Pathophysiological significance of impaired KAT7-dependent histone H3K14 acetylation during zinc deficiency

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

Abstract
Zinc is an indispensable micronutrient for optimal physiological functions, and zinc deficiency has been implicated in the pathogenesis of various human diseases. One potential mechanism underlying such pathogenic effects is the alteration of gene expression caused by zinc deficiency; however, the details of this process remain largely unexplored. Here, we show that during zinc deficiency, the histone acetyltransferase KAT7 loses its enzymatic activity, leading to the attenuated acetylation of histone H3 at Lys14 (H3K14ac) at enhancer regions. Physiologically, the decrease in H3K14ac leads to the upregulation of the expression of ZIP10, a plasma membrane-localized zinc transporter, thereby facilitating the import of extracellular zinc to maintain cellular zinc homeostasis. Moreover, prolonged zinc deficiency in mice induced by a zinc-deficient diet or high-fat diet, accompanied by decreased H3K14ac levels in the liver, upregulated the expression of genes associated with intracellular lipid droplet formation, leading to the accumulation of lipids within liver tissue. Our findings demonstrate that cells respond to zinc deficiency by converting it into an epigenetic signal that drives physiological or pathophysiological biological processes.

Introduction
Zinc is an essential micronutrient that plays a pivotal role in maintaining the structural integrity and enzymatic activities of numerous proteins. Computational analysis revealed that as much as 10% of the human genome encodes a zinc-binding proteins1, suggesting the importance of maintaining zinc homeostasis in the human body. Nevertheless, zinc deficiency occurs, arising from various factors, such as inadequate zinc intake, impaired zinc absorption, and aging2. The clinical manifestations of zinc deficiency in humans were first reported in the 1960s3,4, and since then, numerous studies have shown that zinc deficiency is strongly associated with the development of various diseases. The classical symptoms of zinc deficiency include skin diseases, mental disorders, immune dysfunction, and growth retardation5. Recently, emerging evidence has highlighted correlations between reduced zinc levels and diseases such as cancer6–12, inflammatory bowel disease13, and chronic kidney disease14. However, the role of zinc in the pathogenesis of these conditions and the underlying molecular mechanisms remain to be fully elucidated.

To overcome zinc-deficient conditions, cells need to import zinc from extracellular sources. The Zrt-/Irt-like protein (ZIP) family is a group of zinc ion transporters15. Among the 14 human ZIP family proteins, 10 ZIPs (ZIP1-6,8,10,12, and 14) are thought to be localized on the plasma membrane and to be involved in the import of extracellular zinc. Upon zinc deficiency, the abundance of ZIPs on the plasma membrane increases to facilitate zinc uptake as a stress response to maintain cellular zinc homeostasis. One related mechanism involves the upregulation of gene expression through transcriptional control. For example, our previous research demonstrated that zinc deficiency activates the transcription factor ATF6 via the endoplasmic reticulum (ER) stress response, thereby promoting the transcription of ZIP1416. In addition, it has been reported that ZIP10 expression increases during zinc deficiency through transcriptional control mediated by the transcription factor MTF117. However, the detailed molecular mechanisms remain largely unclear.

Histone posttranslational modifications (PTMs) are among the key regulators that govern transcriptional control18. Each histone PTM is balanced via the regulation of a myriad of enzymes referred to as “writers” and “erasers”; writers catalyse the formation of specific types of PTMs, and erasers remove the PTMs. Interestingly, multiple writers and erasers possess zinc-binding domains19, suggesting the critical role of zinc in regulating histone PTMs. Nevertheless, little is known about the dynamics of these writers and erasers during zinc deficiency.

In this study, we observed a drastic decrease in histone H3K14ac during zinc deficiency. Mechanistically, this decrease was driven by the loss of activity of the zinc-dependent writer KAT7, which catalyses H3K14ac formation. In cultured cells, a decrease in H3K14ac led to the transcriptional upregulation of ZIP10, thereby importing zinc from extracellular sources to maintain cellular zinc homeostasis. At the organismal level in mice, chronic zinc deficiency in the liver induced the upregulation of the expression of genes involved in lipid droplet synthesis pathways and promoted lipid accumulation in liver tissue via a reduction in KAT7 activity and H3K14ac. Additionally, we showed that a high-fat diet led to a decrease in hepatic zinc and H3K14ac levels, suggesting that these reductions may more universally contribute to hepatic lipid accumulation. Our data demonstrate that cells respond to zinc deficiency stress by converting it into an epigenetic signal to drive cellular responses.

Results
Global decrease in histone H3K14ac during zinc deficiency
We previously reported that the activation of the transcription factor ATF6 elicits the upregulation of ZIP14 expression in HepG2 cells during zinc deficiency16. To investigate the impact of ATF6 on global expression changes in genes other than ZIP14, we examined the presence of ATF6-binding motifs on the promoters of genes whose expression was upregulated under zinc-deficient conditions. The Gene Expression Omnibus (GEO) repository contains four datasets reporting gene expression profiles in various types of cells during zinc deficiency, including a dataset from our laboratory16,20–22. We found that, across all the datasets, fewer than 2% of the genes presented an ATF6-binding motif23 (ER stress-responsive element, ERSE) within their promoter regions (Fig. S1a). These findings indicate that mechanisms other than the ATF6 axis contribute to the alterations in gene expression provoked by zinc deficiency.

In this study, we focused on the role of histone PTMs, which are critical factors responsible for gene expression changes whose association with zinc deficiency remains unexplored. First, we systematically analysed whether zinc deficiency stress affects the major histone PTMs implicated in gene expression in HEK293A cells (promoter marks: H3K4me3, H4K5ac, and H4K12ac; enhancer marks: H3K4me, H3K14ac, and H3K27ac; gene body marks: H3K36me3 and H3K79me3; and heterochromatin marks: H3K9me3 and H3K27me3).

We found that treatment with N,N,N’,N’-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), a cell-permeable zinc chelator, selectively attenuated the signal for H3K14ac; however, no other marks were affected (Fig. 1a). This reduction in H3K14ac was observed across diverse cellular contexts (Fig. S1b, c); this reduction was also evident when cells were cultured in zinc-deficient medium (Fig. 1b), where the reduction was reversed by the addition of zinc and remained unaltered by the addition of other metals (Fig. 1c, d). Consistently, treatment with zinc-saturated TPEN failed to reduce H3K14ac levels (Fig. S1d), highlighting that zinc availability is indispensable for maintaining the H3K14ac level. Furthermore, the H3K14ac signal was restored in a time-dependent manner by the resupplementation of zinc under conditions of zinc deficiency (Fig. 1e). Collectively, these results suggest that zinc reversibly regulates H3K14ac.

HDACs deacetylate H3K14ac during zinc deficiency
We next investigated the molecular mechanisms underlying the reduction in H3K14ac during zinc deficiency. In general, the histone acetylation status is balanced by histone deacetylases (HDACs) and histone acetyltransferases (HATs). We hypothesized that the activation of HDACs or the inactivation of HATs results in a decrease in H3K14ac during zinc deficiency.

First, we investigated the involvement of HDACs. HDACs are classified into two distinct categories: the classic HDAC family (HDAC1-11) and the sirtuin family of NAD-dependent HDACs (SIRT1-7). To identify which HDACs are responsible for the deacetylation of H3K14ac during zinc deficiency, we treated cells with specific inhibitors of each HDAC family. Treatment with nicotinamide (NAM), a pan inhibitor of the sirtuin family, failed to inhibit the reduction in H3K14ac (Fig. 2a). In contrast, treatment with trichostatin A (TSA), a pan inhibitor of the HDAC family, completely prevented this decrease (Fig. 2b), suggesting that the classic HDAC family deacetylates H3K14ac during zinc deficiency.

Next, we attempted to identify the specific member of the HDAC family responsible for the deacetylation of H3K14ac during zinc deficiency. However, the knockdown of individual members of the HDAC family did not prevent the TPEN-induced deacetylation of H3K14ac (Figs. 2c, S2a). In addition, SAHA, a pan inhibitor of the HDAC family, but not other subtype-specific HDAC inhibitors (TMP195: HDAC4/5/7/9; tubastatin: HDAC6; UF010: HDAC1/2/3/6/8/10; MS275: HDAC1/2/3/8) effectively inhibited the TPEN-induced deacetylation of H3K14ac (Fig. S2b, c). These data suggest that several HDAC family members cooperatively contribute to the deacetylation of H3K14ac during zinc deficiency.

To examine the possibility that zinc-deficient stress enhances the deacetylation activity of members of the HDAC family towards H3K14ac, we measured HDAC activity via a cell lysate–based HDAC activity assay using purified histones. The basal HDAC activity for H3K14ac in cell lysates did not differ in the presence or absence of TPEN, suggesting that zinc deficiency in cells enhances the deacetylation of H3K14ac through an independent mechanism that regulates HDAC activity (Fig. 2d). To further investigate whether zinc deficiency directly alters HDAC enzymatic activity, we next examined the activity of individual HDACs toward H3K14ac using an in vitro HDAC assay. While HDAC1 and HDAC6 exhibited measurable deacetylase activity, their enzymatic functions were not enhanced under zinc-deficient conditions (Fig. 2e), which was further supported by a more detailed time-course analysis (Fig. 2f). Moreover, simultaneous knockdown of HDAC1 and HDAC6 did not prevent the reduction of H3K14ac induced by zinc-deficient stress (Fig. S2d). These data suggest that certain members of the classic HDAC family are needed to deacetylate H3K14ac during zinc deficiency but are not direct regulatory targets of zinc deficiency.

Loss of KAT7 activity during zinc deficiency
Given that HDAC activity is not enhanced under zinc-deficient conditions, we hypothesized that zinc deficiency suppresses the activity of HATs towards H3K14. First, to identify the specific HAT responsible for H3K14ac in HEK293A cells, we performed an siRNA screen using siRNAs targeting 17 human HATs and found that the knockdown of K (lysine) acetyl transferase 7 (KAT7) led to a significant decrease in H3K14ac (Figs. 3a, S3a). This decrease was also observed when the cells were treated with WM-3835, a specific inhibitor of KAT724 (Fig. S3b), indicating the crucial role of KAT7 in the acetylation of H3K14. Thus, we focused on the regulatory mechanisms of the KAT7 HAT activity during zinc deficiency.

Previous reports have shown that KAT7 undergoes degradation in response to DNA damage and lipopolysaccharide stimulation via the ubiquitin‒proteasome pathway25,26. However, our investigation indicated that the effect of TPEN on the protein level of KAT7 was limited (Fig. S3c). While treatment with the proteasome inhibitor MG132, but not the autophagy inhibitor bafilomycin A1, inhibited the TPEN-induced slight decrease in the protein amount of KAT7, both inhibitors had little effect on the decrease in H3K14ac. These results suggest that the degradation of KAT7 is not involved primarily in the reduction in H3K14ac during zinc deficiency.

Several reports have demonstrated that KAT7 localizes to the nucleus to acetylate histones27,28. We investigated the possibility that the subcellular localization of KAT7 changes during zinc deficiency. However, immunofluorescence analysis of endogenous KAT7 revealed that KAT7 localized to the nucleus after TPEN treatment (Fig. S3d), suggesting that a change in subcellular localization is not involved in the regulation of KAT7 during zinc deficiency.

Interactome analyses demonstrated that KAT7 forms a multi-subunit complex that includes BRPF1/2/3, JADE1/2/3, ING4/5, and MEAF629–35. Given that these interactors have been reported to directly bind to KAT7 to increase its HAT activity, we hypothesized that zinc deficiency leads to the dissociation of KAT7 from the complex. The systematic knockdown of each interactor revealed that BRPF2 and MEAF6 play critical roles in the acetylation of H3K14 in HEK293A cells (Fig. S3e). However, treatment with TPEN did not dissociate KAT7 from each component (Fig. S3f). These findings suggest that the disruption of the KAT7 complex is not a cause of the reduction in H3K14ac during zinc deficiency.

Since working hypotheses based on previous reports were not applicable in this case, we next investigated whether zinc deficiency directly affects KAT7 activity by employing an in vitro HAT assay. The treatment of cells with TPEN strongly inhibited the HAT activity of immunoprecipitated KAT7 to a similar level as that of the HAT-dead EQ mutant24 (Fig. 3b). In addition, the KAT7 HAT activity in cells cultured in low-zinc-medium was attenuated in a time-dependent manner, which was followed by the deacetylation of H3K14ac (Fig. 3c). Consistently, no reduction in KAT7 activity was observed upon zinc-saturated TPEN treatment (Fig. S3g). These results suggest that KAT7 loses its enzymatic activity under zinc deficiency.

Zinc coordination in the KAT7 MYST domain regulates HAT activity towards H3K14
KAT7 possesses two zinc finger domains: one is located within the MYST-type HAT domain (MYST domain), and the other is in the N-terminal region (Fig. 3d). We speculated that the loss of zinc coordination during zinc deficiency affects KAT7 HAT activity. To test this possibility, we examined whether the zinc finger domains in KAT7 are needed for KAT7 HAT activity. Depleting KAT7 using the CRISPR‒Cas9 system resulted in decreased H3K14ac levels in HEK293A cells (Fig. S3h). Adding back KAT7 wild-type (WT) or KAT7 mutants with mutations in the N-terminal zinc finger (C185A, C190A, H203A, and C209A) into the KAT7 knockout HEK293A cell line restored the H3K14ac level (Fig. S3h). In contrast, the reintroduction of KAT7 mutants with mutations in the zinc finger within the MYST domain (C368A, C371A, H384A, and C388A) failed to do so. In support of that finding, immunoprecipitated KAT7 mutants harbouring a zinc finger mutation in the MYST domain showed no HAT activity for H3K14 in vitro, whereas KAT7 mutants with a zinc finger mutation in the N-terminal region presented HAT activity similar to that of WT KAT7 (Fig. 3e). These results suggest that the zinc finger within the MYST domain and zinc-binding amino acid residues are needed for KAT7 HAT activity.

Next, we analysed the properties of recombinant proteins purified from E. coli. We attempted to obtain full-length KAT7 but were unsuccessful. Thus, we purified the recombinant KAT7 HAT domain (recKAT7HAT), which includes the MYST domain. Consistent with the results above, recKAT7HAT WT, but not the recKAT7HAT EQ mutant or recKAT7HAT C371A mutant, exhibited HAT activity towards H3K14 (Fig. 3f). Notably, a ZnAF-2 assay, a method to quantify the zinc content coordinated within recombinant proteins, demonstrated that recKAT7HAT C371A coordinates with substantially less zinc than recKAT7HAT WT does (Fig. 3g), suggesting the critical role of proper zinc coordination in the MYST domain for KAT7 HAT activity.

To further assess the role of zinc in KAT7, we manipulated the zinc metalation status of KAT7 in vitro and examined the effect on HAT activity. First, we attempted to demetallate zinc from KAT7 in vitro. However, even when recKAT7HAT WT was treated with TPEN in vitro, recKAT7HAT WT still coordinated with zinc and exhibited HAT activity (Fig. S3i, j). Consistent with these findings, in vitro TPEN treatment of KAT7 immunoprecipitated from HEK293A cells did not affect HAT activity (Fig. S3k). These results suggest that KAT7 coordinates zinc much more strongly than TPEN does and that the elimination of zinc from KAT7 in vitro was unsuccessful.

Given that in vitro TPEN treatment did not change the KAT7 activity, we considered that improper zinc coordination during the production or folding of KAT7 may lead to a decrease in its intracellular activity. To investigate this possibility, we treated cells with the proteasome inhibitor MG132 and the protein synthesis inhibitor cycloheximide (CHX) to block protein degradation and synthesis, respectively, and then subjected the cells to zinc depletion. Although the differences were not statistically significant, both H3K14ac levels and KAT7 activity showed a slight increase at 1 h after TPEN treatment when cells were co-treated with MG132 and CHX compared with the control, but by 2 h both parameters had declined to control levels despite the inhibitor treatment (Fig. S3l). These observations suggest that improper zinc coordination during KAT7 production or folding contributes partly to the observed activity loss, yet the predominant contribution likely comes from zinc dissociation in mature KAT7.

