MASLD progression in vivo and promotes lipid accumulation in hepatocytes in vitro.

Hepatocyte-specific GCN5 deletion ameliorates MASLD/MASH
To investigate the role of GCN5 in MASLD pathogenesis, hepatocyte-specific Kat2a-knockout (Kat2aHKO) mice were generated by crossing Kat2afl/fl mice with Alb-Cre mice and feeding them an HFD (Fig. 3A and Supplementary Fig. S3A, B). qRT-PCR and western blotting confirmed successful knockout of Kat2a in hepatocytes (Fig. 3B and Supplementary Fig. S3C–D). Hepatocyte-specific Kat2a deletion did not affect body weight or food intake under HFD conditions (Supplementary Fig. S3E, F). Importantly, compared with WT littermates, Kat2aHKO mice had lower LW and LW/BW (Fig. 3C), as well as less severe hepatic steatosis after 10 weeks of HFD feeding, as shown by H&E and Oil Red O staining (Fig. 3D and Supplementary Fig. S3G). Kat2aHKO mice also developed less severe hypertriglyceridemia and hypercholesterolemia than Kat2afl/fl mice (Fig. 3E). Hepatic TG and TC contents were markedly reduced in Kat2aHKO mice compared with Kat2afl/fl mice (Fig. 3F). Hepatocyte ballooning was also attenuated in Kat2aHKO mice (Fig. 3D). Serum AST and ALT levels were significantly lower in Kat2aHKO mice than in Kat2afl/fl mice (Fig. 3G). To further assess the role of GCN5 in MASLD progression, mice were fed a high-fructose diet (HFrD) for 6 weeks (Supplementary Fig. S4A–K) or an Amylin liver NASH (AMLN) diet for 16 weeks (Supplementary Fig. S5A–K). Results from both models were consistent with those obtained in HFD-fed mice.

We next fed Kat2afl/fl and Kat2aHKO mice a fructose–palmitate–cholesterol–trans-fat (FPC) diet for 16 or 24 weeks to investigate the involvement of GCN5 in MASLD to MASH (Fig. 3H, I, and Supplementary Fig. S6A). Body weight and food intake did not differ significantly between Kat2aHKO and Kat2afl/fl mice (Supplementary Fig. S6B, C). After 16 and 24 weeks of FPC feeding, Kat2aHKO mice showed significantly reduced LW and LW/BW (Fig. 3J), lower fasting serum TG, TC, and LDL-c levels, and higher HDL-c levels (Fig. 3K). H&E and Oil Red O staining and hepatic TG and TC measurements confirmed reduced hepatic steatosis in Kat2aHKO mice compared with Kat2afl/fl mice (Fig. 3L–N and Supplementary Fig. S6D, E). In addition, Kat2aHKO ameliorated FPC diet-induced liver injury, as indicated by decreased plasma AST and ALT levels (Fig. 3O). Furthermore, Kat2aHKO mice showed lower degrees of inflammation and fibrosis than Kat2afl/fl mice, as evidenced by F4/80, and Sirius Red staining (Fig. 3P and Supplementary Fig. S6F, G). These phenomena were accompanied by reduced expression of MASH-related inflammatory factors, such as IL6, IL16, Cxcl1, Cxcl5, and Cxcl10, as well as lower expression of profibrotic and MASH-related profibrotic genes, including Acta2, α-Sma, ctgf, Tgfβ, Col3a1, and Col1a1 (Fig. 3Q). To investigate the role of GCN5 deficiency in a specific MASH model, we used a mouse model of a high-fat and methionine-choline-deficient refined diet (HFMRCD)-induced MASH, in which the animals exhibited more profound inflammatory responses and fibrosis symptoms (Fig. 3R, S and Supplementary Fig. S6H). Body weight and food intake were not significantly different between Kat2aHKO and Kat2afl/fl mice (Supplementary Fig. S6I, J). However, the LW and LW/BW of Kat2aHKO mice were significantly decreased (Fig. 3T). Kat2aHKO mice had lower fasting blood lipid levels, including TG, TC, and LDL-c, and higher HDL-c levels (Fig. 3U). Furthermore, H&E staining, Oil Red O staining, and hepatic TG and TC concentrations showed that Kat2aHKO mice had reduced hepatic steatosis in response to the HFMRCD diet (Fig. 3V, W and Supplementary Fig. S6K). In addition, Kat2aHKO mitigated HFMRCD-induced liver injury, as indicated by decreased plasma AST and ALT levels (Fig. 3X). Kat2aHKO mice also exhibited lower degrees of inflammation and fibrosis than Kat2afl/fl mice, as evidenced by F4/80 and Sirius Red staining (Fig. 3W and Supplementary Fig. S6L, M). These effects were accompanied by reduced expression of MASH-related inflammatory factors, such as IL6, IL16, Cxcl1, Cxcl5, and Cxcl10 (Fig. 3Y), and lower expression of profibrotic and MASH-related profibrotic genes, including Acta2, α-Sma, ctgf, Tgfβ, Col3a1, and Col1a1 (Fig. 3Z). Moreover, hepatic GCN5 deficiency markedly decreased HFMRCD-induced overactivation of signaling pathways involving the transcription factor NF-κB by promoting the phosphorylation of the NF-κB inhibitor α (Iκbα) and the p65 NF-κB subunit (Supplementary Fig. S6N). The TGF-β1/Smad signaling pathway is of great importance in the development of liver fibrosis25. As expected, the marked increase in phosphorylated SMAD2/3 protein levels was reduced in Kat2aHKO mice (Supplementary Fig. S6N). Meanwhile, protein expression of Collagen I and α-SMA was also reduced in Kat2aHKO mice (Supplementary Fig. S6N). Furthermore, we generated a KAT2A knockout HL-7702 cell line (KAT2AKO) using the CRISPR-Cas9 system to investigate the function of GCN5 (Supplementary Fig. S7A, B). Knockout of KAT2A reduced cellular TG by 21% and TC by 33% (Supplementary Fig. S7C). Nile Red and Bodipy staining of hepatocytes showed that lipid droplet accumulation, including droplet size and number, was significantly decreased in KAT2AKO cells (Supplementary Fig. S7D–F). Moreover, cellular TG and TC levels were not significantly decreased in KAT2AKO cells replenished with KAT2A WT, but not with KAT2A MUT (E575Q) (Supplementary Fig. S7C). Nile Red and Bodipy staining yielded similar results (Supplementary Fig. S7D–F). Collectively, these data demonstrate that hepatocyte-specific GCN5 deficiency protects against diet-induced hepatic