Next, we examined the effect of in vitro zinc repletion. As expected, in vitro zinc addition to KAT7 WT immunoprecipitated from HEK293A cells treated with TPEN, but not KAT7 C371A, restored the HAT activity in a dose-dependent manner (Fig. 3h). Collectively, these results suggest that KAT7 loses zinc from the MYST-type zinc finger domain during zinc deficiency, which leads to decreased HAT activity towards H3K14.

Loss of H3K14ac on the enhancer induces ZIP10 expression to maintain cellular zinc homeostasis
Next, we investigated the physiological role of transcriptional changes through the drastic reduction in H3K14ac during zinc deficiency. We and others have previously shown that H3K14ac regulates gene expression at enhancers36–38 and at promoters39,40. ChIP–seq analysis revealed that H3K14ac signals were markedly decreased at enhancer and promoter regions in cells cultured under zinc-deficient conditions (Figs. 4a, S4a). The zinc deficiency–sensitive loci showed substantial overlap with those exhibiting H3K14ac loss following KAT7 knockdown, indicating that zinc deficiency attenuates KAT7 activity (Fig. S4b). Notably, a greater reduction of H3K14ac at enhancer regions was associated with larger gene expression changes, involving both up- and down-regulated genes (Fig. 4b). In contrast, for genes with H3K14ac loss at promoters or at both promoters and enhancers, stronger H3K14ac reduction correlated with transcriptional down-regulation (Fig. S4c). These correlations highlight the functional importance of KAT7-mediated H3K14 acetylation in regulating gene expression.

Next, we speculated that genes responsible for maintaining cellular zinc homeostasis are regulated by H3K14ac. The Molecular Signatures Database (MSigDB) lists genes involved in zinc homeostasis (GO: 0006829, zinc ion transport)41,42. We systematically integrated our gene expression profile data with H3K14ac ChIP-seq data under conditions of zinc deficiency and KAT7 knockdown. This analysis revealed that multiple genes were transcriptionally altered, with decreased H3K14ac signals at their enhancer regions under zinc-deficient conditions and upon KAT7 knockdown (Fig. 4c), suggesting that these genes may be regulated by a reduction in H3K14ac. Among these genes, we became interested in plasma membrane-localized ZIPs, which play a key role in importing zinc from extracellular sources. Quantitative PCR (qPCR) analysis results confirmed our previous microarray findings (Fig. 4d). In particular, the notable upregulation of ZIP10 expression led us to focus on the physiological role of upregulated ZIP10 expression during zinc deficiency.

In HeLa cells, zinc supplementation suppressed the decrease in H3K14ac during zinc deficiency, which was strongly correlated with KAT7 HAT activity (Fig. S4d, e). Moreover, treatment with TSA also suppressed the decrease in H3K14ac but did not restore KAT7 HAT activity. Under these experimental conditions, we examined whether the zinc-dependent regulation of H3K14ac regulates ZIP10 expression. Zinc supplementation or TSA treatment counteracted the upregulation of ZIP10 expression during zinc deficiency (Fig. 4e). Using a membrane protein biotinylation assay, we confirmed that zinc deficiency led to the expression of ZIP10 at the plasma membrane, an effect that was suppressed by treatment with zinc or TSA (Fig. 4f). In addition, ChIP-seq and ChIP‒qPCR analyses revealed that the H3K14ac signal in the enhancer region of ZIP10, which was predicted by EnhancerAtlas 2.043, significantly decreased during zinc deficiency, whereas the signal in the promoter region did not decrease (Figs. 4g, S4f). This decrease in the enhancer regions was completely suppressed by treatment with zinc or TSA. Moreover, this decrease in the enhancer region of H3K14ac and increase in ZIP10 mRNA were dependent on KAT7, as confirmed after KAT7 knockdown (Fig. S4g-i). These results suggest that ZIP10 expression is regulated by KAT7-catalysed H3K14ac at enhancer regions.

To investigate whether the upregulation of ZIP10 expression contributes to maintaining zinc homeostasis, we assessed cellular zinc levels indirectly by measuring MT2A mRNA, the expression of which is well known to change depending on the concentration of cellular zinc44,45. Consistent with previous reports, zinc supplementation drastically upregulated MT2A expression in cells cultured in low-zinc medium, an effect that was suppressed by the knockdown of ZIP10 (Fig. S4j). Notably, the knockdown of ZIP10 but not other ZIPs significantly attenuated the upregulation of MT2A expression (Fig. S4k), suggesting the critical role of ZIP10 in the import of zinc during zinc deficiency. To directly assess zinc uptake through ZIP10, we employed ZnDA-3H, a cell-permeable fluorescent Zn2+ probe that can be localized to specific subcellular compartments, via HaloTag technology46. ZnDA-3H was localized in the cytosol when Halo-NES was expressed, and zinc influx was measured under these experimental conditions. The import of zinc (Δ[Zn2+]=80.8±3.0 pM) was significantly greater in the cells cultured in the low-zinc medium than in the cells cultured in the normal medium (42.4±4.0 pM) (Fig. 4h). In contrast, TSA treatment suppressed zinc uptake (44.5±8.4 pM), suggesting a critical role for H3K14ac in zinc influx. Notably, zinc uptake was significantly lower after ZIP10 knockdown (siRNA#1, 18.7±5.3 pM; siRNA#2, 16.2±4.3 pM) than after control knockdown (91.9±6.2 pM) (Fig. 4i). Furthermore, zinc replenishment after deficiency led to recovery of H3K14ac levels in the control knockdown condition, whereas no such recovery was observed under ZIP10 knockdown (Fig. 4j). Collectively, these data suggest that the zinc influx induced by the addition of zinc to zinc-deficient cells is driven primarily by the H3K14 deacetylation-dependent increase in ZIP10 expression.

Next, we examined the biological role of upregulated ZIP10 expression during zinc deficiency. A previous study showed that zinc deficiency induces cell cycle arrest and that subsequent zinc replenishment facilitates cell cycle re-entry47. By employing an EdU assay, we confirmed that zinc deficiency indeed arrests the cell cycle and that subsequent zinc supplementation induces cell cycle re-entry (Fig. 4k, siCtrl). Notably, ZIP10 knockdown inhibited the process of cell cycle re-entry (Fig. 4k, siZIP10#1 and siZIP10#2). These findings reinforce the importance of upregulated ZIP10 expression during zinc deficiency in cellular biological processes (Fig. 4l).

Lipid accumulation in the liver during zinc deficiency
Next, we investigated the biological significance of the zinc deficiency-induced reduction in H3K14ac in vivo. Given the central role of the liver in zinc metabolism, we examined the expression of ZIP10 in the livers of mice fed a zinc-deficient diet. Consistent with the findings in HeLa cells, ZIP10 expression was significantly upregulated (Fig. S5a), suggesting that zinc uptake through the increased expression of ZIP10 may also contribute to maintaining zinc homeostasis in vivo. Moreover, if zinc deficiency persists due to a prolonged unbalanced diet, the limited availability of zinc for cellular uptake may disrupt cellular zinc homeostasis, potentially leading to abnormalities in biological functions. Thus, we investigated the possibility that prolonged zinc deficiency contributes to disease development by inducing sustained changes in gene expression associated with reduced H3K14ac levels in vivo.

We utilized the gene expression database Expression Atlas48 to investigate diseases potentially linked to a reduction in zinc levels. Focusing on the metallothionein 2 (Mt2) gene, whose gene expression changes depending on the cellular zinc level, we examined the gene expression profile datasets of the mice with reduced Mt2 expression. There were 174 datasets in which Mt2 expression was significantly decreased. Among them, we focused on the “control vs. high-fat diet” data for the liver, which reported the most significant decrease in Mt2 expression (Fig. 5a). On the basis of these publicly available data, a high-fat diet in mice may lead to a significant reduction in hepatic zinc levels. This finding was confirmed by inductively coupled plasma‒mass spectrometry (ICP‒MS) analysis, which revealed a marked reduction in hepatic zinc concentrations in mice fed a high-fat diet (Fig. 5b). To explore the molecular mechanisms underlying this reduction, we examined the expression of zinc transporters. Specifically, we analyzed ZIP10, ZIP14—which primarily mediates zinc uptake in the liver—and ZNT1, which is responsible for zinc efflux. However, none of these transporters showed significant changes in expression (Fig. S5b). Thus, while the molecular mechanism regarding the cause of zinc reduction remained unresolved, we shifted our focus to the pathological consequences of hepatic zinc reduction. Since a high-fat diet induces hepatic lipid accumulation and promotes the development of fatty liver, we investigated the possibility that the reduction in liver zinc levels is involved in hepatic steatosis.

First, to examine the effect of zinc deficiency on hepatic steatosis, mice fed a normal or zinc-deficient diet were fed a high-fat diet. Comparative analysis revealed a pronounced exacerbation of hepatic lipid accumulation in mice fed the zinc-deficient diet, as demonstrated by oil red O staining and BODIPY staining (Figs. 5c, S5c). Conversely, dietary zinc supplementation in mice fed a high-fat diet attenuated hepatic lipid accumulation (Figs. 5d, S5d). These findings implicate zinc deficiency as a contributing factor to the development of fatty liver.

To elucidate the mechanisms underlying the contribution of zinc deficiency to hepatic steatosis, we investigated the hepatic characteristics of a zinc-deficient mouse model. Mice fed a zinc-deficient diet presented a reduction in hepatic zinc levels (Fig. 5e). This reduction was accompanied by notable hepatic lipid accumulation (Figs. 5f, S5e) and elevated triglyceride levels to a similar extent as those observed with a high-fat diet (Fig. 5g). Collectively, these results suggest that diminished hepatic zinc levels facilitate lipid accumulation in the liver, thereby contributing to the development of hepatic steatosis.

Loss of H3K14ac promotes lipid accumulation in liver tissue via gene expression upregulation
Next, we investigated the mechanism underlying hepatic steatosis induced by zinc deficiency. Mice fed a zinc-deficient diet presented reduced levels of H3K14ac (Fig. 6a, b). Similarly, mice fed a high-fat diet presented reduced H3K14ac levels, whereas zinc supplementation counteracted this reduction (Figs. 6c, S6a). Notably, KAT7 immunoprecipitated from mouse livers fed a zinc-deficient diet presented significantly decreased HAT activity (Fig. 6d). Moreover, the intraperitoneal treatment of mice with the KAT7 inhibitor WM-3835 resulted in lipid accumulation in the liver (Fig. 6e). These results suggest that a reduction in KAT7 activity and the subsequent decrease in H3K14ac levels in the liver may induce hepatic steatosis. To assess whether zinc deficiency in the liver promotes hepatic steatosis in a cell-autonomous manner, we conducted experiments using mouse primary hepatocytes. TPEN treatment of these cells resulted in a decrease in H3K14ac levels and an increase in lipid droplets, effects that were suppressed by TSA treatment (Fig. 6f, g). Furthermore, treatment with WM-3835 or the knockdown of KAT7 led to increased lipid droplet formation (Fig. S6b-e). Taken together, these results suggest that zinc deficiency contributes at least in part to hepatic steatosis through decreased KAT7 activity and a reduction in H3K14ac.

Next, we investigated whether zinc deficiency in the liver alters the expression of genes associated with lipid accumulation. RNA-seq analysis revealed that zinc deficiency in the liver induced a decrease in the expression of 1,193 genes and an increase in the expression of 1,781 genes (Fig. 6h). We then conducted ChIP-seq analysis for H3K14ac, the results of which revealed a global reduction in H3K14ac at enhancer regions during zinc deficiency (Fig. 6i). In line with the findings shown in Fig. 4, the RNA-seq analysis demonstrated an upregulation of ZIP14, a zinc transporter reported to be critical for maintaining hepatic zinc homeostasis (Fig. S6f). Moreover, the ChIP-seq analysis revealed a reduction of H3K14ac signals at the enhancer regions of both ZIP10 and ZIP14 (Fig. S6g). Together, these results suggest that the decrease in H3K14ac also contributes to hepatic zinc homeostasis through the regulation of zinc transporters under zinc-deficient conditions. In addition to the zinc transporters, the expression of several genes associated with lipid droplet synthesis pathways, such as triglyceride synthesis, intracellular lipid transport, and lipid droplet budding, was upregulated during zinc deficiency, along with a decrease in H3K14ac at enhancer regions (Fig. 6j, k). Additionally, the treatment of primary hepatocytes with WM-3835 increased the expression of key genes involved in lipid droplet formation (Fig. S6h). Collectively, these findings suggest that zinc deficiency drives lipid accumulation by upregulating the expression of genes involved in lipid droplets synthesis, a process mediated by reduced KAT7 activity and diminished H3K14ac levels (Fig. 6l).

Finally, we conducted a clinical literature review to examine the association between zinc and fatty liver diseases in humans. Several clinical studies have measured hepatic zinc concentrations in both healthy individuals and patients with diseases accompanied by hepatic steatosis. A meta-analysis of these studies revealed significantly lower hepatic zinc levels in the patient group than in the healthy control group (Fig. 6m). This finding suggests that zinc deficiency in the liver may serve as a potential risk factor for fatty liver diseases, including NAFLD.

Methods
A list of the primers, antibodies, and siRNAs used in this study is provided in Table S1-S7.

Plasmids
The cDNAs encoding human KAT7, BRPF1, BRPF2, BRPF3, JADE1, JADE2, JADE3, ING4, ING5, MEAF6, and HDACs were amplified by PCR using KOD One PCR Master Mix -Blue- (TOYOBO, KMM-201) and inserted into pcDNA3/GW with an HA or a FLAG tag (Invitrogen) or into pMAL-c6T (New England Biolabs). The mutated constructs were prepared by PCR-mediated site-directed mutagenesis. A single guide RNA (sgRNA) targeting KAT7 (5’-AGCCGCCGGCAATGCCGCGA-3’) was designed using CHOPCHOP. The sgRNA was inserted into pX459 (Addgene, 62988). The primer lists used for cloning and mutagenesis are provided in Tables S1 and S2.

Cell culture
HEK293A cells, U2OS cells, HCT116 cells, and Neuro2A cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma‒Aldrich, D5796; Wako, 044-29765). HeLa cells were maintained in low-glucose DMEM (Wako, 041-29775). Each type of culture medium was supplemented with 10% fetal bovine serum (FBS) (Gibco, 10270-106) and 100 units/ml penicillin G (Meiji Seika, 01028-85). All cell lines were grown in 5% CO2 at 37°C.

Preparation of zinc-deficient medium
FBS was incubated with Chelex-100 resin (Bio-Rad, 1422832) at a final concentration of 0.05 g/mL at 4°C with rotation. After 12 h, the mixture was centrifuged at 500 × g for 5 min. The supernatant was passed through a Millex-GV 0.22 µm PVDF filter (Millipore, Cat# SLGVR33RS). Cell culture medium supplemented with Chelex-100 resin-treated FBS at a final concentration of 10% was used as zinc-deficient medium.

Preparation of Zn-saturated TPEN
Preparation of Zn-saturated TPEN was performed by mixing 10 µM TPEN with an equimolar concentration of ZnCl2 (10 µM) and incubating the mixture overnight. The resulting solution was used as Zn-saturated TPEN for cell treatment.

Transfection
Plasmid transfection was performed using Polyethylenimine “MAX” (Polysciences, 24765) or Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) according to the manufacturer’s instructions. siRNA-mediated knockdown was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778500) at a final siRNA concentration of 10 or 20 nM by reverse transfection, according to the manufacturer’s instructions. The list of siRNAs used in this study is provided in Tables S3 and S4.

Generation of knockout cell lines
To generate KAT7 knockout HEK293A cells or HeLa cells, cells were transfected with pX459 encoding a sgRNA targeting KAT7. Forty-eight hours after transfection using Lipofectamine 2000, the cells were selected by 1.0 µg/mL puromycin (Gibco, A11138-03) treatment for another 48 h. The selection medium was replaced with fresh standard medium, and the cells were grown for 3 days. Limiting dilution was performed to obtain single-cell clones. The knockout status of the clones was confirmed by immunoblotting.

Immunoblotting
Cell were lysed in a cell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 5 µg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 nM TSA) for 10 min at 4°C. The lysates were sonicated with 2 sets of 10 s pulses using an ultrasonic homogenizer (SMT, UH-50). The cell debris was cleared by centrifugation at 17,700 × g for 15 min. The supernatant was mixed with a sample buffer (80 mM Tris-HCl pH 8.8, 80 µg/mL bromophenol blue, 28.8% glycerol, and 4% SDS) supplemented with 10 mM DTT and incubated for 3 min at 98°C. The samples were then resolved by SDS–PAGE and transferred onto Immobilon-P PVDF membranes (Millipore, IPVH00010). The SDS–PAGE gels used were either self-prepared polyacrylamide gels or NuPAGE® Bis-Tris Precast Gels (Thermo Fisher Scientific, WG1403BOX). Blocking was performed with 5% skim milk (Megmilk Snow Brand) in TBS-T (50 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.05% Tween 20) for 30 min at room temperature. After incubation of the membranes with the indicated primary antibodies for 12–36 h at 4°C and corresponding HRP-linked secondary antibodies for 1–2 h at room temperature, the chemiluminescent signals enhanced by ECL Select (Cytiva, RPN2235) were detected using a Fusion Solo 7S instrument (M&S Instruments). Quantification of the bands was performed using Fiji software83. The list of antibodies used for immunoblotting is provided in Table S5.

Coimmunoprecipitation analysis
Cell lysates were incubated with anti-DYKDDDDK-tagged antibody beads (Wako, 016-22784) at 4°C for 10 min. The beads were washed with washing buffer 1 (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 5 mM EGTA and 1% Triton X-100) and washing buffer 2 (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM EGTA). The washed beads were mixed with sample buffer (80 mM Tris-HCl pH 8.8, 80 µg/mL bromophenol blue, 28.8% glycerol, and 4% SDS) supplemented with 20 mM DTT for 5 min at room temperature and then incubated for 3 min at 98°C. The samples were separated by SDS–PAGE and immunoblotted with antibodies.

Immunofluorescence analysis
For confocal microscopy, cells were grown on glass coverslips (Matsunami Glass, C015001). For imaging with a high-content imaging system, cells were grown on a 96-well plate (Corning, 353072). The cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After blocking with 1% BSA (Iwai Chemicals, A001) in TBS-T for 1 h, the cells were incubated with the indicated primary antibodies overnight at 4°C. After washing three times with PBS, the cells were incubated with fluorophore-conjugated secondary antibodies at room temperature for 2 h and then with Hoechst 33342 (Dojindo, 346-07951) for 20 min. After washing three times with PBS, the cells were mounted with Fluoromount (Diagnostic BioSystems, K024) for confocal microscopy. Images were acquired using an LSM880 with Airyscan (Zeiss) with a 63×/1.40 oil immersion objective or a CellInsight NXT HCS system (Thermo Scientific). The contrast and brightness of the images were adjusted using Fiji software. Quantification of the images was performed using Fiji software or CellProfiler84.

Histone purification
HEK293A cells or HEK293A KAT7 KO cells were incubated in hypotonic lysis buffer for 30 min at 4°C with rotation. After centrifugation at 10,000 × g for 10 min at 4°C, the cell pellets were resuspended in 0.4 N H2SO4 and incubated for 30 min at 4°C with rotation. After centrifugation at 15,100 × g for 10 min at 4°C, the supernatant was transferred to a fresh 1.5 mL tube. Then, trichloroacetic acid (TCA) was added to the supernatant dropwise and incubated for 30 min on ice. After centrifugation at 15,100 × g for 10 min at 4°C, the pelleted histones were washed twice with ice-cold acetone. The pellet histones were air-dried at room temperature and dissolved in TBS at a final concentration of 3 µg/µL.

Cell lysate–based HDAC activity assay
HEK293A cells treated with DMSO or 10 µM TPEN for 1 h or 2 h were lysed in a cell lysis buffer for in vitro HDAC assay (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 5 µg/mL leupeptin, and 1 mM PMSF) for 10 min at 4°C. Purified histones were mixed with the cell lysates and incubated at 37°C for 0.5, 1 or 2 h with DMSO or 10 nM TSA. Then, the samples were mixed with an equal volume of 2x sample buffer supplemented with 10 mM DTT and incubated for 3 min at 98°C, followed by SDS‒PAGE and immunoblotting.

In vitro HDAC assay
HEK293A cells transfected with HDAC-FLAG were lysed in cell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 5 µg/mL leupeptin, and 1 mM PMSF). For HDAC1, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC10, cell lysates derived from 1.0 × 10^6 cells were used, whereas for HDAC2, HDAC3, and HDAC11, lysates from 1.0 × 10^8 cells were prepared. The cell lysates were incubated with anti-FLAG M2-agarose gel (Sigma‒Aldrich, A2220) at 4°C for 5 min. The beads were washed twice with washing buffer 3 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100) and once with TBS. Purified histones at a final concentration of 0.5 µM were mixed with the beads incubated for 5–15 min at 37°C with frequent mixing (1200 rpm) using a Thermomixer C (Eppendorf). The samples were mixed with an equal volume of 2x sample buffer supplemented with 10 mM DTT and incubated for 3 min at 98°C, followed by SDS‒PAGE and immunoblotting.

In vitro HAT assay
To measure the activity of KAT7 immunoprecipitated from cells, HEK293A cells transfected with FLAG-KAT7 WT or mutants were lysed in cell lysis buffer for an in vitro HAT assay (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 5 µg/mL leupeptin, and 1 mM PMSF). The cell lysates were incubated with anti-FLAG M2-agarose gel (Sigma‒Aldrich, A2220) at 4°C for 5 min. The beads were washed twice with washing buffer 3 (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100) and once with TBS. Purified histones at a final concentration of 0.5 µM were mixed with the beads and with 50 µM acetyl-CoA in TBS and incubated for 30 min at 30°C with frequent mixing (1200 rpm) using a Thermomixer C (Eppendorf). The samples were mixed with an equal volume of 2x sample buffer supplemented with 10 mM DTT and incubated for 3 min at 98°C, followed by SDS‒PAGE and immunoblotting. To measure the activity of the recombinant KAT7 HAT domain, 5 µM recombinant protein was used. To measure the activity of the KAT7 from mice liver, the organ lysates were incubated with KAT7 antibody at 4°C for 16 h. Then, the lysates were incubated with Protein G Sepharose™ 4 Fast Flow (GE healthcare, 17061802) at 4°C for 2 h.

Recombinant protein purification
The KAT7 HAT domain (a.a. 336-611) WT or C371A mutants encoded in pMAL-c6T were expressed in E. coli BL21 cells. Expression was induced by incubation in LB medium supplemented with 0.3 mM IPTG at 25°C for 1.5 h. The cells were pelleted and resuspended in PBS supplemented with 1% Triton X-100 and 1 mM PMSF. After sonication with 4 sets of 30 s pulses using an ultrasonic homogenizer, the cell debris was cleared by centrifugation at 26,800 × g for 30 min. The supernatants were incubated with Ni Sepharose 6 Fast Flow (Cytiva, 17531806) at 4°C for 8 h. The beads were then washed twice with PBS and twice with TBS and eluted with a 250 mM imidazole solution in TBS using Poly-Prep Chromatography Columns (Bio-Rad, 7311550). The eluates were loaded onto a Superose 6 Increase 10/300 GL column on an AKTA purifier (GE Healthcare). The purified His-MBP-KAT7 was incubated with TEV protease (NEB, P8118) at 30°C for 4 h. The cleaved His-MBP fragment was removed by filtration through Ni Sepharose 6 Fast Flow resin. The purified KAT7 was dialyzed in TBS overnight at 4°C. The purity and concentration of the final products were estimated by Coomassie blue staining of SDS–PAGE gels.

qPCR analysis
Total RNA was isolated from cells using Isogen (Nippongene, 319-90211) and reverse transcribed with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, FSQ-301) according to the manufacturer’s instructions. Samples of the qPCR mixture were prepared using the KAPA SYBR Fast qPCR Kit (Kapa Biosystems, KK4602). qPCR was then performed on a LightCycler 96 (Roche) or QuantStudio 1 (Thermo Fisher Scientific) instrument. Data for each mRNA were normalized to that of RPS18. The list of primers used for qPCR is provided in Table S6.

ChIP‒qPCR
Chromatin samples for ChIP‒qPCR were prepared using a SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003) according to the manufacturer’s instructions. The chromatin samples were incubated with anti-H3K14ac antibody or anti-histone H3 antibody (Cell Signaling Technology, 4499) at 4°C for 8 h with rotation. Then, the samples were incubated with Dynabeads Protein G (VERITAS, DB10004) at 4°C for 2 h with rotation. After washing three times with low-salt buffer (1% SDS, 1% Triton-X, 2 mM EDTA, 20 mM Tris-HCl pH 8.0 and 150 mM NaCl) and once with high-salt buffer (1% SDS, 1% Triton-X, 2 mM EDTA, 20 mM Tris-HCl pH 8.0 and 500 mM NaCl), the chromatin was eluted with elution buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 8.0) for 30 min at 65°C with frequent mixing (1,200 rpm) using a Thermomixer C. The purified DNA was analysed as described in the “qPCR analysis” section. ChIP-seq data used for designing primers were downloaded from the ENCODE portal85 with the following identifier: ENCFF002QJL. Data visualization was performed with Integrative Genomics Viewer (Broad Institute). The list of primers used for qPCR is provided in Table S7.

ZnAF2 assay
Recombinant proteins or ZnCl2 solution for normalization was incubated with 10 µM ZnAF-2 (Goryo Chemical, SK2001-01) in TBS for 30 min at room temperature. The fluorescence signal was detected with a Varioskan microplate reader (Ex/Em=492/515 nm, Thermo Scientific).

ERSE sequence analysis
ERSE sequences, CCAAT-(N)9-CCAC[A/G], on the human genome GRCh/hg38 were retrieved from the GGGenome ultrafast sequence search browser. Among the sequences obtained (plus strand, 3764 sequences; minus strand, 3874 sequences), 1897 sequences were annotated as sequences on gene promoters by employing HOMER. The number of ERSE sequences on genes that were upregulated during zinc deficiency was counted programmatically. Gene expression profile data analysed by GEO2R were downloaded from the GEO repository (GSE49657, GSE99204, GSE108923, and GSE135873). Genes with a fold change in expression greater than 2 were considered upregulated.

EdU assay
HeLa cells were grown on a 96-well plate and analysed with a Click-iT EdU imaging kit (Invitrogen, C10340). The cells were incubated with 10 µM EdU for 1 h, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, and washed twice with PBS containing 3% BSA. The cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature and washed twice with PBS containing 3% BSA. Then, Click-iT reaction cocktail was added and allowed to react for 30 min at room temperature. After washing twice with PBS containing 3% BSA, the cells were incubated with Hoechst 33342 (1:2000) in PBS for 30 min at room temperature. Images were acquired with a CellInsight NXT automated microscope and analysed with CellProfiler.

Membrane protein biotinylation assay
Cells were incubated with 0.4 mg/mL biotin (EZ-Link Sulfo-NHS-SS-Biotin, Thermo Fisher Scientific, 21331) in PBS(+) (PBS containing 50 mM MgCl2 and 100 mM CaCl2) and washed twice with 150 mM glycine in PBS(+). Cells were lysed as described in the “Immunoblotting” section. The cell lysates were pulled down with streptavidin agarose resin (Thermo Fisher Scientific, 20353). The resin samples were washed twice with washing buffer 1 and once with washing buffer 2. The samples were subjected to immunoblotting.

Zinc imaging by ZnDA-3H
Zinc measurement with ZnDA-3H was performed as described previously with minor optimization46,86. HeLa cells were reverse-transfected with 10 µM siRNA. The cells were then transfected with Halo-NES and cultured for 24 hours. The cells were replated on 35 mmφ glass bottom dishes (Matsunami, D11130H) that were coated with 1% Cellmatrix Type I-C (Nitta Gelatin, KP-4100). After 24 hours, the cells were incubated with zinc-normal medium or zinc-deficient medium for an additional 48 hours. Cells were washed with Chelex-treated HEPES-buffered HBSS (30 mM HEPES-NaOH, pH 7.4, 5.36 mM KCl, 137 mM NaCl), and incubated with serum-free DMEM containing 250 nM ZnDA-3H and 5 nM HTL-TMR for 30 min. After labelling, the cells were incubated for an additional 30 minutes. After incubation, the cells were washed twice with Chelex-treated HEPES-buffered HBSS, and the medium was exchanged for Chelex-treated HBSS buffer. Multichannel time-lapse images were acquired with 4 averages per frame in 30-second intervals. ZnDA-3H or HTL-TMR was excited at 488 nm with an argon laser or at 561 nm with a DPSS laser and detected by a HyD detector (Leica). Images were acquired for 5 minutes, and then the cells were incubated with containing Chelex-treated HBSS 10 µM ZnCl2 for an additional 10 min. To obtain additional images under zinc-deprived and zinc-saturated conditions, the cells were incubated with Chelex-treated HBSS containing 10 µM TPEN for 5 minutes, and then, the cells were incubated with Chelex-treated HBSS containing 400 µM ZnCl2 and 2.0 µM zinc pyrithione for an additional 10 minutes. For presentation, the pseudocolour fluorescence ratio images of ZnDA-3H/HTL-TMR were generated using Fiji software. For quantification of the intensity, we used CellProfiler.

Meta-analysis
A systematic literature search was conducted in multiple electronic databases (PubMed, Web of Science, and Embase) using the pre-defined search terms “zinc” and “liver disease.” From the identified articles, the means of and variances in the liver zinc content in healthy subjects and patients with liver disease were extracted for analysis. All the statistical analyses were conducted using R (R Foundation for Statistical Computing, Vienna, Austria). For studies reporting only median and IQR data, the means and variances were estimated using the “estmeansd” package87. A multilevel meta-analysis was performed to address possible correlations among several outcome statistics using the “meta” package88.

Animal studies
All the experiments were performed following the experimental protocol approved by the animal ethics committee of the University of Tokyo. We complied with all relevant ethical regulations for animal use. Male and female C57BL/6J mice were used. C57BL/6J mice were purchased from Japan SLC Inc. The mice were maintained in a specific pathogen-free facility and housed under 25°C, 40–60% humidity, 12/12 light/dark cycle. Age (8–10 weeks) and sex (both male and female)-matched mice were used for the experiments. Sample sizes determination was based on previous reports89,90.

The mice were fed a ND (Zn: 6 mg/100g, 13% calories from fat; CE-2, Clea Japan), ZD (Zn: 0 mg/100g, 13% calories from fat; Clea Japan), HFD (Zn: 6 mg/100g, 60% calories from fat; CE-2, Clea Japan), or HFD+ZD (Zn: 0 mg/100g, 60% calories from fat; Clea Japan). Mouse livers were perfused and immersed O/N in 4% paraformaldehyde/PBS for fixation and then cleared with 30% sucrose solution. Fixed livers were embedded in CryoMount I (Muto Pure Chemicals), and 8 µm-thick sections were cut using a cryostat (Leica), followed by oil red O staining and BODIPY staining.

Primary hepatocyte isolation
Primary hepatocytes were isolated from male and female mice as previously described75. Briefly, primary hepatocytes were isolated by two-step collagenase perfusion. After isolation, the cells were resuspended in low-glucose DMEM supplemented with 5% FBS and 100 units/mL penicillin G (Meiji Seika) and then seeded in a six-well plate precoated with rat collagen type I (Sigma, C3867). The culture medium was changed to maintenance medium after three hours. The maintenance medium contained William’s E medium (Gibco, 12551-032), 1% L-glutamine (Wako, 076-00521), and 1% penicillin G. After an overnight incubation, the hepatocytes were used for subsequent experiments.