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors proposed a MASLD mechanism linked with increased GCN5 (KAT2), a histone\nacetyltransferase. The authors claimed that GCN5 directly interacts with LXR which subsequently induces SREBP1F\nexpression and leads to fat accumulation in the liver. While the manuscript is interesting, some of the data especially the\nmechanistic parts may require a bit validation using more unbiased approaches.\nMajor comments:\n1) Earlier studies have reported that GCN5 controls gluconeogenesis in the liver through PGC1a and the high level of GCN5\nis associated with improved glucose control in mice. Did the author check the body weight, insulin sensitivity and glucose\ncontrol in their over-expression and KO mice?\n2) For the correlation of GCN5 expression in the human cohort (Figure 1F-L), it is not clear where the data comes from. Also\na correlation in the human cohort with R2<0.5 is not considered significant even p is less than 0.05. It might also be\nimportant to validate such results in other published datasets, one of the datasets (PMID: 33268509) is widely used by other\nresearchers. Other datasets include GSE89632 and GSE48452, both were used by the authors in Figure 6G. Is GCN5\nreduction also seen in the MASLD/MASH subjects in those datasets? It might also be interesting to directly check the\ncorrelation of GCN5 mRNA with a group of LXR target genes in the manuscript.\n3) GCN5 has important enzymatic independent functions in many cell and disease contexts. While in this manuscript, the\nauthors tend to prove that the lipid metabolic function of GCN5 in the liver is mainly through its enzymatic activities. It is\ninteresting to confirm this using a more unbiased approach, for example by comparing the RNAseq between AAV-LacZ with\nAAV-KAT2A WT and MUT transfected livers. This will provide unbiased data to dissect the enzymatic dependent and\nindependent metabolic functions of GCN5 in the liver. Could the author also compare the GCN5 KO RNAseq results\nbetween mice (not done yet) and human cells to prove that the GCN5 metabolic function is species conserved.\n4) In Figure 7 and 8 the authors proposed a model that GCN5 directly interacts with LXR and controls SREBF expression\nvia H3K9ac, mainly with ChIP qPCR and luciferase assays, which are biased and heavily depend on the specific promoter\nand the primers selected.\nCould the author present an unbiased RNAseq result in LXR agonist treated cells with or without GCN5? How many of the\naltered genes are directly controlled by GCN5 (with GCN5 recruitment at the gene promoters or enhancers). Do those genes\ncontain some of the key bile acid regulatory candidates such as Cyp7a1 and Cyp8b1, etc?\nGenome-widely, does LXR colocalize with GCN5 in human or mouse liver cells? It is also unclear how the authors chose\nthe ChIP qPCR primers. Could the authors present an LXR ChIPseq or H3K9ac/H3K14ac ChIPseq genome browser results\nat SREBF locus and label the location where the primers are picked?\n5) GCN5 does not have a DNA binding domain, its interaction with LXR may suggest that LXR recruits GCN5 to the SREBF\nlocus. Could the author explain why GCN5 KO abrogates LXR ChIP (Figure 8N). Could the authors use more unbiased\ntechniques (such as ChIPseq) to validate the key results. For example, does LXR knockdown lead to global recruitment\nchanges of GCN5 in the liver cells? Is GCN5 KO induced gene expression associated with H3K9ac/H3K14ac changes?", "reviewer_2": "Jin and colleagues presented a study which highlights the role of novel role of GCN5 in NAFLD. Using a range of\nexperimental techniques on both in vitro and in vivo models, they were able to provide compelling evidence that the\nacetyltransferase function of GCN5 is involved in NAFLD. Furthermore, mechanistic evidence that GCN5 promotes the gene\nexpression of SREBP1c via interaction of H3K9ac and LXRα have been provided in this study. These findings suggest that\ninhibition of GCN5 may serve as a therapeutic avenue against NAFLD as it not only downregulates SREBP1c mediated\nlipid synthesis but also maintain LXRα stimulated RCT.\nOverall, the manuscript is well written and the approach as well as validation of GCN5 role in NAFLD progression through\nLXRα/SREBP1c signalling has been well described. The methods are appropriate and well described and the discussion\nand conclusions are well balanced, although a bit long.\nMajor comments\n1. A nuclei marker (DAPI or Hoechst) should be included in the immunofluorescence (IF) images in order to differentiate the\ncells from the background noise. Furthermore, the presence of a nuclei marker can serve as a form of normalization in regard\nto the quantification of neutral lipid (Nile red and Bodipy).\n2. GCN5 staining should be included in the IF images. This will provide further evidence that hepatocytes which display\nreduced Nile red or Bodipy staining is associated with reduced GCN5.\n3. While Kat2A knockout mice display markedly improved characteristics such as decreased liver weight, TG and TC levels\nrelative to the controls under HFD, it will be interesting to age these mice even further and measure the percentage of them\nwhich progresses from NAFLD to NASH. This would provide a strong proof of concept regarding GCN5 role in NAFLD\nprogression and severity.\n4. The data regarding interaction of GCN5 and LXRα is limited to overexpression systems. Native protein interactions should\nalso be demonstrated as should colocalization by immunofluorescence. Directly addressing the subcellular colocalization of\nboth proteins (potentially nuclear) should also be evaluated.\n5. Although nothing to be done about it at present (we are not asking that this experiment be done) it will be interesting to do\nan ATAC sequencing in order to investigate the genes that become more/less accessible upon GCN5 knockout in mice.\nThis is based on the findings that “GCN5 promotes gene expression of SREBF1c by altering the acetylation state of histone\nH3”,\n6. Most of the western blot results should be quantified and made into a graph in order to show the significance of the data.\nAlso, the inclusion of uncropped western blot images in supplementary data is recommended.