Oil red O staining
Oil red O stock solution (oil red O (Wako, 154-02072) 0.03 g/10 mL isopropanol) was diluted 3:2 with Milli-Q water to make an Oil red O working solution. Frozen blocks were sectioned using a cryostat (Leica), and the sections were stained with Oil Red O working solution for 20 min. The sections were then washed three times for 5 min with Milli-Q water and transferred to haematoxylin solution (Sakura, 6187-2) for 1 min, followed by three washes with Milli-Q water. The sections were mounted with Fluoromount (Diagnostic BioSystems, K024) for confocal microscopy. Images were acquired using a DM2500B (Leica) with a 40× objective.

BODIPY staining
Mouse primary hepatic cells were seeded on glass coverslips, fixed with 4% paraformaldehyde in PBS for 30 min, and incubated with 0.5 µM BODIPY 493/503 (Cayman Chemical, 25892) for 20 min. The coverslips were mounted with Fluoromount (Diagnostic BioSystems, K024) for confocal microscopy. Frozen blocks were sectioned using a cryostat (Leica), and the sections were stained with 0.5 µM BODIPY 493/503 for 3 h. The sections were subsequently washed twice in 1x PBS and then mounted with Fluoromount (Diagnostic BioSystems, K024) for confocal microscopy. Images were acquired using a TCS SP8 confocal microscope with a 63× oil immersion objective. Lipid droplet counts were quantified using CellProfiler software.

Liver triglyceride quantification
Liver samples ranging from 40 to 60 mg were washed three times with 1x PBS. The samples were subsequently lysed with 10% NP-40. The livers were then homogenized using a homogenizer. The samples were boiled at 98°C for 3 min with intermittent vortexing, cooled to room temperature, and then boiled again for complete solubilization of triglycerides. The samples were centrifuged at 10,000 × g for 10 min at 4°C, after which the supernatant (including the lipid layer) was collected. The samples were diluted 1:20 in water for triglyceride measurements. Liver triglycerides were quantified using a triglyceride assay kit (Abcam, ab653363) in accordance with the manufacturer’s instructions.

ICP‒MS
The zinc concentration in liver tissues was measured via ICP‒MS (Agilent 7800, Agilent Technologies). Liver tissues were dried at 100°C for 16 h. Thirty milligrams of dried liver was decomposed using concentrated nitric acid at 150°C for 15 h. The decomposed product was dissolved in 0.08 M nitric acid. Zinc (m/z 66) was used to measure the zinc concentration, and indium (m/z 115) was used as an internal standard to correct the measurement results.

RNA-seq
Total RNA isolated by ISOGEN was used for library construction for RNA-Seq analysis. Library preparation was conducted using an NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB, E6420). Deep sequencing was performed on the Illumina NextSeq 2000 platform to obtain 36-bp paired-end reads. Approximately 15 million sequences were obtained and mapped to the reference human genome (hg38) or mouse genome (mm10) via HISAT2 (v.2.2.1). Transcript read counts were determined using featureCounts (v.2.0.6). Differential gene expression analysis was performed using the R (v.4.3.2) package DESeq2 (v.1.42.1).

ChIP-seq
Chromatin samples were prepared using a SimpleChIP Enzymatic Chromatin IP Kit following the manufacturer’s instructions. For normalization, the samples were mixed with spike-in chromatin (Active Motif, 53083). The chromatin samples were then incubated with an anti-H3K14ac antibody (Abcam, ab52946) and a spike-in antibody (Active Motif, 61686) for 8 h at 4°C with rotation. The samples were subsequently purified as described in the “ChIP‒qPCR” section. Library preparation was conducted using an NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (NEB, E6420). Deep sequencing was performed on the Illumina NextSeq 2000 platform to obtain 36-bp paired-end reads. Approximately 30 million sequences were obtained and mapped to the reference human genome (hg38) or mouse genome (mm10) via HISAT2 (v.2.2.1). The H3K14ac signals on the enhancers were calculated programmatically. BED files for enhancer regions were downloaded from the EnhancerAtlas 2.043 and FANTOM591,92 databases. The H3K14ac signals on the enhancer regions were analysed via wigglescout, an R package (https://github.com/cnluzon/wigglescout/) on a supercomputer. The supercomputing resource was provided by the Human Genome Center (the Univ. of Tokyo) and the Institute of Statistical Mathematics.

Correlation analysis between H3K14ac ChIP-seq signal and gene expression
To investigate the relationship between transcriptional changes and histone modification alterations, microarray data were retrieved from the Gene Expression Omnibus (GEO) dataset GSE49657. This dataset comprises samples subjected to TPEN treatment or FBS starvation (FBS(−)), both of which represent zinc-deficient stimuli. Genes exhibiting a fold change > 1.5 under these zinc-deficient conditions compared to controls were defined as up-regulated and down-regulated. The change in H3K14ac ChIP-seq signal intensity was calculated for enhancer regions associated with these genes. Subsequently, genes were ranked based on the magnitude of H3K14ac signal reduction at their associated enhancers and classified into four quartiles (bins 1–4). Bin 1 comprises genes associated with enhancers showing the largest decrease in H3K14ac signal, whereas Bin 4 represents those with the smallest decrease.