\n7. This study showcases an impressive amount of work pointing to the involvement of GCN5 in NAFLD through\nLXRα/SREBP1c signalling. It is recommended to include a graphical model in Figure 9 which summarizes the findings from\nthis study regarding GCN5 involvement in SREBP1c signalling thereby leading to NAFLD.\n8. In reference to Figure 5B-D and Suppl line 248, both NEFA and EFA were measured in the lipidomic analysis. However it\nwas not mentioned specifically how the lipids were processed. For example, in EFA which is made up of fatty acid and a\nlipid (head)group component, were they hydrolysed to obtain just the fatty acids for the analysis? Please kindly describe the\nmethod.\n9. Suppl line 267: Since a(n untargeted) screen was used for lipid analysis, please indicate how were lipids identified—\nusing authenticated standards or otherwise? The SPLASH lipid standard was used, but were specific standards used for the\nidentification? Please describe the method especially for EFA, which led eventually to the summing of EFA and represented\nby the individual FAs.\n10. While an untargeted LC-MS method was used as described in the methods (Suppl line 226: The data were repeatedly\nobtained in data- independent mode over a mass range of m/z: 100 to 1,250 Da.), the abstract described the method as\n“targeted lipidomics” (line 43). Please clarify.\n11. Please kindly describe how were metabolites from Fig 5 E and F (Acyl CoAs, DHAP, Citric acid etc) measured. These\nglycolytic intermediates are very polar and cannot be separated well on the usual reverse phase columns.\n12. Given that (from Fig 5) fatty acid synthesis transcripts are down in Kat2aHKO, and fatty acid oxidation/transport\ntranscripts are not changed, how does this explain that only shorter chained fatty acids are reduced in Kat2aHKO? Since an\nuntargeted LC-MS screen was performed, did the authors notice whether acylcarnitines, reflective of fatty acid oxidation\nwere changed?\n13. Suppl line 342 to 355: Could authors please provide details on software (if any) and parameters used for the extraction of\nstable isotopic data, as well as isotopic background correction. It is also important to clarify whether a targeted or\nuntargeted approach was used to obtain these isotopic information. These details are necessary for experimental replication as well we\nensuring accuracy of data.\nMinor comments\n1. In Figure S1K-S, the inclusion of a negative control where PAOA is not added to the cells (HL-7702/HEPG2 or Primary\nhepatocytes) will show that the increase in GCN5 protein and mRNA levels is due to PAOA rather than the time course.\n2. In Line 209-213 of manuscript, it seems contradictory that “Kat2aHKO mice had more severe hepatic steatosis,” and\n“Kat2aHKO mice developed more severe hypertriglyceridemia and hypercholesterolemia than the Kat2afl/fl mice” given that\nFigure 3D-F shows the knockout mice displaying reduced oil red staining as well as TG and TC levels.\n3. Figure legends should be included in Figure S3G and Oil red is misspelt on the y-axis for the graph\n4. Size of the scale bars in IHC and IF images (e.g Figure 1C, 2P) are not mentioned anywhere in the manuscript\n5. In Line 232-233 “Kat2aHKO mice showed greater degrees of inflammation, and fibrosis than Kat2afl/fl mice, as evidenced\nby IHC, and Sirus Red staining”. Figure 3L seems to show otherwise where the knockout mice seem to display reduced\nSirus red and F4/80 staining. Furthermore, there is no quantification data of the Sirus red and F4/80 staining to substantiate\nthis statement.\n6. In line 286 “When the KAT2A KO HL-7702 cells were replenished the KAT2A”. Sentence structure should be revised.\n7. In Figure S6J, the “indicated genes” measured via the luciferase assay was not mentioned. Furthermore, normalized has\nbeen spelt incorrectly on the y-axis of the graph.\n8. Co-IP experiment in Figure 8D needs to be revised. IP:IgG which is supposed to be the negative control is able to pull\ndown both Myc-LXRα and Flag-GCN5. Furthermore, flag has been misspelt in some of the graphs in Figure 8.\n9. Please check language in abstract.", "reviewer_3": "The authors show that hepatic expression of GCN5 increases with NAFLD progression in humans and mice fed a HFD, and\nthat GCN5 contributes to NAFLD progression via transcriptional activation of SREBP-1c, a master regulator of de novo\nlipogenesis (DNL), using loss- and gain-of-function approaches in mouse liver and cultured human hepatocyte cell lines.\nMechanistically, GCN5 is shown to recruit LXRE to the SREBP-1c gene promoter through interaction with LXRα, and\nacetylate histone H3K9 and H3K14, leading to transcriptional activation. The authors also show that pharmacological\ninhibition of GCN5 does not inhibit LXR ligand-dependent cholesterol reverse transport (RCT) in macrophage-like cell lines\nbut inhibits LXR ligand-induced NAFLD, suggesting the possibility of treating dyslipidemia and NAFLD with a combination\nof LXR ligands and GCN5 inhibitors.\nThis study presents a seeming plethora of data and some potentially interesting aspects, but it lacks key data that support\nthe authors' points. Although GCN5 has been shown to promote fat accumulation in mouse liver and cultured hepatocyte cell\ntypes, there is no causal link between fat accumulation, induction of SREBP-1c expression, and LXR activation. It is unclear\nwhether the expression of SREBP-1c and its target lipogenic genes, as well as hepatic DNL, is upregulated in the liver of\nmice fed a HFD as well as in hepatocyte cell lines. The specificity of GCN5 inhibitor is also questionable, and its strong\ninhibitory effect on histone H3 acetylation makes it essential to examine its effects on gene expression in extrahepatic\norgans when administered systemically. There were also many problems involving the design of the experiments and\npresentation of the data, as well as discrepancies between the data presented and the explanations offered, and many\nmisstatements in the Figures. My criticisms are as follows:\nSpecific concerns\n1. The mechanisms by which GCN5 expression is induced in the livers of HFD-fed mice and palmitate and oleate(PAOA)-\ntreated hepatocytes need to be elucidated.\n2. In the livers of HFD-fed mice compared with normal chow-diet-fed mice, the expression of SREBP-1c and its target\nlipogenic genes are augmented, and SREBP-1c-driven de novo lipogenesis is upregulated?\n3. In primary hepatocytes, HepG2 cells, and HL-7702 cells, does exposure to PAOA induce SREBP-1c and its target\nlipogenic enzymes as well as SREBP-1c-driven de novo lipogenesis in a GCN5 dependent manner? The authors should\nalso investigate the effects of gain- and loss-of-GCN5 on uptake, re-esterification, and β oxidation of fatty acid in PAOA-\ntreated cells.\n4. In Figures 1N, 1Q, and 1R, the time-course analysis of hepatic GCN5 and PCAF is shown in one panel.\n5. As shown in Figure 2, the authors mentioned that overexpression of Kat2a WT, but not Kat2a MUT, promoted hepatic lipid\naccumulation. However, the hepatic expression of Kat2a WT and MUT is shown separately (Figures 2B and 2I). Therefore, it\nwas not possible to assess the effect of Kat2a MUT overexpression to the same extent as that of Kat2a WT on hepatic fat\naccumulation.\n6. In Figures 5E, 5D, and S5A, the authors mentioned that fatty acid synthesis was specifically suppressed, as the hepatic\nabundance of the precursors for DNL, such as acetyl-CoA and malonyl-CoA, as well as lipogenic intermediates such as\ncitric acid, DHAP, glycerol, and glycerol-3P, were significantly reduced in GCN5 knockouts. However, such a reduction may\nbe derived from the suppression of glycolysis and the TCA cycle upstream of DNL, resulting in the suppression of DNL.\nA discussion should be held on this matter.\n7. As shown in Figure 5, the hepatic expression of SREBP-1c and its target lipogenic genes and the activity of hepatic DNL\nwere assessed in normal chow-fed and HFD-fed animals. HFD promoted SREBP-1c-driven hepatic lipogenesis?\n8. As shown in Figure 7, hepatic GCN5 depletion or systemic administration of a GCN5 inhibitor suppressed histone H3K9\nand H3K14 acetylation, whereas hepatic overexpression of GCN5 promoted it. Systemic administration of a GCN5 inhibitor\nappears to alter gene expression in extrahepatic organs. As shown in Figure S6J, this inhibitor also had a GCN5-\nindependent effect that induced regulatory element (SRE)-luc activity in GCN5-deficient cells. Therefore, analysis of\nextrahepatic organs during systemic administration is mandatory.\n9. As shown in Figures 6 and S6, Forced expression of SREBP-1c in GCN5-deficient liver or hepatocytes recovered lipid\naccumulation. Is acetylation of histones H3K9 and H3K14 dispensable for a SREBP-1c-mediated lipogenic gene induction?\nMinor points\n1. Inconsistencies between the data and descriptions in Figures 3D, 3E, 3L, 3N, 3O, and 3P must be corrected.\n2. At lines 399-400, “Although GCN5 down-regulates SREBF1c mRNA and precursor form protein levels”need to be\ncorrected.\n3. In Figures 5A-5F, liver samples were collected from HFD-fed animals under ad libitum-fed conditions.\n4. Figure S6A provides detailed information on the ChREBP and USF promoter-reporter constructs.\n5. In Figures 8D and 8E: correction of erroneous description is needed.\n6. Expression of anti-inflammatory genes was not evaluated in line 235.\n7. In line 235, “the activity of SREBP was changed by KAT2A MUT overexpression”should be corrected.\n8. At lines 146-148, the inconsistency with reference #22 has been clarified.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We sincerely appreciate the opportunity to resubmit our work to Nature Communications, a journal we hold in high regard for its broad impact and recognition in the interdisciplinary scientific community. We are particularly grateful to the editor and reviewers for their exceptionally professional and constructive feedback, which has been invaluable in improving the quality of our manuscript.\nWe have taken every comment with the utmost seriousness and have dedicated substantial time to addressing each point comprehensively. Our responses reflect a thorough consideration of the reviewers' suggestions, including additional experiments where warranted. Given the journal's prestigious standing and our deep respect for the peer review process, we sincerely hope the editorial team will reconsider our revised manuscript for further review. We believe these revisions have significantly strengthened our study, and we would be honored to have it reevaluated for potential publication in Nature Communications.\nOur initial research aimed to investigate the expression and function of GCN5 in non-alcoholic fatty liver disease (NAFLD)/metabolic-associated fatty liver disease (MAFLD) (In our revision manuscript, we believe that MAFLD is more appropriate. Therefore, we have changed all instances of NAFLD to MAFLD in the following text) (doi:10.1016/j.jhep.2020.03.039). Therefore, when investigating the function of GCN5 in the progression of MAFLD, we selected a commonly used MAFLD animal model induced by high-fat diet (HFD) diet, and used the liver tissues of this animal model to explore the mechanism by which GCN5 regulates MAFLD. Of course, initially we were not aware that GCN5 would affect the progression of MAFLD by regulating the process of de novo lipogenesis (DNL) in the liver.\nIndeed, as the reviewers and editors have said, DNL does not represent the core pathogenic mechanism in our selected MAFLD model induced by HFD diet (doi: 10.1038/nrgastro.2010.191). As you rightly noted, models characterized by hyperinsulinemia and excessive carbohydrate intake (particularly fructose) would be more physiologically relevant for investigating DNL-driven MAFLD pathogenesis (doi:10.1002/hep.31897).