Quantification and statistical analysis
All the statistical data were analysed via R (ver. 4.0.5) with RStudio (ver. 2022.12.0+353). P values < 0.05 were considered to indicate statistical significance. Detailed statistical information for each experiment, including the number of biological replicates (n), is shown in the figures and corresponding legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors conducted their work investigating how zinc deficiency alters gene expression at the molecular level using cell lines and animal models. In this study, they focused on histone posttranslational modifications (PTMs), which are key regulators of transcriptional control. Their findings demonstrated that zinc deficiency reversibly affects acetylation of histone H3 at lysine 14 (H3K14ac). Specifically, they showed that the HDAC family members contribute to the deacetylation of H3K14ac under zinc-deficient conditions. Additionally, they demonstrated that the histone acetyltransferase KAT7 loses its enzymatic activity during zinc deficiency. To identify genes potentially regulated by the reduction in H3K14ac, the authors integrated transcriptomic data with H3K14ac ChIP-seq data under conditions of zinc deficiency and KAT7 knockdown. This integrative analysis highlighted ZIP10 as a zinc transporter gene whose expression is upregulated during zinc deficiency. Functional experiments revealed that ZIP10 knockdown inhibited cell cycle re-entry, underscoring its potential role in cellular responses to zinc deficiency. The authors extended their investigation to in vivo models and observed increased ZIP10 expression in the livers of mice fed a zinc-deficient diet—paralleling the in vitro results in HeLa cells. These findings suggest that ZIP10-mediated zinc uptake might contribute to maintaining zinc homeostasis also in vivo. Furthermore, the authors propose a model in which ZIP10-dependent zinc import is necessary for KAT7 activity, thereby linking zinc status to epigenetic gene regulation.\nThis study is well-executed and provides compelling mechanistic insights based on in vitro data. However, there appears to be a significant disconnect between the in vitro findings and the in vivo validation. While data from Figures 1 through 4 align with their proposed model, Figures 5 and 6 do not convincingly demonstrate a functional link with data shown in Figures 1 through 4, in terms of the mechanisms of zinc-mediated epigenetic regulation in vivo. If ZIP10 is indeed essential for epigenetic gene regulation during zinc deficiency or high-fat diet t (HFD) model, it would be critical to assess the consequences of ZIP10 loss specifically in the liver. Thus, generating and analyzing liver-specific conditional ZIP10 knockout mice is necessary to validate the physiological relevance of the proposed mechanism in vivo.\nIn fact, the authors present a well-structured series of in vitro experiments through Figure 4, which effectively support their proposed model. However, the interpretation of the in vivo data related to zinc deficiency with or without high-fat diet -induced epigenetic regulation via ZIP10 raises various concerns, as these complex pathophysiological conditions involve multiple confounding factors. Furthermore, the discussion gives only limited attention to the physiological role of ZIP10, which weakens the overall impact of the conclusions. As a result, a conceptual disconnect emerges between the in vitro findings and the in vivo observations, leading to an overall impression of inconsistency in the study’s central claims. Taken together, the current evidence appears insufficient to fully substantiate the authors’ conclusions, and the mechanistic link remains unconvincing at this stage.\nIn addition, given that another zinc importer ZIP14 is predominantly expressed in the liver, it is surprising that the authors neither addressed nor investigated the potential link between ZIP14 and zinc-mediated epigenetic gene regulation in hepatic tissue, especially in vivo model. This also represents a significant gap in the current study. Exploring this connection would substantially strengthen the biological relevance and impact of the work.\nSpecific comment:\nIn Fig.3C:\nThere appear to be two identical descriptions for the conditions labeled as ZN medium cultured for 48 hours. The reviewer would appreciate clarification on whether there is a distinction between these two conditions, or if this is a duplication error.\nIn Fig.4H:\nThe Zip10 siRNA treatment resulted in a reduction of zinc content in HeLa cells. Given this observation, the reviewer is interested to know whether H3K14ac levels were also affected under the same conditions. Furthermore, since zinc pyrithione—a known zinc ionophore—can enhance intracellular zinc levels, the reviewer suggests evaluating whether zinc pyrithione treatment could rescue H3K14ac status or restore KAT7 activity in the context of Zip10 knockdown. This would provide valuable insight into the functional consequences of zinc depletion on histone acetylation and its potential reversibility.\nIn Fig.5B:\nThe authors report a decrease in hepatic zinc content during HFD treatment, accompanied by reduced Mt2 mRNA expression. However, the underlying mechanism driving the reduction in liver zinc content remains unclear. The manuscript presents only minor changes in Zip10 expression in Figure S5A, without addressing the potential involvement of other zinc transporters or regulatory mediators. The reviewer suggests that additional analysis regarding other zinc transport pathways would strengthen the interpretation of these findings.\nFigure S5A:\nThe authors report a statistically significant increase in Zip10 mRNA levels under zinc-deficient conditions. However, the corresponding protein levels were not provided. To strengthen the conclusion, it is important to include data on ZIP10 protein expression under the same conditions. Additionally, as noted above, assessing the expression of a broader panel of zinc transporters and other zinc-related mediators would help determine whether ZIP10 is uniquely or predominantly affected in response to zinc deficiency.\nDiscussion:\nThe Discussion section would benefit from a more comprehensive analysis of the role of ZIP10, particularly regarding its physiological relevance in the context of epigenetic regulation. Currently, the discussion centers largely on zinc-dependent epigenetic changes observed in vitro. To provide deeper mechanistic insight, it is important to address how zinc may influence epigenetic modifications in vivo, and to discuss the potential involvement of ZIP10 within this broader physiological context.\nAdditionally, given that ZIP14 is known to be predominantly expressed in the liver, the authors should consider discussing its potential contribution to zinc-mediated epigenetic gene regulation in hepatic tissue. This could help clarify whether ZIP10 plays a unique or complementary role in this process.", "reviewer_2": "Fujisawa et al present a clear, elegantly designed and thorough analysis, assessing the role of H3K14ac in zinc deficiency. The authors show that zinc deficiency impairs the histone acetyltransferase function of KAT7, resulting in gene expression changes in zinc transporters. This novel work gives clear mechanistic insight into the pathological effects of zinc deficiency and nicely links this finding to intracellular lipid droplet formation in a mouse model, demonstrating its relevance to health and liver disease. This provides a novel understanding in the field and clearly links nutrient conditions to changes in epigenetic histone marks. The data is clearly presented, thoroughly investigated and convincing and has been performed to a high standard. There is also sufficient detail in the methods to allow this work to be reproduced. There are a couple of points, however, that I feel are missing and the authors should consider adding:\nMajor Points:\n1. H3K14ac and Gene Expression Inhibition:\no Generally, H3K14ac and histone acetylation are associated with increased gene expression. The role for H3K14ac in marking inactive enhancers seems to be limited to cancer cells and not otherwise healthy cells. However, the model in Fig. 4J suggests that H3K14ac at the promoter inhibits expression of ZIP10, counter to the current dogma. The authors should expand the discussion to explain why they believe H3K14ac exhibits an inhibitory effect in their study and consider recreating this model to better reflect that this is an enhancer not a promoter effect of H3K14ac.\no Additionally, correlating the RNA-seq (GSE49647) changes in gene expression between ZN and ZD conditions and changes in H3K14ac ChIP-seq between ZN and ZD conditions, could help demonstrate whether H3K14ac in this case inhibits gene expression. Is this inhibitory effect genome-wide in ZD conditions, or is it specific to ZIP genes such as ZIP10? Correlations, such as log2 fold changes in gene expression (ZN vs. ZD) with H3K14ac abundance at enhancers or promoters between ZN vs ZD, could reveal specific sets of genes impacted by H3K14ac loss. If ZIP10 is one of only a few genes showing this effect, its uniqueness should be highlighted.\n2. Promoter-Specific Retention of H3K14ac:\no The western blots presented in this study show a complete loss of H3K14ac in zinc deficiency and KAT7 knockdown conditions, consistent with ChIP-seq findings. However, Fig. 4F reveals that this loss does not extend to promoters. Since promoters constitute only a small portion of the genome, the sensitivity of western blots may be insufficient to detect the retention of H3K14ac at promoters, however, the authors should discuss why promoter regions appear to be spared from loss of H3K14ac in zinc deficiency, as KAT7 is solely responsible for H3K14ac.\no Heatmaps for H3K14ac across promoters should be included in the main or supplemental figures to reinforce the claim that zinc deficiency spares loss of H3K14ac at promoters in ZD conditions. Presumably siRNA knockdown of Kat7 causes a complete loss of H3K14ac both at enhancers and promoters.\nMinor Points:\n1. The authors may consider assessing whether KAT7 knockdown under normal zinc nutrient conditions affects ZIP10 expression, possibly through RT-qPCR. While the link between zinc deficiency and KAT7-mediated acetylation loss is compelling, it remains unclear if KAT7 knockdown alone recapitulates the effects of zinc deficiency. Investigating this could uncover whether zinc deficiency triggers additional cellular responses leading to increased ZIP10 expression that loss of KAT7 alone cannot explain.\n2. On page 10, the authors state that \"TSA treatment suppressed zinc uptake, suggesting a critical role for H3K14ac in zinc influx.\" As TSA inhibits class I and II HDACs, this claim may not be entirely accurate since TSA elevates all histone acetylation marks. This statement should be revised or removed.\n3. In Fig. S3E, TPEN treatment after 2 hours appears to increase the association of KAT7 with BRPF3. Do the authors have any thoughts as to why this may be the case? If so, this should be added to the discussion or results section.\n4. Fig. 4I is missing a Y-axis label. I assumed this graph is \" %EDU+ cells \".\n5. Uncropped western blot images should be provided in the supplemental data.\n6. The degree of KAT7 knockdown achieved by siRNA should be shown through either western blotting or RT-qPCR.", "reviewer_3": "In this manuscript, the authors demonstrate that zinc is essential for the histone acetyltransferase (HAT) activity of KAT7, leading to genome-wide epigenetic changes, particularly at enhancer regions. They employ a variety of experimental approaches to determine whether the observed reduction in H3K14 acetylation results from enhanced histone deacetylase (HDAC) activity or reduced HAT activity. Their data indicate that zinc deficiency does not affect HDAC activity in vitro, but it does impair KAT7-mediated H3K14 acetylation in both in cellulo and in vitro HAT activity assays. The authors identify two zinc finger domains in KAT7, with the zinc finger within the MYST domain responsible for zinc coordination. Mutations in this domain reduce enzymatic activity. Furthermore, the authors show that reduced KAT7 activity leads to epigenetic changes that elevate Zip10 expression and alter lipid homeostasis in the mouse liver. These findings are original and potentially significant for the fields of zinc/metal homeostasis and metabolic disorders.\nHowever, several concerns should be addressed to strengthen the manuscript:\nWhile the authors suggest that multiple HDAC family members may cooperatively contribute to H3K14 deacetylation during zinc deficiency—based on the observation that TSA (a pan-inhibitor of classical HDACs) rescues H3K14ac levels—this conclusion lacks direct support. The individual siRNA knockdowns of HDAC1–9 do not replicate the TSA effect. Although TSA clearly affects H3K14 acetylation, the evidence does not directly demonstrate that H3K14ac is regulated by one or more HDACs. The authors should consider including speculative discussion on how multiple HDACs might act cooperatively in this context.\nAdditionally, after establishing KAT7 as the primary HAT affecting H3K14 acetylation, the continued use of TSA in downstream experiments (e.g., Figure 6F) complicates data interpretation. The data suggest a possible secondary but functionally relevant role for HDACs—or potentially off-target effects of TSA—in histone deacetylation under zinc-deficient conditions.\nSpecific Comments:\n1. Figure 3A: The siRNA screen targets several HATs, including KAT6A and KAT6B, which also contain zinc-binding domains and are part of the MYST family. However, only KAT7 knockdown significantly affects H3K14ac levels. This raises the question of whether all siRNA knockdowns were equally efficient. Data validating the knockdown efficiency for each siRNA used in Figure 3A would enhance interpretation.\n2. Figure 2: A similar concern applies to the HDAC siRNA screen. Validation of knockdown efficiency for each siRNA would be valuable.\n3. The manuscript refers to a HAT-dead EQ KAT7 mutant. The 'EQ' mutation should be clearly described in both the figure legend and the Methods section.\n4. Figure S3E: Two recombinant proteins are expressed in HEK293A cells, but the antibody used for immunoprecipitation is not indicated in the figure or legend. Proper annotation is necessary for clarity.\n5. Figure 4B: The figure legend is inadequate to explain the data presented. Please revise for clarity and completeness.\n6. Figure 6D: Is there an explanation for the reduced total H3 levels observed in zinc-deficient (ZD) samples? According to the Methods section, purified histones (final concentration: 0.5 µM) were added for the in vitro HAT assay, yet the H3 band intensity appears lower in ZD compared to normal diet (ND) conditions.\n7. The manuscript provides a rationale for zinc deficiency-induced lipid droplet accumulation in the liver, but additional discussion on how a high-fat diet induces zinc deficiency mechanistically would be helpful.\n8. Figure 4I: Please clarify and include the units for the Y-axis.", "reviewer_4": "Review of manuscript NCOMMS-25-16751 by Fujisawa et al. for Nature Communications\nThe manuscript combines four stories in an inaccurate way and makes conclusions, which cannot be derived from the experiments done. However, some parts of the manuscript are well done, but not the combination of the four parts HDAC-activity, KAT7-activity, Zip10-regulation, and fatty liver in zinc deficiency (ZD). The major problem of the manuscript is the first part of the manuscript, from which the authors conclude that HDAC activity is not regulated by zinc homeostasis, although they show that HDAC inhibitors show the same effect than ZD. From here they went into the story with a wrong assumption. For this conclusion, the authors must have analyze the HDAC activity in vitro in ZD. The second part about KAT7 activity is once more not correct since the activity is also not measured. The third part about Zip10 regulation is interesting and new. The last part has a weak connection, since parts one and two are flawed.\nCritique in detail:\n1. The statistic in figures 1D and 1E is wrong or wrongly described. If the authors compare ZD with ZN as described in the figure legend, then the significances must be at low concentrations (1D) or short times (1E) and disappear at high concentrations and longer times. Now it looks like a statistics in the concentration or time course.\n2. Figure 2: The authors did not show that HDACs disappear after siRNA treatment, like they do for KAT7 in figure S4. This is a must.\n3. Figure 2: From the “knock-down” (see point 2) the authors conclude that HDAC activity is not important. However, they don´t measure the activity itself with recombinant HDACs. It has been already shown that some HDACs are inhibited by zinc and show an increased activity in ZD. For this an HDAC activity assay must be done to follow the conclusion of the authors. Now the conclusion is not supported by the experiments. The effect in figures 6F/6G supports this assumption.\n4. Figure 3: The authors show, that KAT7 loses its activity after mutation of the cysteines/histidine of the zinc-finger-motif important for its structure. This is new, but nothing fancy, since the activity is gone after braking the structure. This has been shown for various proteins with zinc-finger-motifs. However, an in vitro HAT activity assay was done (3F), but not under different zinc conditions. In fact, this would be difficult with a His-tagged protein since histidine has a high zinc affinity. Therefore, the authors have to do KAT7 activity assay with a non His-tagged recombinant KAT7.\n5. At least for key experiments a control with zinc-saturated TPEN should be run.\nIn conclusion, the manuscript needs additional experimentation to support the assumptions and some additional controls.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point response to the reviewers’ comments\nWe would like to begin by thanking all the reviewers for providing insightful feedback\nand constructive suggestions. We have addressed all concerns from the reviewers by\nperforming the requested experiments. Furthermore, in the mouse experiments, we have\nnow specified the sex of the animals, and for key experiments we have included results\nfrom both male and female mice. With respect to the western blot (WB) data, because\nhistone WBs often produce similar patterns, we have prepared uncropped data as Source\nData to provide a more reliable and transparent presentation. In addition, the sections that\nhave been modified compared with the initial version are highlighted in red text. We hope\nthat the reviewers’ concerns are relieved in the revised manuscript.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors conducted their work investigating how zinc deficiency alters gene\nexpression at the molecular level using cell lines and animal models. In this study, they\nfocused on histone posttranslational modifications (PTMs), which are key regulators of\ntranscriptional control. Their findings demonstrated that zinc deficiency reversibly affects\nacetylation of histone H3 at lysine 14 (H3K14ac). Specifically, they showed that the\nHDAC family members contribute to the deacetylation of H3K14ac under zinc-deficient\nconditions. Additionally, they demonstrated that the histone acetyltransferase KAT7 loses\nits enzymatic activity during zinc deficiency. To identify genes potentially regulated by\nthe reduction in H3K14ac, the authors integrated transcriptomic data with H3K14ac\nChIP-seq data under conditions of zinc deficiency and KAT7 knockdown. This\nintegrative analysis highlighted ZIP10 as a zinc transporter gene whose expression is upregulated\nduring zinc deficiency. Functional experiments revealed that ZIP10 knockdown inhibited\ncell cycle re-entry, underscoring its potential role in cellular responses to zinc deficiency.\nThe authors extended their investigation to in vivo models and observed increased ZIP10\nexpression in the livers of mice fed a zinc-deficient diet—paralleling the in vitro results\nin HeLa cells. These findings suggest that ZIP10-mediated zinc uptake might contribute\nto maintaining zinc homeostasis also in vivo. Furthermore, the authors propose a model\nin which ZIP10-dependent zinc import is necessary for KAT7 activity, thereby linking\nzinc status to epigenetic gene regulation.\nResponse:\nWe sincerely thank the reviewer for the generous and thoughtful comments on our work.