\nFructose has been identified as the most potent inducer of DNL due to its ability to bypass rate-limiting steps and utilize insulin-independent metabolic pathways (doi: 10.1530/JOE-22-0270). The underlying mechanisms involve: 1) Fructose enters hepatocytes via GLUT5 transporters and is rapidly phosphorylated by fructokinase to fructose-1-phosphate. This bypasses the glycolysis rate-limiting enzyme phosphofructokinase-1 (PFK-1), leading to uncontrolled production of acetyl-CoA, the primary substrate for DNL (doi: 10.1007/s10620-016-4054-0). 2) Intestinal microbiota metabolize fructose into acetate, which enters the liver via the portal vein. Acetate directly fuels DNL while simultaneously upregulating ChREBP expression (doi: 10.1038/s41586-020-2101-7).\nWhile our HFD model primarily investigates GCN5’s role in lipid dysregulation under insulin-resistant conditions, we acknowledge that fructose-based models could provide additional mechanistic insights into DNL-specific pathology. To better model MAFLD under conditions of hyperinsulinemia and excessive fructose intake, we employed the following three dietary regimens: (1) FPC diet: 52 kcal% fat, 34.5 kcal% sucrose, and 1.25% cholesterol, combined with 42 g/L sugar solution (55% glucose + 45% fructose) in drinking water (doi: 10.1126/science.abf8271). (2) AMLN diet: A compositionally defined diet containing 40 kcal% fat, 20 kcal% fructose, and 2% cholesterol (doi:10.1126/science.abf8271). (3) HFrD diet: an isocaloric diet where 60% of total calories are derived from fructose. This extreme model maximizes DNL stimulation through both hepatic and intestinal microbiota-mediated pathways described earlier (doi: 10.1016/j.cmet.2022.08.005).\nWe first evaluated the functional role of GCN5 in MAFLD progression in Kat2aHKO mice fed an FPC diet for 16 weeks (Fig. R1-1A-C). Body weight and food intake of mice were not significantly influenced in Kat2aHKO mice compared with those of Kat2afl/fl mice (Fig. R1-1D-E). However, the LW and LW/BW of Kat2aHKO mice were significantly decreased (Fig. R1-1F). Kat2aHKO mice had lower fasting blood lipids, including TG, TC, and LDL-c levels, and increased HDL-c levels (Fig. R1-1G). Furthermore, H&E staining, Oil Red O staining, and hepatic TG and TC concentrations in Kat2aHKO mice indicated that these mice had reduced hepatic steatosis in response to an FPC diet (Fig. R1-1H-L). Additionally, Kat2aHKO deteriorated FPC diet-induced liver injury, as indicated by decreased plasma AST and ALT levels (Fig. R1-1M). Considering that MASH is a severe form of MAFLD, we continued to feed the Kat2afl/fl and Kat2aHKO mice induced by FPC diets, extending the feeding period from 16 weeks to 24 weeks (Fig. R1-1A), to investigate the involvement of GCN5 in MASH further. As shown in Fig. R1-1N, significant inflammatory responses and fibrotic symptoms were observed in the livers of the Kat2afl/fl mice fed with the FPC diet for 24 weeks. Kat2aHKO mice showed lower degrees of inflammation and fibrosis than Kat2afl/fl mice, as evidenced by IHC F4/80 and Sirius Red staining (Fig. R1-1N-P). These phenomena were accompanied by a lower expression of MASH-related inflammatory factors, such as IL6, IL16, Cxcl1, Cxcl5, and Cxcl10, lower profibrotic genes, as well as MASH-related profibrotic genes, such as Acta2, α-Sma, ctgf, Tgfβ, Col3a1, and Col1a1 (Fig. R1-1Q).\nFig. R1-1 Liver-specific KO of GCN5 protects mice from FPC diet-induced MAFLD and MASH. Related to Fig. 3H-Q and Fig. S6A-G in the revised manuscript.\nThe deficiency of GCN5 also demonstrated a significantly beneficial effect in improving MAFLD induced by the HFrD diet (Fig. R1-2) and AMLN diet (Fig. R1-3).\nFig. R1-2 Liver-specific KO of GCN5 protects mice from HFrD diet-induced MAFLD. Related to Fig. S4 in the revised manuscript.\nFig. R1-3 Liver-specific KO of GCN5 protects mice from AMLN diet-induced MAFLD. Related to Fig. S5 in the revised manuscript.\nSimilarly, we investigated the effect of the GCN5 inhibitor CPTH2 in the FPC diet-induced MAFLD model. MAFLD was established in mice fed an FPC diet for 10 weeks, and then the mice were orally administered CPTH2 (20 mg/kg/d) while continuing the FPC diet for an additional 6 weeks (Fig. R1-4A-B). During the process of the experiment, the body weights were no different between CPTH2-treated mice and FPC diet-fed mice (Fig. R1-4C), without food intake change (Fig. R1-4D). The increased LW and LW/BW in HFD-fed mice were decreased by CPTH2 treatment (Fig. R1-4E). Moreover, H&E and Oil-Red O staining showed that CPTH2 remarkably decreased the lipid accumulation in HFD-fed mice (Fig. R1-4F), which was sustained by liver and serum TG and TC, and serum HDL-c and LDL-c levels (Fig. R1-4G-H). Additionally, the hepatocyte ballooning was also reduced in FPC diet-fed mice treated with CPTH2 (Fig. R1-4F). The serum AST and ALT were largely lower in CPTH2-treated mice than in control-FPC diet mice (Fig. R1-4I).\nFig. R1-4 CPTH2 ameliorates mice from FPC diet-induced MAFLD. Related to Fig. 4H-N in the revised manuscript.\nTo further investigate whether the anti-MAFLD effect of GCN5 deficiency is dependent on SREBP1 in vivo. We conducted the same verification on the FPC diet-induced MAFLD model, m-SREBP1 was specifically overexpressed in the liver by tail vein injection of the adeno-associated virus (AAV)-m-SREBP1c or AAV-LacZ, after administration of the FPC diet for 8 weeks (Fig. R1-5A). The weight gain and food intake were not significantly changed in each group (Fig. R1-5B-C). Kat2a-deficient-decreased LW and LW/BW were disrupted in liver-specific overexpression of SREBP1c (Fig. R1-5D). The reduction of lipid accumulation by H&E and Oil-Red O staining in liver-specific Kat2a-deficient mice was reversed by SREBP1c overexpression (Fig. R1-5E), which was consistent with liver and serum TG and TC, and serum LDL-c and HDL-c levels (Fig. R1-5F and G). Consequently, the AST and ALT were not largely reduced by SREBP1c overexpression in liver-specific Kat2a-deficient mice (Fig. R1-5H). These data collectively demonstrated that SREBP1c is critical for the MAFLD progression effect of GCN5.\nFig. R1-5 Overexpression of SREBP1c eliminates the protective effect of liver-specific KO of GCN5 on MAFLD induced by FPC diet. Related to Fig. 6O-T in the revised manuscript.\nTo measure DNL directly, we force-fed mice 13C-fructose, a rich source of carbons for DNL in the liver, and quantified 13C incorporation into liver fatty acids. DNL was decreased by about one-half in Kat2aHKO mice fed a short-term FPC diet, in the absence of changes in body weight (Fig. R1-6A-C). Similar results were observed in mice injected with deuterium oxide, followed by measuring incorporation of 2H into liver fatty acids, an alternate method of measuring DNL (Fig. R1-6D-F). Significant suppression of DNL was also evident in Kat2aHKO mice fed HFD (Fig. R1-6B and E).\nFig. R1-6 Significant suppression of DNL was evident in Kat2aHKO mice fed FPC diet. Related to Fig. S10E-H and K-L in the revised manuscript.\nIn summary, our multi-model validation with FPC, AMLN, and HFrD dietary models confirmed the conserved role of GCN5 in hepatic lipogenesis, answering the most concerned questions of reviewers and editors.\nHere, we have attempted to address all the reviewers’ concerns from the original submission point to point. The reviewers’ comments are provided in bold italics below, with our response following. We believe the revised manuscript can be significantly improved."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my major questions with substantial amount of experiments. I am convinced the current version\nof the manuscript is of sufficient quality to publish in Nat Com.\nMinor:\nThe new nomenclature of NAFLD is Metabolic dysfunction-associated steatotic liver disease (MASLD). It needs to be\nchanged in the manuscript.", "reviewer_2": "The rebuttal to all reviewers were done very well, impressive work. I have no further comments to my part.", "reviewer_3": "1. The authors argued that hepatic GCN5 deficiency in high-fat diet-induced obese mice suppressed hepatic steatosis\nmainly through the transcriptional suppression of SREBP-1c. I think that To clearly illustrate the central contribution of\nSREBP-1c, the authors must show the improvement of MASLD by the loss of SREBP-1c, as I raised specific concerns 2 and\n5.\n2. The involvement of KLF15 in the lipid-dependent induction of GCN5 expression is interesting. However, KLF15 is a\nfasting-induced transcription factor that promotes the expression of gluconeogenic genes and simultaneously suppresses\nSrebp1c gene expression via the recruitment of the nuclear transcription co-repressor RIP40 (Takeuchi et al. Cell Rep\n16:2378, 2016).The authors' in vitro data showing that KLF15 also activates SREBP-1c gene transcription via GCN5\ninduction are confusing to this reviewer as well as the readership. Therefore, it was necessary to demonstrate the KLF15-\nmediated induction of GCN5, which activates the SREBP-1c-DNL pathway in vivo. Does KLF15-dependent induction of\nGCN5 expression occur in vivo during fasting?\n3. The authors should also describe the process by which they identified candidate transcription factors that bind to the\nKAT2A promoter in silico in more detail, enabling bioinformaticians to verify its reproducibility.\n4. In reference 22, the authors showed that hepatic expression of GCN5 was upregulated in obese diabetic mice compared\nwith that in control lean mice, and that in primary mouse hepatocytes, cAMP induces GCN5 expression. However, they did\nnot conduct a human study or report a correlation between GCN5 expression and blood glucose levels. This must be\ndescribed accurately.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Dear Editor and Reviewers,\nThank you for our manuscript \"GCN5 drives MAFLD progression through\nLXRα/SREBP1c signaling pathway–mediated de novo lipogenesis\" (NCOMMS-24-\n64897A-Z) review and valuable advice. We have given serious consideration to all the\ncomments. In response to the specific requests for additional animal experiments, and\ndespite the challenge of the standard breeding timeline, we made every effort to\nexpedite the process by procuring the necessary animal models to meet the four-week\nrevision deadline. The following are our point-by-point responses to each question. All\nmodifications have been reflected in the revised manuscript. The reviewers' comments\nare displayed in bold and italic below, followed by our responses. We believe that these\nrevisions have significantly enhanced the scientific rigor and persuasiveness of the\nmanuscript.\nReviewers' Comments:\nReviewer #1:\n1. The new nomenclature of NAFLD is Metabolic dysfunction-associated steatotic\nliver disease (MASLD). It needs to be changed in the manuscript.\nResponse: Thank you very much for your insightful comment regarding the\nnomenclature update from MAFLD to MASLD. We fully agree with your suggestion\nand have thoroughly revised our manuscript to reflect this change in accordance with\nthe latest international consensus, particularly the 2023 AASLD/EASL guidelines,\nwhich recommend using \"metabolic dysfunction-associated steatotic liver disease\n(MASLD)\" to better emphasize the metabolic underpinnings of the disease and ensure\nconsistency with contemporary standards. Specifically, we have systematically\nreplaced all instances of \"MAFLD\" with \"MASLD\" across the entire manuscript,\nincluding the title, abstract, introduction, methods, results, discussion, and reference\nsections, while retaining \"MASH\" (metabolic dysfunction-associated steatohepatitis)\nas it already aligns with current terminology. This update was carefully implemented to\nmaintain contextual coherence and scientific accuracy, and we have verified that the\nchanges do not alter the study's conclusions or data interpretation. We believe these\nrevisions enhance the clarity and relevance of our work, and we greatly appreciate your\nguidance in this matter. Please let us know if any further adjustments are needed.