\nThis study is well-executed and provides compelling mechanistic insights based on in\nvitro data. However, there appears to be a significant disconnect between the in vitro\nfindings and the in vivo validation. While data from Figures 1 through 4 align with their\nproposed model, Figures 5 and 6 do not convincingly demonstrate a functional link with\ndata shown in Figures 1 through 4, in terms of the mechanisms of zinc-mediated\nepigenetic regulation in vivo. If ZIP10 is indeed essential for epigenetic gene regulation\nduring zinc deficiency or high-fat diet t (HFD) model, it would be critical to assess the\nconsequences of ZIP10 loss specifically in the liver. Thus, generating and analyzing liver-\nspecific conditional ZIP10 knockout mice is necessary to validate the physiological\nrelevance of the proposed mechanism in vivo.\nResponse:\nWe thank the reviewer for this valuable comment. We acknowledge that we did not\nsufficiently consider the in vivo role of ZIP10. The primary aim of our study was to\nelucidate the mechanisms underlying gene expression changes induced during zinc\ndeficiency, and therefore we mainly focused on the significance of ZIP10 upregulation in\nHeLa cells and on lipid metabolism–related genes in the mouse liver. Nevertheless, since\nwe also found that dietary zinc deficiency led to increased ZIP10 mRNA expression in\nthe mouse liver (Fig. S5A), we should have discussed the significance of this observation\nin more detail, which was missing from our initial discussion.\nAs the reviewer pointed out, hepatic lipid accumulation could potentially be\ninfluenced by ZIP10 depletion. Motivated by this important suggestion, we therefore\nperformed additional experiments to examine whether ZIP10 depletion in the liver affects\nlipid accumulation under zinc-deficient conditions. Because whole-body ZIP10 knockout\nis embryonic lethal (He et al., Adv. Sci., 2023 PMID:37068188), we employed an\nalternative approach by introducing shRNA against ZIP10 into the liver by employing the\nadeno-associated virus (AAV)–mediated delivery. Given that ZIP10 expression is\nupregulated under zinc deficiency, we reasoned that it may play a functional role\nspecifically in this setting and therefore assessed its impact on lipid accumulation during\ndietary zinc deficiency.\nIn this experiment, AAV-shRNA was administered via tail vein injection, and\n96 hours later, the mice were placed on a zinc-deficient diet. After one week of feeding,\nthe animals were sacrificed, and liver samples were collected, PFA-fixed, sucrose-\nsubstituted, and stained with BODIPY to visualize lipid droplets. Under these conditions,\nhowever, we did not observe significant differences in lipid accumulation between control\nand ZIP10 knockdown livers (Figure for reviewers 1A,B).\nAlthough these results might appear to suggest that hepatic ZIP10 is not directly\ninvolved in linking zinc homeostasis to lipid accumulation, we do not interpret the data\nin this way. Rather, we believe that this outcome reflects limitations inherent to our\nexperimental system. In particular, under the severe zinc-deficient diet used in this study,\nserum zinc levels were markedly reduced (Figure for reviewers 1C). Consequently, the\ndegree of ZIP10 upregulation and the corresponding increase in zinc uptake were likely\ninsufficient to generate measurable differences in lipid accumulation, thereby masking\nthe potential contribution of ZIP10.\nAlthough the results were not as expected, this experiment has deepened our\nunderstanding of the in vivo role of ZIP10. We are sincerely grateful for the reviewer’s\ninsightful comments.\nFigure for reviewers 1\nA. Lipid staining analysis of liver samples from mice treated with shRNA for 96 hours,\nfollowed by a zinc-deficient diet for one week. Scale bar, 50 µm\nB. Quantitative PCR analysis of liver samples from female mice treated with the indicated\nshRNAs for 96 hours followed by a zinc-deficient diet for two weeks. The data are\npresented as means ± SEM (shCtrl, n=4; shZIP10, n=5). *P < 0.05 by Student’s t-test.\nC. ICP‒MS analysis of serum samples from mice fed the indicated diets for two weeks.\nThe data are presented as means ± SEM (n=6). *P < 0.05 by Student’s t test.\nIn fact, the authors present a well-structured series of in vitro experiments through Figure\n4, which effectively support their proposed model. However, the interpretation of the in\nvivo data related to zinc deficiency with or without high-fat diet -induced epigenetic\nregulation via ZIP10 raises various concerns, as these complex pathophysiological\nconditions involve multiple confounding factors. Furthermore, the discussion gives only\nlimited attention to the physiological role of ZIP10, which weakens the overall impact of\nthe conclusions. As a result, a conceptual disconnect emerges between the in vitro\nfindings and the in vivo observations, leading to an overall impression of inconsistency\nin the study’s central claims. Taken together, the current evidence appears insufficient to\nfully substantiate the authors’ conclusions, and the mechanistic link remains\nunconvincing at this stage.\nIn addition, given that another zinc importer ZIP14 is predominantly expressed in the\nliver, it is surprising that the authors neither addressed nor investigated the potential link\nbetween ZIP14 and zinc-mediated epigenetic gene regulation in hepatic tissue, especially\nin vivo model. This also represents a significant gap in the current study. Exploring this\nconnection would substantially strengthen the biological relevance and impact of the\nwork.\nResponse:\nWe thank the reviewer for highlighting the important point regarding ZIP14, which is\nindeed predominantly expressed in the liver. In response, we re-analyzed both our RNA-\nseq and ChIP-seq datasets to specifically examine the role of ZIP14 under zinc-deficient\nconditions. This analysis revealed that ZIP14 expression was upregulated during zinc\ndeficiency. Moreover, we observed distinct H3K14ac signals at the enhancer region of\nthe ZIP14 locus, which were markedly reduced under zinc-deficient conditions (Fig. S6F,\nG). These findings suggest that, similar to ZIP10, ZIP14 expression in the liver might be\nregulated through changes in H3K14ac. Importantly, this additional analysis of ZIP14,\nprompted by the reviewer’s suggestion, has broadened our perspective on the mechanisms\nby which zinc homeostasis is regulated in the liver through H3K14ac modulation. We are\nsincerely grateful for this valuable comment, which has allowed us to further strengthen\nour discussion.\nSpecific comment:\nIn Fig.3C:\nThere appear to be two identical descriptions for the conditions labeled as ZN medium\ncultured for 48 hours. The reviewer would appreciate clarification on whether there is a\ndistinction between these two conditions, or if this is a duplication error.\nResponse:\nWe sincerely thank the reviewer for carefully pointing out the issue. In the initial version,\nthe figure contained two lanes labeled as “ZN,” which may have appeared redundant.\nHowever, these lanes were intended to represent two distinct conditions: one in which\nFLAG-KAT7 was absent and one in which FLAG-KAT7 was present. We acknowledge\nthat this difference was not clearly represented in the original figure, which may have\nobscured the intended distinction. To address this, we have revised both the figure\nlabeling and the legend to explicitly indicate the presence or absence of FLAG-KAT7.\nWe greatly appreciate the reviewer’s valuable feedback, which has allowed us to improve\nthe clarity and accuracy of our data presentation.\nIn Fig.4H:\nThe Zip10 siRNA treatment resulted in a reduction of zinc content in HeLa cells. Given\nthis observation, the reviewer is interested to know whether H3K14ac levels were also\naffected under the same conditions. Furthermore, since zinc pyrithione—a known zinc\nionophore—can enhance intracellular zinc levels, the reviewer suggests evaluating\nwhether zinc pyrithione treatment could rescue H3K14ac status or restore KAT7 activity\nin the context of Zip10 knockdown. This would provide valuable insight into the\nfunctional consequences of zinc depletion on histone acetylation and its potential\nreversibility.\nResponse:\nWe are grateful to the reviewer for this thoughtful suggestion. Indeed, as shown in the\nrevised Fig. 4I (corresponding to Fig. 4H in the initial version), the zinc content under\nz under\nz under\nz under\nz under\nzinc-deficient conditions was further decreased when ZIP10 was knocked down,\ncompared to siCtrl. This observation suggested that H3K14ac might be more strongly\naffected under siZIP10 conditions. To test this possibility, we carried out western blot\nanalysis (Fig 4J). The results showed that H3K14ac levels remained largely unchanged\nbetween siCtrl and siZIP10 conditions. We therefore conclude that the reduction in zinc\ncontent caused by zinc deficiency under siCtrl is already sufficient to decrease H3K14ac,\nand that the further reduction caused by ZIP10 knockdown does not alter H3K14ac levels\nany further. Moreover, when zinc was replenished after deficiency, H3K14ac levels\nrecovered in the siCtrl condition but not under ZIP10 knockdown (Fig 4J). These results\nsuggest that ZIP10 contributes to the reversibility of H3K14ac levels through zinc uptake.\nIn addition, we sincerely thank the reviewer for the valuable suggestion\nregarding the use of zinc pyrithione. Following this advice, we examined whether zinc\npyrithione could restore H3K14ac status. However, when zinc pyrithione was added after\n48 hours of zinc depletion, HeLa cells became completely rounded and exhibited marked\ntoxicity (Figure for reviewers 2). We consider that the prolonged zinc deprivation\nrendered the cells highly vulnerable, thereby sensitizing them to the toxic effects of zinc\npyrithione. Unfortunately, under our experimental conditions, it was not feasible to utilize\nzinc pyrithione effectively. Nevertheless, we greatly appreciate this insightful comment,\nwhich provided us with an important opportunity to further consider the underlying\nmechanisms.\nFigure for reviewers 2\nImages of HeLa cells subjected to the indicated stimulation with 10 µM ZnCl₂ after being\ncultured in zinc-deficient medium for 48 hours. Scale bar, 100 µm\nIn Fig.5B:\nThe authors report a decrease in hepatic zinc content during HFD treatment, accompanied\nby reduced Mt2 mRNA expression. However, the underlying mechanism driving the\nreduction in liver zinc content remains unclear. The manuscript presents only minor\nchanges in Zip10 expression in Figure S5A, without addressing the potential involvement\nof other zinc transporters or regulatory mediators. The reviewer suggests that additional\nanalysis regarding other zinc transport pathways would strengthen the interpretation of\nthese findings.\nResponse:\nWe greatly appreciate the reviewer’s insightful comment regarding the mechanisms\nunderlying the reduction in hepatic zinc content. In response, we examined the expression\nof several zinc transporters under HFD conditions and did not observe significant changes\n(Fig. S5B). Nevertheless, we agree that the potential involvement of other zinc transport\npathways, as well as additional regulatory mechanisms beyond zinc transporters, is an\nimportant point to consider. We have therefore added the following discussion to the\nrevised manuscript;\n“We demonstrated that hepatic zinc levels are reduced in mice fed a HFD. The molecular\nmechanisms behind the reduction remain to be fully elucidated. One hypothesis that can\nbe postulated is that HFD has the potential to compromise zinc absorption in the\nduodenum. Zinc is primarily absorbed from the intestinal lumen into enterocytes via the\nzinc importer ZIP4(PMID: 22737083). It is subsequently exported to the circulation\nthrough ZNT1 for distribution to peripheral tissues (PMID: 19325039). It is hypothesised\nthat the modulation of the transcription or membrane trafficking of ZIP4 or ZNT1 by\nHFD could result in a reduction of zinc absorption and release into the bloodstream,\nwhich would lead to a decrease in hepatic zinc level. An alternative hypothesis is that the\nenhanced secretion of zinc-binding proteins, including specific apolipoproteins, induced\nby HFD contributes to the reduction in hepatic zinc content. The secretion of protein by\nhepatocytes is known to be subject to regulation by hormonal and peptide signals\noriginating from the pancreas and adipose tissue (PMID: 33743013). Such signals might\nbe augmented under conditions of an HFD. Taken together, these observations suggest\nthat a combination of altered zinc absorption, redistribution, and excretion pathways may\nunderlie the reduction in hepatic zinc content induced by an HFD. Further investigation\nof these possibilities will advance a more comprehensive understanding of the\nmechanisms linking dietary fat intake to systemic zinc homeostasis.”\nFigure S5A:\nThe authors report a statistically significant increase in Zip10 mRNA levels under zinc-\ndeficient conditions. However, the corresponding protein levels were not provided. To\nstrengthen the conclusion, it is important to include data on ZIP10 protein expression\nunder the same conditions. Additionally, as noted above, assessing the expression of a\nbroader panel of zinc transporters and other zinc-related mediators would help determine\nwhether ZIP10 is uniquely or predominantly affected in response to zinc deficiency.\nResponse:\nWe thank the reviewer for drawing our attention to this significant issue. To address this,\nwe attempted to examine ZIP10 protein levels by immunoblotting using two different\ncommercial antibodies. However, both antibodies detected multiple bands (Figure for\nreviewers 3). Based on the expected molecular weight, we considered the band around\n100 kDa to correspond to ZIP10, while the additional bands likely represented non-\nspecific signals. Under zinc-deficient dietary conditions, this band appeared to show a\nmodest increase, consistent with the mRNA data. Nevertheless, because we could not\ndefinitively confirm that this band represented ZIP10, we decided not to include these\ndata in the manuscript. In addition, we also attempted to examine ZIP14 protein levels.\nHowever, we could not detect a clear band at the expected molecular weight (~55 kDa),\nand therefore were unable to reliably assess protein expression levels for ZIP14.\nFigure for reviewers 3\nImmunoblot analysis of liver samples from female mice fed the indicated diets for two\nweeks.\nDiscussion:\nThe Discussion section would benefit from a more comprehensive analysis of the role of\nZIP10, particularly regarding its physiological relevance in the context of epigenetic\nregulation. Currently, the discussion centers largely on zinc-dependent epigenetic\nchanges observed in vitro. To provide deeper mechanistic insight, it is important to\naddress how zinc may influence epigenetic modifications in vivo, and to discuss the\npotential involvement of ZIP10 within this broader physiological context.\nResponse:\nWe thank the reviewer for this constructive suggestion. We acknowledge that, due to the\nlimitations of our current experimental system, we were not able to directly demonstrate\nthe in vivo significance of ZIP10 upregulation. Nevertheless, our data indicate that both\nZIP10 and ZIP14 are upregulated under zinc-deficient conditions in the liver. This\nsuggests that these zinc transporters may function cooperatively to maintain hepatic zinc\nhomeostasis during zinc deficiency. Given the essential role of zinc as a cofactor for key\nhepatic enzymes, including those involved in alcohol metabolism and ammonia\ndetoxification, the coordinated regulation of ZIP10 and ZIP14 is likely to contribute to\nthe maintenance of liver function under zinc-deficient conditions. In line with the\nreviewer’s comment, we have added the discussion part as below;\n“We found that reduced H3K14ac in HeLa cells drives the upregulation of the zinc\ntransporter ZIP10, thereby contributing to the maintenance of intracellular zinc\nhomeostasis. Furthermore, in the mouse liver, zinc deficiency stress induced the\nexpression of both ZIP10 and ZIP14 (Fig. S5A, Fig. S6F,G). Given that zinc serves as an\nessential cofactor for liver enzymes such as alcohol dehydrogenase and those involved in\nammonia metabolism (PMID: 19011745,PMID: 1505922), its adequate regulation is\nindispensable for hepatic function. Thus, the upregulation of ZIP10 and ZIP14 expression\nin response to decreased H3K14 acetylation under zinc-deficient conditions is likely to\nfunction cooperatively to safeguard hepatic zinc homeostasis and thereby maintain liver\nfunction.”\nReviewer #1 (Remarks on code availability):\nThe code used in this study is publicly available at the indicated link. It includes detailed\ndocumentation and instructions for reproducing the analyses.\nResponse:\nWe thank the reviewer for this kind and supportive comment. We have also uploaded the\nsource code for the newly added data in the revised manuscript to our GitHub repository.\nReviewer #2 (Remarks to the Author):\nFujisawa et al present a clear, elegantly designed and thorough analysis, assessing the role\nof H3K14ac in zinc deficiency. The authors show that zinc deficiency impairs the histone\nacetyltransferase function of KAT7, resulting in gene expression changes in zinc\ntransporters. This novel work gives clear mechanistic insight into the pathological effects\nof zinc deficiency and nicely links this finding to intracellular lipid droplet formation in\na mouse model, demonstrating its relevance to health and liver disease. This provides a\nnovel understanding in the field and clearly links nutrient conditions to changes in\nepigenetic histone marks. The data is clearly presented, thoroughly investigated and\nconvincing and has been performed to a high standard. There is also sufficient detail in\nthe methods to allow this work to be reproduced. There are a couple of points, however,\nthat I feel are missing and the authors should consider adding:\nResponse:\nWe sincerely thank the reviewer for the generous and thoughtful comments on our work.\nMajor Points:\n1. H3K14ac and Gene Expression Inhibition:\no Generally, H3K14ac and histone acetylation are associated with increased gene\nexpression. The role for H3K14ac in marking inactive enhancers seems to be limited to\ncancer cells and not otherwise healthy cells. However, the model in Fig. 4J suggests that\nH3K14ac at the promoter inhibits expression of ZIP10, counter to the current dogma. The\nauthors should expand the discussion to explain why they believe H3K14ac exhibits an\ninhibitory effect in their study and consider recreating this model to better reflect that this\nis an enhancer not a promoter effect of H3K14ac.\no Additionally, correlating the RNA-seq (GSE49647) changes in gene expression between ZN and ZD conditions and\nchanges in H3K14ac ChIP-seq between ZN and ZD conditions, could help demonstrate whether H3K14ac in this case inhibits gene\nexpression. Is this inhibitory effect genome-wide in ZD conditions, or is it specific to ZIP genes such as ZIP10?\nCorrelations, such as log2 fold changes in gene expression (ZN vs. ZD) with H3K14ac abundance at enhancers or promoters between ZN vs ZD, could reveal specific sets of genes impacted by H3K14ac loss. If ZIP10 is one of only a few genes showing this effect, its uniqueness should be highlighted.\nResponse:\nWe sincerely thank the reviewer for this important and insightful comment. In our study,\nwe have primarily focused on genes such as ZIP10, where zinc deficiency led to\ndecreased H3K14ac at enhancer regions accompanied by increased gene expression.\nHowever, we had not extended this analysis to other genes showing transcriptional or\nepigenomic changes under zinc deficiency. Following the reviewer’s valuable suggestion,\nwe conducted a genome-wide correlation analysis between changes in gene expression\nunder zinc deficiency (GSE49657) and zinc-dependent alterations of H3K14ac at\nenhancer or promoter regions. The promoter-specific results are addressed separately in\nour response to “2. Promoter-Specific Retention of H3K14ac.” Here, we summarize our\nfindings at enhancer regions.\nAs shown in the newly added Fig. 4B, we categorized zinc-responsive genes\ninto upregulated and downregulated sets and examined whether the extent of enhancer\nH3K14ac reduction correlated with the magnitude of transcriptional changes. To focus on\nenhancer-specific effects, we restricted our analysis to 3,048 genes in which H3K14ac\nwas markedly reduced at enhancers but minimally changed at promoters. This analysis\nrevealed that stronger enhancer H3K14ac loss was associated with larger transcriptional\nchanges (Fig. 4B). This suggests that zinc deficiency–induced reduction of H3K14ac at\nenhancers plays a significant role in regulating genome-wide gene expression dynamics.