\nReviewer #3:\n1. The authors argued that hepatic GCN5 deficiency in high-fat diet-induced obese\nmice suppressed hepatic steatosis mainly through the transcriptional suppression of\nSREBP-1c. I think that To clearly illustrate the central contribution of SREBP-1c,\nthe authors must show the improvement of MASLD by the loss of SREBP-1c, as I\nraised specific concerns 2 and 5.\nResponse: We thank the reviewer for this critical point. We fully agree that direct\ngenetic evidence is paramount to unequivocally establish the central contribution of\nSREBP-1c.\nIn fact, anticipating the importance of this functional validation, we had already\ninitiated the requisite experiments prior to the initial submission of our previous\nrevision. While the existing pharmacological and literature evidence was compelling at\nthe time, we recognized that experimental data from a genetic model would provide the\nmost definitive proof. We are now pleased to present these new results, completed\nduring this revision period, which directly address your concern.\nAs the reviewer suggested, we generated hepatocyte-specific Srebp1c-knockdown\nmice using an adeno-associated virus serotype 8 (AAV-shSrebp1c) and fed them an\nHFD (Fig. R1A). Western blot analysis confirmed the successful knockdown of\nSREBP-1c in hepatocytes (Fig. R1B). Consistent with its proposed central role, loss of\nSREBP-1c significantly protected mice from HFD-induced MASLD. This was\nevidenced by a marked reduction in hepatic TG content (Fig. R1C) and alleviated\nhepatic steatosis, as shown by H&E and Oil Red O staining (Fig. R1D, E). Furthermore,\nthe mRNA levels of key SREBP-1c target genes involved in lipogenesis (Acc, Fasn,\nand Scd1) were significantly downregulated in the AAV-shSrebp1c group (Fig. R1F).\nThese findings are consistent with previous reports using genetic ablation of SREBP-\n1c (Thuy T.P. et al., Molecular Cell, 2021; Lee, Eun-Ho et al., EXP MOL MED, 2024),\nand pharmacological inhibition (Kamisuki, Shinji et al., Chemistry & biology , 2009;\nTang, Jing-Jie et al., Cell metabolism , 2011).\nTogether with the previously cited literature, our new experimental data from\nhepatocyte-specific Srebp1c knockdown provide direct functional evidence that loss of\nSREBP-1c ameliorates MASLD, thereby solidifying its central role in the GCN5-\ndriven pathogenesis we propose.\nFig. R1 Loss of SREBP-1c protects mice from HFD diet-induced MASLD.\n2. The involvement of KLF15 in the lipid-dependent induction of GCN5 expression\nis interesting. However, KLF15 is a fasting-induced transcription factor that\npromotes the expression of gluconeogenic genes and simultaneously suppresses\nSrebp1c gene expression via the recruitment of the nuclear transcription co-repressor\nRIP40 (Takeuchi et al. Cell Rep 16:2378, 2016).The authors' in vitro data showing\nthat KLF15 also activates SREBP-1c gene transcription via GCN5 induction are\nconfusing to this reviewer as well as the readership. Therefore, it was necessary to\ndemonstrate the KLF15-mediated induction of GCN5, which activates the SREBP-\n1c-DNL pathway in vivo. Does KLF15-dependent induction of GCN5 expression\noccur in vivo during fasting?\nResponse: Thank you for your insightful guidance and your affirmation of the\ninteresting phenomena we have found. We fully understand the source of the potential\nconfusion. The classical study by Takeuchi et al. elegantly demonstrated that in the\nfasting state, KLF15 acts to suppress SREBP1c expression by recruiting the co-\nrepressor RIP140, and in the fed state, direct transcriptional regulation of SREBP1c by\nKLF15 was lost (Takeuchi et al., Cell Reports, 2016).\nOur discovery that KLF15 can activate the SREBP-1c-DNL pathway via GCN5\ninduction under conditions of nutrient excess (PAOA treatment in vitro, HFD in vivo),\ntherefore, may appear paradoxical at first glance. Rather than a contradiction, we\nbelieve this highlights a critical layer of metabolic regulation: the functional output of\nKLF15 is exquisitely context dependent. This plasticity is not without precedent, as\nsuggested by Jung DY et al. (Jung DY et al., PLoS ONE, 2013), where KLF15\nknockout improved hepatic steatosis under HFD, implying a potential lipogenic role for\nKLF15 in the over-nutrition state.\nTo directly address your questions and demonstrate the in vivo relevance and state-\nspecificity of this pathway, we performed the following key experiments:\nTo validate the in vivo induction of the GCN5–SREBP-1c axis by KLF15 under HFD\nconditions within the 4-week revision period, we purchased C57BL/6J mice that had\nbeen maintained on an HFD for 7 weeks (beginning at 8 weeks of age) and an adeno-\nassociated virus (AAV) vector enabling hepatocyte-specific overexpression of Klf15\n(AAV-Klf15). The mice then received a tail-vein injection of AAV-Klf15 to achieve\nliver-specific overexpression of KLF15 while continuing on the HFD regime (Fig. R2-\n1A). Consistent with our in vitro data, KLF15 overexpression significantly increased\nthe protein levels of hepatic GCN5 and SREBP-1 and upregulated the mRNA\nexpression of DNL target genes (Fig. R2-1B-D). This provides direct evidence that\nKLF15-mediated induction of GCN5, activating the SREBP-1c-DNL pathway, occurs\nin vivo.\nFig. R2-1 Klf15-mediated induction of GCN5, which activates the SREBP1c-DNL\npathway in vivo under HFD diet.\nTo verify whether KLF15-dependent induction of GCN5 expression occurs in the\nfasting state, we analyzed mice after a 24-hour fast. As expected, western blot analysis\nconfirmed fasting strongly induced KLF15 expression (Fig. R2-2A, B). However, the\nprotein level of GCN5 was not elevated (Fig. R2-2A, B). These results indicated that\nunder fasting conditions, although KLF15 was strongly induced, the expression level\nof GCN5 did not change significantly.