\nWe are deeply grateful for this constructive feedback, which allowed us to strengthen our\ninterpretation and highlight the genome-wide relevance of enhancer-associated H3K14ac\nloss.\n2. Promoter-Specific Retention of H3K14ac:\no The western blots presented in this study show a complete loss of H3K14ac in zinc deficiency and KAT7 knockdown conditions, consistent with ChIP-seq findings.\nHowever, Fig. 4F reveals that this loss does not extend to promoters. Since promoters constitute only a small portion of the genome, the sensitivity of western blots may be insufficient to detect the retention of H3K14ac at promoters, however, the authors should discuss why promoter regions appear to be spared from loss of H3K14ac in zinc deficiency, as KAT7 is solely responsible for H3K14ac.\nResponse:\nWe sincerely thank the reviewer for this important and insightful comment. Given the\naccumulated evidence from previous studies on H3K14ac and KAT7, we recognize that\nwe should have conducted a deeper analysis of the relationship between zinc deficiency–\ninduced changes in H3K14ac at promoter regions and the associated transcriptional\nchanges.\nAs noted above, we have identified 3,048 genes, such as ZIP10, in which\nH3K14ac was reduced specifically at enhancers. To expand on this, as shown in the newly\nadded Fig. S4C, we further examined genes showing H3K14ac reduction at promoters or\nat both promoters and enhancers, integrating our ChIP-seq data with the publicly\navailable transcriptomic dataset (GSE49657). This analysis revealed that 2,854 genes\ndisplayed zinc deficiency–dependent reduction of H3K14ac exclusively at promoters,\nand 4,752 genes showed reduction at both promoters and enhancers (Fig. S4C). Next, we\ndivided genes into those upregulated or downregulated under zinc deficiency and\nassessed the correlation between transcriptional changes and the degree of H3K14ac\nreduction. We found that genes with greater H3K14ac reduction at promoters, or at both\npromoters and enhancers, tended to show stronger transcriptional downregulation (Fig.\nS4C, bottom). Conversely, for genes upregulated under zinc deficiency, no such\ncorrelation was observed (Fig. S4C, upper). These results suggest an important role for\npromoter-associated H3K14ac reduction in mediating zinc deficiency–dependent\ntranscriptional repression. Our comprehensive analysis also highlighted the existence of\ngenes controlled primarily through enhancer-specific H3K14ac, as well as those regulated\nby promoter-associated H3K14ac. We are grateful to the reviewer for this valuable\nsuggestion, which enabled us to gain deeper mechanistic insights into the regulatory\nlandscape of H3K14ac under zinc deficiency.\no Heatmaps for H3K14ac across promoters should be included in the main or supplemental figures to reinforce the claim that zinc deficiency spares loss of H3K14ac at promoters in ZD conditions. Presumably siRNA knockdown of Kat7 causes a complete loss of H3K14ac both at enhancers and promoters.\nResponse:\nWe sincerely thank the reviewer for this important suggestion. As noted above, our re-\nanalysis of the ChIP-seq data revealed that zinc deficiency also induces a decrease in\nH3K14ac at promoter regions. In order to provide a more comprehensive visualization of\nthese changes, we have added heatmaps across promoters in the supplemental figures\n(Fig. S4A). To further investigate the possibility that zinc deficiency and KAT7\nknockdown regulate a common set of genes, we examined the degree to which H3K14ac\npeaks at enhancer and promoter regions were commonly reduced under these two\nconditions. As shown in Fig. S4B, a considerable number of peaks exhibited overlapping\nreductions. This observation reinforces the hypothesis that zinc deficiency compromises\nKAT7 activity and thereby contributes to the observed decrease in H3K14ac. We\nappreciate the reviewer’s thoughtful recommendation.\nMinor Points:\n1. The authors may consider assessing whether KAT7 knockdown under normal zinc nutrient conditions affects ZIP10 expression, possibly through RT-qPCR. While the link between zinc deficiency and KAT7-mediated acetylation loss is compelling, it remains unclear if KAT7 knockdown alone recapitulates the effects of zinc deficiency. Investigating this could uncover whether zinc deficiency triggers additional cellular responses leading to increased ZIP10 expression that loss of KAT7 alone cannot explain.\nResponse:\nWe thank the reviewer for this important suggestion. Compared to the marked\nupregulation of ZIP10 expression under zinc deficiency (Fig. 4E), the increase observed\nupon KAT7 suppression was relatively modest (S4I). This suggests that mechanisms other\nthan the reduction of H3K14ac contribute to the induction of ZIP10 under zinc-deficient\nconditions. Interestingly, when TPEN was applied to KAT7 knockout HeLa cells, we\nobserved a further increase in ZIP10 expression (Figure for reviewers 4). This result\nsupports the reviewer’s view that other regulatory mechanisms, in addition to KAT7, are\ninvolved in the induction of ZIP10 under zinc deficiency. Indeed, transcription factors\nsuch as STAT3/5 have been reported to promote ZIP10 expression (Miyai et al., PNAS,\n2013). However, zinc deficiency has been associated with conflicting reports regarding\nSTAT3 activity regulation (Zhao et al., Free Radic. Biol. Med., 2024; Supasao et al.,\nRedox Biol., 2017), and it remains unclear whether this pathway contributes to ZIP10\nupregulation in the present context. Further investigation into the involvement of\nSTAT3/5 and other transcription factors will therefore be required.\nFigure for reviewers 4\nQuantitative PCR analysis of HeLa cells or KAT7 KO HeLa cells treated with or without\n10 µM TPEN for 4hr.\n2. On page 10, the authors state that \"TSA treatment suppressed zinc uptake, suggesting a critical role for H3K14ac in zinc influx.\" As TSA inhibits class I and II HDACs, this claim may not be entirely accurate since TSA elevates all histone acetylation marks. This statement should be revised or removed.\nResponse:\nWe thank the reviewer for this valuable comment. We apologize for the overstatement in\nour original text. We fully agree that TSA elevates histone acetylation marks beyond\nH3K14ac. In light of this, we have revised the sentence as follows:\n“TSA treatment suppressed zinc uptake, suggesting that global elevation of histone\nacetylation, potentially including H3K14ac, may influence zinc influx.”\nWe are grateful for the reviewer’s valuable suggestion, which has improved the accuracy\nof our description.\n3. In Fig. S3E, TPEN treatment after 2 hours appears to increase the association of KAT7 with BRPF3. Do the authors have any thoughts as to why this may be the case? If so, this should be added to the discussion or results section.\nResponse:\nWe greatly appreciate the reviewer’s thoughtful observation. Indeed, we observed that\nTPEN treatment clearly enhances the association of KAT7 with BRPF3. The precise\nbiological meaning of this enhanced association is not yet understood. However, one\npossible explanation is that zinc deficiency triggers a reorganization of KAT7-containing\ncomplexes, with increased incorporation of BRPF3, potentially altering KAT7’s\nregulatory functions. Although these mechanistic implications are beyond the scope of\nthe current manuscript, we are keen to pursue them in our future studies.\n4. Fig. 4I is missing a Y-axis label. I assumed this graph is \" %EDU+ cells \".\nResponse:\nWe sincerely thank the reviewer for pointing out this important issue. We have now\ncorrected this error by adding the label “EDU positive cells [%]” to the Y-axis.\n5. Uncropped western blot images should be provided in the supplemental data.\nResponse:\nWe thank the reviewer for the valuable suggestion. In response, we have now provided\nuncropped western blot images for all experiments as Source Data, compiled into an\nExcel file in the supplemental materials.\n6. The degree of KAT7 knockdown achieved by siRNA should be shown through either western blotting or RT-qPCR.\nResponse:\nWe sincerely thank the reviewer for this important comment. In the initial manuscript, the\nefficiency of KAT7 knockdown was not presented. In the revised manucript, we have\nnow demonstrated the knockdown efficiency by RT-qPCR (Fig. S3A). In addition, to\nconfirm the validity of our siRNA screening, we have also provided the knockdown\nefficiencies for other HATs in the revised manuscript. We appreciate the reviewer’s\nvaluable suggestion, which has helped us improve the clarity of our data presentation.\nReviewer #3 (Remarks to the Author):\nComments for the authors:\nIn this manuscript, the authors demonstrate that zinc is essential for the histone\nacetyltransferase (HAT) activity of KAT7, leading to genome-wide epigenetic changes,\nparticularly at enhancer regions. They employ a variety of experimental approaches to\ndetermine whether the observed reduction in H3K14 acetylation results from enhanced\nhistone deacetylase (HDAC) activity or reduced HAT activity. Their data indicate that\nzinc deficiency does not affect HDAC activity in vitro, but it does impair KAT7-mediated\nH3K14 acetylation in both in cellulo and in vitro HAT activity assays. The authors\nidentify two zinc finger domains in KAT7, with the zinc finger within the MYST domain\nresponsible for zinc coordination. Mutations in this domain reduce enzymatic activity.\nFurthermore, the authors show that reduced KAT7 activity leads to epigenetic changes\nthat elevate Zip10 expression and alter lipid homeostasis in the mouse liver. These\nfindings are original and potentially significant for the fields of zinc/metal homeostasis\nand metabolic disorders.\nResponse:\nWe express our sincere gratitude to the reviewer for the generous and thoughtful\nevaluation of our work.\nHowever, several concerns should be addressed to strengthen the manuscript:\nWhile the authors suggest that multiple HDAC family members may cooperatively\ncontribute to H3K14 deacetylation during zinc deficiency—based on the observation that\nTSA (a pan-inhibitor of classical HDACs) rescues H3K14ac levels—this conclusion\nlacks direct support. The individual siRNA knockdowns of HDAC1–9 do not replicate\nthe TSA effect. Although TSA clearly affects H3K14 acetylation, the evidence does not\ndirectly demonstrate that H3K14ac is regulated by one or more HDACs. The authors\nshould consider including speculative discussion on how multiple HDACs might act\ncooperatively in this context.\nResponse:\nWe sincerely thank the reviewer for this important comment. We agree that our initial\nmanuscript did not provide direct evidence that classical HDACs deacetylate H3K14ac.\nTo address this, we performed additional experiments in which classical HDACs were\nexpressed in HEK293A cells, immunoprecipitated, and examined for their deacetylation\nactivity against recombinant histones. As shown in the revised manuscript (Fig. 2E),\nwe found that at least HDAC1 and HDAC6 exhibited deacetylase activity toward H3K14ac.\nHowever, for HDAC2, HDAC3, and HDAC11, we were unable to obtain sufficient\namounts of protein despite immunoprecipitation from 1.0×10^8 cells, which yielded\nlower protein levels than other HDACs immunoprecipitated from only 1.0×10^6 cells.\nTherefore, we speculate that these HDACs might also contribute to H3K14 deacetylation.\nSupporting this idea, the double knockdown of HDAC1 and HDAC6 did not suppress the\nTPEN-induced reduction of H3K14ac (Fig. S2D), suggesting that additional mechanisms\nbeyond HDAC1 and HDAC6 are involved. While the present study primarily focuses on\nHAT function, and further extensive HDAC analyses are beyond the current scope, we\nhave added the following discussion on potential cooperative roles of multiple HDACs\nto the revised Discussion section:\n“In this study, employing in vitro HDAC assays, we found that HDAC1 and HDAC6 are\ncapable of deacetylating H3K14ac (Fig. 2E). However, for HDAC2, HDAC3, and\nHDAC11, we were unable to obtain sufficient amounts of protein, as their\nimmunoprecipitation yielded markedly lower levels compared with other HDACs.\nTherefore, we speculate that these HDACs might also contribute to H3K14 deacetylation.\nSupporting this idea, the double knockdown of HDAC1 and HDAC6 did not suppress the\nTPEN-induced reduction of H3K14ac (Fig. S2D), suggesting that additional mechanisms\nbeyond HDAC1 and HDAC6 are involved. Interestingly, although HDACs are known to\nbe zinc-dependent enzymes, there are also reports indicating that zinc deficiency can\nenhance HDAC activity. Future studies investigating the activity of individual HDACs\nunder zinc-deficient conditions will be crucial to elucidate the mechanisms underlying\nthe reduction of H3K14ac during zinc deficiency.”\nAdditionally, after establishing KAT7 as the primary HAT affecting H3K14 acetylation,\nthe continued use of TSA in downstream experiments (e.g., Figure 6F) complicates data\ninterpretation. The data suggest a possible secondary but functionally relevant role for\nHDACs—or potentially off-target effects of TSA—in histone deacetylation under zinc-\ndeficient conditions.\nResponse:\nWe greatly appreciate the reviewer’s insightful comment on the use of TSA in\ndownstream experiments. At present, TSA remains the only means by which we can\nsuppress the reduction of H3K14ac in this context. We fully acknowledge that, as a pan-\nHDAC inhibitor, TSA may also affect acetylation at sites beyond H3K14, and this\nlimitation must be considered. As also noted in our response to Reviewer 2’s Minor point\n2, we have accordingly revised the text to avoid any overstatement.\nSpecific Comments:\n1. Figure 3A: The siRNA screen targets several HATs, including KAT6A and KAT6B,\nwhich also contain zinc-binding domains and are part of the MYST family. However,\nonly KAT7 knockdown significantly affects H3K14ac levels. This raises the question of\nwhether all siRNA knockdowns were equally efficient. Data validating the knockdown\nefficiency for each siRNA used in Figure 3A would enhance interpretation.\nResponse:\nWe sincerely thank the reviewer for this important comment. In response, we have now\nprovided the knockdown efficiency of each HAT included in the siRNA screen (Fig. S3A).\nAll HATs were sufficiently knocked down for the purpose of the screening experiment,\nand thus we believe the screening system was valid. As the reviewer noted, knockdown\nof KAT6B, another member of the MYST family, appeared to cause a modest reduction\nin H3K14ac. However, this reduction was considerably weaker than that observed upon\nKAT7 knockdown. Therefore, we conclude that in our experimental system, KAT7\nrepresents the major acetyltransferase responsible for H3K14 acetylation. We appreciate\nthe reviewer’s constructive suggestion, which has helped us improve the clarity of our\ndata presentation.\n2. Figure 2: A similar concern applies to the HDAC siRNA screen. Validation of\nknockdown efficiency for each siRNA would be valuable.\nResponse:\nWe fully agree with the suggestion that validation of knockdown efficiency is essential.\nAccordingly, we have now included the knockdown efficiency of each HDAC siRNA\nused in the screening (Fig. S2A). We believe that all HDACs were sufficiently knocked\ndown for the purpose of this screening assay. We greatly appreciate the reviewer’s\nconstructive feedback, which has helped us to improve the clarity and reliability of our\ndata presentation.\n3. The manuscript refers to a HAT-dead EQ KAT7 mutant. The 'EQ' mutation should be\nclearly described in both the figure legend and the Methods section.\nResponse:\nWe would like to thank the reviewer for this valuable suggestion. We have now included\na precise description of the HAT-dead EQ KAT7 mutant in both the figure legend in Fig.\n3B and the Methods section, as indicated below.\nIn figure legend:\n“KAT7 EQ mutant is reported to be catalytically inactive.”\nIn Methods section:\n“The catalytically inactive EQ mutant and zinc finger domain mutants were constructed\nusing PCR-mediated site-directed mutagenesis.”\n4. Figure S3E: Two recombinant proteins are expressed in HEK293A cells, but the\nantibody used for immunoprecipitation is not indicated in the figure or legend. Proper\nannotation is necessary for clarity.\nResponse:\nWe thank the reviewer for pointing out the lack of annotation in Fig. S3E. In the revised\nfigure, we have clearly indicated “IP: FLAG” to specify the antibody used for\nimmunoprecipitation, and we have also updated the figure legend accordingly.\n5. Figure 4B: The figure legend is inadequate to explain the data presented. Please revise\nfor clarity and completeness.\nResponse:\nWe apologize that the original figure legend of Figure 4B was inadequate to fully explain\nthe data presented. In the revised manuscript, we have rewritten the legend to ensure\nclarity and completeness, as follows:\n(Initial manuscript)\nHeatmaps showing gene expression changes during zinc deficiency and the H3K14ac\nsignal in enhancer regions. Genes in red are possible candidates regulated by the decrease\nin H3K14ac during zinc deficiency and upon KAT7 knockdown, as defined by the\nfollowing criteria: a fold change in expression > 1.5 (upregulation or downregulation,\ndata from GSE49657). ND, not determined (no enhancer information on FANTOM5).\n(Revised manuscript)\nHeatmaps showing gene expression changes during zinc deficiency and the H3K14ac\nsignal in enhancer regions. The left heatmap depicts log2 fold changes in mRNA\nexpression following TPEN treatment or FBS starvation (FBS(–)), both of which\nrepresent zinc-deficient stimuli, highlighting upregulated (red) and downregulated (blue)\ngenes. Gene names shown in red characters represent possible candidates regulated by\nthe decrease in H3K14ac during zinc deficiency and upon KAT7 knockdown, as defined\nby the following criteria: a fold change in expression > 1.5 (upregulation or\ndownregulation, data from GSE49657). The right heatmaps show H3K14ac ChIP-seq\nsignals at enhancer regions under different conditions (ZN: zinc-normal condition; ZD:\nzinc-deficient condition; siCtrl: control knockdown; siKAT7: KAT7 knockdown). ND,\nnot determined (no enhancer information available from FANTOM5).\n6. Figure 6D: Is there an explanation for the reduced total H3 levels observed in zinc-\ndeficient (ZD) samples? According to the Methods section, purified histones (final\nconcentration: 0.5 µM) were added for the in vitro HAT assay, yet the H3 band intensity\nappears lower in ZD compared to normal diet (ND) conditions.\nResponse:\nWe thank the reviewer for this careful observation. The reduced total H3 levels in the\noriginal Figure 6D were due to a technical issue in immunoblotting, which gave the\nappearance of unequal H3 loading. We have repeated the experiment and replaced the\npanel with a corrected image, in which total H3 levels are consistent across conditions.\nImportantly, the reduction of H3K14ac under ZD conditions remains evident in the\nrevised figure.\n7. The manuscript provides a rationale for zinc deficiency-induced lipid droplet\naccumulation in the liver, but additional discussion on how a high-fat diet induces zinc\ndeficiency mechanistically would be helpful.\nResponse:\nWe greatly appreciate the reviewer’s insightful comment regarding the mechanisms\nunderlying the reduction in hepatic zinc content. In response, we examined the expression\nof several zinc transporters under HFD conditions and did not observe significant changes\n(Fig. S5B). Nevertheless, we agree that the potential involvement of other zinc transport\npathways, as well as additional regulatory mechanisms beyond zinc transporters, is an\nimportant point to consider. We have therefore expanded the Discussion to include these\npossibilities in the revised manuscript.\n8. Figure 4I: Please clarify and include the units for the Y-axis.\nResponse:\nWe thank the reviewer for this helpful suggestion. We have added the appropriate units\nto the Y-axis in Figure 4I in the revised version of the manuscript.\nReviewer #4 (Remarks to the Author):\nReview of manuscript NCOMMS-25-16751 by Fujisawa et al. for Nature\nCommunications\nThe manuscript combines four stories in an inaccurate way and makes conclusions, which\ncannot be derived from the experiments done. However, some parts of the manuscript are\nwell done, but not the combination of the four parts HDAC-activity, KAT7-activity,\nZip10-regulation, and fatty liver in zinc deficiency (ZD). The major problem of the manuscript is the first part of the manuscript, from which the authors conclude that HDAC\nactivity is not regulated by zinc homeostasis, although they show that HDAC inhibitors\nshow the same effect than ZD. From here they went into the story with a wrong\nassumption. For this conclusion, the authors must have analyze the HDAC activity in\nvitro in ZD. The second part about KAT7 activity is once more not correct since the\nactivity is also not measured. The third part about Zip10 regulation is interesting and new.\nThe last part has a weak connection, since parts one and two are flawed.\nResponse:\nWe sincerely thank the reviewer for this comprehensive and critical evaluation of our\nmanuscript. We carefully considered the points raised in the “Critique in detail” section\nand have addressed each concern experimentally in a point-by-point manner. We hope\nthat these additional data and revisions adequately resolve your concerns.\nCritique in detail:\n1. The statistic in figures 1D and 1E is wrong or wrongly described. If the authors\ncompare ZD with ZN as described in the figure legend, then the significances must be at\nlow concentrations (1D) or short times (1E) and disappear at high concentrations and\nlonger times. Now it looks like a statistics in the concentration or time course.\nResponse:\nWe thank the reviewer for this thoughtful and constructive comment. As noted, the\nstatistics presented in Figures 1D and 1E were indeed conducted within the concentration\nor time course, which was intended to reveal the extent to which H3K14ac levels recover\nstatistically in response to zinc. However, we agree that direct comparisons between ZN\nand ZD are also important. Therefore, we have performed additional statistical analyses.\nIn Fig. 1D, we observed a significant difference in H3K14ac levels between ZN and ZD\nat 0 μM zinc concentration, but this difference was no longer significant after\nsupplementation with 5 μM zinc. In Fig. 1E, a significant difference was seen between\nZN and ZD at time point 0, but the difference disappeared by 12 hours. These findings\nprovide supportive evidence for the reversible regulation of H3K14ac by zinc. We\nsincerely thank the reviewer for drawing our attention to this important issue and for\nhelping us improve the clarity of our results.\n2. Figure 2: The authors did not show that HDACs disappear after siRNA treatment, like\nthey do for KAT7 in figure S4. This is a must.\nResponse:\nWe thank the reviewer for raising this critical issue. In response, we have now included\nthe knockdown efficiencies for all HDACs in Figure S2A. We believe that the degree of\nknockdown achieved is sufficient for the purpose of the screening experiments. We\nappreciate the reviewer’s valuable suggestion, which has helped us improve the clarity of\nour data presentation.\n3. Figure 2: From the “knock-down” (see point 2) the authors conclude that HDAC\nactivity is not important. However, they don´t measure the activity itself with recombinant\nHDACs. It has been already shown that some HDACs are inhibited by zinc and show an\nincreased activity in ZD. For this an HDAC activity assay must be done to follow the\nconclusion of the authors. Now the conclusion is not supported by the experiments. The\neffect in figures 6F/6G supports this assumption.\nResponse:\nWe sincerely thank the reviewer for this critical and constructive comment. As the\nreviewer pointed out, previous noteworthy reports have demonstrated that zinc deficiency\nor TPEN treatment can enhance HDAC activity (Kloubert et al., Clin Nutr., 2021, PMID:\n33183881; Puppa et al., J Trace Elem Med Biol., 2023, PMID: 37030059). In light of\nthese findings, we performed in vitro HDAC activity assays to address this important\nissue. As described in our response to Reviewer 3, we confirmed that HDAC1 and\nHDAC6 are capable of deacetylating H3K14ac. We therefore examined whether their\nenzymatic activities were altered under zinc-deficient conditions. We compared the\nactivities of HDAC1 and HDAC6 immunoprecipitated from control cells and TPEN-\ntreated cells. However, these experiments revealed no increase in HDAC activity toward\nH3K14ac under zinc-deficient conditions. Thus, in our system, we did not obtain\nevidence supporting enhanced H3K14ac deacetylation by HDACs upon zinc deficiency.\nNevertheless, based on our preliminary findings, we consider it plausible that other\nHDACs—particularly HDAC2, HDAC3, and HDAC11—may be involved, and we plan\nto investigate these candidates in future work. In addition, we have added the following\ndiscussion to the Discussion section to include the possibility that HDAC activity may\nincrease under zinc-deficient conditions.\n“Interestingly, although HDACs are known to be zinc-dependent enzymes, there are also\nreports indicating that zinc deficiency can enhance HDAC activity. Future studies\ninvestigating the activity of individual HDACs under zinc-deficient conditions will be\ncrucial to elucidate the mechanisms underlying the reduction of H3K14ac during zinc\ndeficiency.”\nWe greatly appreciate the reviewer’s constructive feedback, which has guided us toward\na more comprehensive evaluation of HDAC activity in the context of zinc deficiency.\n4. Figure 3: The authors show, that KAT7 loses its activity after mutation of the\ncysteines/histidine of the zinc-finger-motif important for its structure. This is new, but\nnothing fancy, since the activity is gone after braking the structure. This has been shown\nfor various proteins with zinc-finger-motifs. However, an in vitro HAT activity assay was\ndone (3F), but not under different zinc conditions. In fact, this would be difficult with a\nHis-tagged protein since histidine has a high zinc affinity. Therefore, the authors have to\ndo KAT7 activity assay with a non His-tagged recombinant KAT7.\nResponse:\nWe sincerely thank the reviewer for this important advice. Following the suggestion,\nwe performed experiments using recombinant KAT7 without a His-tag. We have replaced the\nprevious data with the new results (Fig. 3F, 3G, Fig. S3I, J). We also appreciate the\nreviewer’s suggestion regarding assays under different zinc conditions. To address this,\nwe attempted to remove zinc from recombinant KAT7 using TPEN and then reconstitute\nits activity by stepwise addition of zinc. However, as shown in Fig. S3J, we were not able\nto extract zinc from KAT7 with TPEN. Therefore, we were unable to evaluate HAT\nactivity under different zinc conditions in this study. While this limitation precluded\nfurther analysis in the present work, we believe that alternative strategies for modulating\nzinc occupancy of KAT7 will be an important direction for future investigation.\n5. At least for key experiments a control with zinc-saturated TPEN should be run.\nIn conclusion, the manuscript needs additional experimentation to support the\nassumptions and some additional controls.\nResponse:\nWe thank the reviewer for this constructive suggestion. As requested, we performed\ncontrol experiments with zinc-saturated TPEN, and the results are now included in the\nrevised manuscript (Fig. S1D and Fig. S3G). These additional controls further strengthen\nthe interpretation of our findings.\nReviewer #4 (Remarks on code availability):\nNot all data are available\nResponse:\nWe sincerely apologize for the incomplete provision of source code in the initial\nsubmission. In response, we have additionally uploaded the datasets and scripts used to\ngenerate each figure to our GitHub repository. We believe that this addition will further\nstrengthen the reproducibility and transparency of our study.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe are deeply grateful to you for kindly reviewing our manuscript.\nPoint-by-point response to the reviewers’ comments\nWe are deeply grateful to the reviewers for their thoughtful and valuable comments on\nour previous revision. We have thoroughly addressed all remaining issues raised and\nmade additional modifications to further improve the clarity and accuracy of the\nmanuscript. Changes made in this version are highlighted in red. We believe that the\ncurrent version satisfactorily addresses all concerns and provides a clearer and more\ncomplete presentation of our findings.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have provided reasonably thorough and generally appropriate responses to\nthe reviewers’ comments in both the revised manuscript and the response letter. While the\nrevisions have improved the overall quality of the manuscript and led to a clearer and\nmore compelling presentation, several issues remain only partially resolved.\nThe in vitro data, which were already valuable and recognized as novel findings in the\noriginal submission, initially required more careful interpretation to avoid overstatement.\nIn the revised version, the newly added data and the expanded discussion strengthen the\nauthors’ main arguments and improve the clarity and broader significance of the work.\nNevertheless, some aspects still appear somewhat overstated and would benefit from a\nmore cautious interpretation.\nWe thank the reviewer for this valuable comment. We apologize for the overstatement in\nour original text and as the reviewer notes, the in vitro results should be interpreted with\nappropriate caution. In particular, in HeLa cells, the experiments using zinc pyrithione\nwere not successful due to its cytotoxicity. Consequently, we were unable to clearly\ndemonstrate whether the intracellular zinc indeed contributed to the restoration of KAT7\nactivity and H3K14ac levels. To avoid overstatements, we added some sentences in the\nDiscussion section as below:\n“We showed that knockdown of ZIP10 suppressed the recovery of H3K14ac levels upon\nzinc replenishment (Fig. 4J). While zinc uptake is reduced under these conditions (Fig.\n4I), it remains to be determined whether these effects result from insufficient ZIP10-\nmediated restoration of intracellular zinc. To address this question, it will be necessary to\ndemonstrate that whether the zinc-transport activity of ZIP10 is required for these\nrecoveries; however, given the limited reports on ZIP10, more detailed characterization\nof the protein is needed. ZIP-family transporters are thought to have conserved histidine\nresidues essential for zinc transport (PMCID: PMC5573306). Although it is still unknown\nwhether the corresponding residues in ZIP10 are similarly required for its transport\nfunction, analyses using ZIP10 mutants with impaired zinc-transport activity would\nenable direct evaluation of the necessity of this activity for the observed recoveries of the\nH3K14ac levels.”\nFurthermore, in the Discussion section, we recognized that our interpretation\nof the in vitro findings in relation to the in vivo context had been overstated. Accordingly,\nwe have clarified and revised the text as follows.\n“We found that reduced H3K14ac in HeLa cells drives the upregulation of the zinc\ntransporter ZIP10, thereby contributing to the maintenance of intracellular zinc\nhomeostasis. Furthermore, in the mouse liver, zinc deficiency stress induced the\nexpression of both ZIP10 and ZIP14 (Fig. S5A, Fig. S6F,G). Given that zinc serves as an\nessential cofactor for liver enzymes such as alcohol dehydrogenase and those involved in\nammonia metabolism63,64, its adequate regulation is indispensable for hepatic function.\nFurthermore, based on our findings that zinc deficiency induces hepatic lipid\naccumulation, ZIP10-mediated zinc uptake may influence the regulation of liver steatosis.\nThus, the upregulation of ZIP10 and ZIP14 expression in response to decreased H3K14\nacetylation under zinc-deficient conditions is likely to function cooperatively to maintain\nhepatic zinc homeostasis and thereby sustaining liver function and modulating hepatic\nlipid accumulation. However, whether ZIP10 contributes to maintaining liver function or\nthe hepatic lipid accumulation during zinc deficiency in vivo remains unresolved, and\nfurther investigation is needed. As the whole-body Zip10 knockout model is lethal\n(PMID:37068188), generating a liver-specific Zip10 knockout mouse and assessing its\neffects on zinc uptake and hepatic lipid accumulation represent an important direction for\nfuture work.”\nRegarding the in vivo experiments, the authors generated liver-specific ZIP10-c\nknockdown mice using shRNA. Although these experiments did not yield the expected\nresults, the authors attributed this outcome to technical limitations. Their attempt to\nestablish and analyze Zip10-cKO mice is commendable and represents a valuable effort.\nHowever, this part of the study remains unsatisfactory, and it will be critical in future\nwork to determine whether the in vivo role of ZIP10 is truly consistent with the functions\nproposed from the in vitro experiments, ideally by analyzing mice in which the Zip10\ngene has been genetically eliminated. I specifically ask that the authors address this point\nmore explicitly in the Discussion.\nResponse:\nWe greatly appreciate the reviewer’s insightful comment regarding the future direction\ninvolving in vivo experiments. We agree that it is important to determine whether the in\nvivo role of ZIP10 is truly consistent with the functions proposed based on our in vitro\nexperiments. Therefore, we have added the following discussion to the revised\nmanuscript:\n“However, whether ZIP10 contributes to maintaining liver function or the hepatic lipid\naccumulation during zinc deficiency in vivo remains unresolved, and further investigation\nis needed. As the whole-body Zip10 knockout model is lethal (PMID:37068188),\ngenerating a liver-specific Zip10 knockout mouse and assessing its effects on zinc uptake\nand hepatic lipid accumulation represent an important direction for future work.”\nReviewer #2 (Remarks to the Author):\nI thank the authors for taking all of my comments on board. I am satisfied that all of my\nconcerns have been clearly and thoroughly addressed.\nResponse:\nWe are deeply grateful for the reviewer’s positive feedback and confirmation that all\nprevious concerns have been adequately addressed. We sincerely appreciate the\nconstructive comments provided during the review process, which have significantly\nstrengthened the manuscript.\nReviewer #3 (Remarks to the Author):\nDear Authors,\nThe authors have made appropriate revisions, including performing additional\nexperiments and updating the figures and legends. The manuscript text has been refined\nto more clearly justify the data interpretation and to provide a thoughtful discussion and\nwell-supported speculation based on the results. I look forward to exciting future studies\nfrom this group and from other laboratories inspired by this work.\nResponse:\nWe thank the reviewer for the encouraging comments and appreciation of our revisions.\nWe are encouraged by the reviewer’s positive outlook on future studies, and we remain\ncommitted to further exploring the molecular and physiological roles of zinc.\nReviewer #4 (Remarks to the Author):\nReview of manuscript NCOMMS-25-16751R1 by Fujisawa et al. for Nature\nCommunications\nThe revision of the manuscript is not convincing. The parts of the story are still not well\nconnected.\nResponse:\nWe thank the reviewer for the detailed and insightful critique, which has been extremely\nvaluable in improving the quality of our manuscript.\nCritique in detail:\n1. Figure 1: The authors just added the significance between ZN and ZD for time point 0\nor no zinc addition. However, the significance stands stable for most of the time and at\nleast up to 5µM zinc. This must be clearly indicated.\nResponse:\nWe agree with the reviewer that the statistical significance between ZN and ZD conditions\nshould be carefully evaluated across different time points and zinc concentrations. To\naddress this point, we conducted additional experiments with zinc supplementation under\nthe ZN condition, revised the figure accordingly, and performed statistical analyses (Fig.\n1D and Fig. 1E). We thank the reviewer for this valuable suggestion.\n2. Figure 2, HDAC KO: The authors answered this point adequate.\nResponse:\nWe thank the reviewer for confirming this point.\n3. Figure 2, HDAC activity and impact of zinc: The addition of Fig. 2E is satisfactory to\nshow activity of HDAC1 and 6. However, the experiments regarding the influence of zinc\nare not shown and can therefore not be evaluated. This is not acceptable.\nResponse:\nWe appreciate the reviewer’s insightful comment. In response, we have examined zinc-\ndependent changes in the activity of all HDACs and have revised Figure 2E accordingly.\nThis valuable feedback has also enhanced our understanding of HDAC regulation.\n4. Figure 3 Kat7 activity and HAT assay: The added figures 3F and G are fine, but not\ninnovative. The new figures S3I and J are not convincing, since the authors showed in\ncontrast to Fig.S3G, that there is no influence of TPEN (i.e. zinc chelation). Therefore,\nthe authors must change their discussion, since the influence seems to be on Kat7\nproduction or folding. Experiments underlining this must be then added to prove the\naltered discussion.\nResponse:\nWe thank the reviewer for this insightful and constructive comment. We agree that under\nzinc-deficient conditions, improper zinc coordination during the production or folding of\nKAT7 may lead to a decrease in its intracellular activity. To investigate this possibility,\nwe treated cells with the proteasome inhibitor MG132 and the protein synthesis inhibitor\ncycloheximide (CHX) to block protein degradation and synthesis, respectively, and then\nsubjected the cells to zinc depletion. We analyzed changes in H3K14ac levels and KAT7\nactivity under these conditions. The results showed that, although not statistically\nsignificant, the average levels of both H3K14ac and KAT7 activity tended to be slightly\nhigher at 1 hour after TPEN stimulation in the presence of MG132 and CHX compared\nwith the control (Fig. 3L). However, at 2 hours after TPEN stimulation, both parameters\ndecreased to levels comparable to the control, even with MG132 and CHX treatment.\nThese findings suggest that the reduction of KAT7 activity under zinc-deficient\nconditions is partly due to improper zinc coordination during KAT7 production or folding,\nbut may also result from zinc dissociation from pre-existing KAT7. We have previously\nencountered the difficulty of removing zinc from zinc-binding proteins in vitro during our\nstudies on SOD1 (Homma et al., Mol. Cell, 2013, PMID: 24076220). In the case of SOD1,\nzinc removal in vitro required harsh conditions, including protein denaturation at pH 3.7.\nBased on our work with both SOD1 and KAT7, we speculate that certain intracellular\nchaperones may facilitate zinc dissociation from zinc-binding proteins. The possibility\nthat such chaperones are involved in removing zinc from KAT7 is described in the\nDiscussion section as follows:\n“Another possibility is that the activity of certain chaperones that help coordinate zinc in\nKAT7 may be reduced under zinc-deficient conditions.”\nWe plan to address this possibility in a future study.\n5. Zinc-saturated TPEN controls were fine, were added (Fig. S1D and S3G), but missing\nfor key experiments in figures 2A andB, 3B and 6F and G.\nIn conclusion, the revised manuscript is not convincing and still needs additional\nexperimentation to support the assumptions and some additional controls still not\nprovided.\nResponse:\nWe thank the reviewer for this valuable comment. In response, we have replaced the data\nin Figures 2A, 2B, 6F, and 6G with those obtained using zinc-saturated TPEN controls.\nThe influence of zinc-saturated TPEN under the experimental conditions corresponding\nto Figure 3B is presented in Figure S3G.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse:\nWe are grateful to both the primary and co-reviewers for their collaborative and\nthoughtful evaluation of our manuscript. The constructive feedback has been invaluable\nin refining our analyses and ensuring greater clarity and reproducibility throughout the\nstudy. We truly appreciate your time and contributions to improving the quality of our\nwork."}, {"round": 2, "reviewer_comments": {"reviewer_4": "The authors changed the manuscript in all points requested. The new additional experiments are now convincing to underline the authors interpretation or to include an alternative discussion."}, "author_response": ""}]

Decision letter