\nTo explain that under fasting conditions, although KLF15 is highly expressed, GCN5\nis not induced, we performed ChIP-qPCR on liver tissue from fasted mice. The results\nshowed no significant enrichment of KLF15 on the Kat2a promoter region in the fasting\nstate (Fig. R2-2C). This indicates that during fasting, KLF15 does not bind to the\nKAT2A promoter, this is similar to the disappearance of the binding of KLF15 to the\npromoter region of SREBP1c during feeding (Takeuchi et al., Cell Reports, 2016).\nThese results show that KLF15-dependent induction of GCN5 expression doesn’t occur\nin vivo during fasting.\nFig. R2-2 Klf15-dependent induction of GCN5 expression doesn’t occur in vivo\nduring fasting.\nIn conclusion, our data collectively demonstrates that the KLF15-GCN5-SREBP-1c\naxis is a pathway specifically activated in the over-nutrition state (HFD) but is not\noperative in the fasting state. We believe that this can resolve your confusion and further\nconfirm the novelty of our discovery.\n3. The authors should also describe the process by which they identified candidate\ntranscription factors that bind to the KAT2A promoter in silico in more detail,\nenabling bioinformaticians to verify its reproducibility.\nResponse: We thank the reviewer for this suggestion to improve the reproducibility of\nour bioinformatic analysis. We have now thoroughly revised the Methods section to\nprovide a detailed, step-by-step description of the in-silico prediction process for\ncandidate transcription factors binding on the KAT2A promoter.\nKey enhancements include: (1) Explicitly defined the promoter regions for human\n(GRCh38/hg38: chr17:40,100,000–40,102,200) and mouse (GRCm39/mm39:\nchr11:69,900,000–69,902,200), spanning 2,000 bp upstream to 200 bp downstream of\nthe transcription start site (TSS). (2) Specified the JASPAR database (2022 release,\nCORE vertebrate collection) and scanning threshold (p-value < 10-5). (3) Detailed the\nintersection strategy, only TFs predicted to bind both human and mouse promoter\nregions were retained as high-confidence candidates, ensuring evolutionary\nconservation.\nThe new method is described as follows: candidate transcription factors (TFs)\ncapable of binding to the KAT2A promoter were predicted using the JASPAR database\n(https://jaspar.genereg.net/; release 2022). The analyzed promoter region was defined\nas 2,000 bp upstream of the transcription start site (TSS) to 200 bp downstream for both\nthe human (GRCh38/hg38: chr17:40,100,000-40,102,200) and mouse\n(GRCm39/mm39: chr11: 69,900,000-69,902,200) KAT2A genes. The search was\nperformed against the JASPAR CORE vertebrate collection of transcription factor\nbinding profiles. Scanning was under the screening condition of p value < 10-5, all\npredicted TFs meeting this threshold in either species were recorded. To prioritize TFs\nwith potential conserved regulatory roles, we intersected the prediction results from the\nhuman and mouse KAT2A promoter sequences. This analysis yielded six candidate TFs.\nSubsequently, we reviewed the existing literature for these six TFs and selected four\nthat have previously documented roles in lipid metabolism for further experimental\nvalidation.\nThese additions provide unambiguous technical details (e.g., exact coordinates,\nversion numbers) that enable bioinformaticians to precisely replicate our analysis. The\nupdated text is located under the new subsection \"Prediction of transcription factors\nbinding to the KAT2A promoter\" (Page 4, lines 72-85 in revised supplementary\nmanuscript).\n4. In reference 22, the authors showed that hepatic expression of GCN5 was\nupregulated in obese diabetic mice compared with that in control lean mice, and that\nin primary mouse hepatocytes, cAMP induces GCN5 expression. However, they did\nnot conduct a human study or report a correlation between GCN5 expression and\nblood glucose levels. This must be described accurately.\nResponse: We fully agree with the reviewer's assessment and thank them for\nhighlighting this important point. In response, we have carefully revised our manuscript\nto ensure accurate description of the limitations of Reference 22 (Sakai, M. et al., Nat\nCommun, 2016).\nFirst, we have removed any implication that Reference 22 establishes a direct\ncorrelation between GCN5 expression and blood glucose levels in humans. In the\nResults section (Page 8, Lines 169-171), we deleted the phrase: \"This discrepancy from\nprevious reports, may be due to our analysis of glucose, and insulin levels in patients\nwith MAFLD rather than in patients with diabetes22.\". And in the Discussion section\n(Page 30, Lines 628-630), we deleted the phrase: \"which may be inconsistent with\nprevious reports, possibly because we analyzed blood glucose, and insulin levels in\npatients with MAFLD rather than in those with diabetes22.,\" as it inaccurately suggested\nthat Reference 22 supported such a correlation.\nInstead, we now explicitly state in the revised text that Reference 22's findings are\nbased solely on mouse models and primary hepatocytes, with no human data or\ncorrelation analysis provided (Page 30, Lines 627-629 in revised manuscript). This\nclarification ensures that readers do not overinterpret the reference's scope.\nThese revisions ensure that the description of Reference 22 is accurate and does not\noverstate its conclusions. We appreciate your guidance in improving the precision of\nour manuscript.\nIn summary, we have comprehensively revised the manuscript based on comments\nfrom reviewers one by one, including supplementing experimental data, refining the\nmethod description, and correcting the text. We believe that the revised manuscript\nbetter illustrates the mechanism of GCN5 in MASLD and meets the reviewer's\nrequirements. Thanks again to the editors and reviewers for their hard work and\nvaluable guidance."}]

Decision letter