Native chromatome profiling reveals hundreds of metabolic enzymes in the nucleus across tissues

Domain: chemistry
Published: 2026-03-06
Editorial outcome: minor_revision

Abstract
Metabolic and epigenetic rewiring are hallmarks of cancer, with increasing evidence suggesting crosstalk between these processes. While previous studies have hinted at the role of metabolic enzymes in the nucleus, the extent and functional relevance of their nuclear localization remain unclear. In this study, we present a comprehensive chromatome proteomic analysis across cancer lineages and healthy samples, revealing that metabolic enzyme moonlighting on chromatin is widespread across various tissues and metabolic pathways. We show that the abundance of metabolic enzymes on chromatin is tissue-specific, with oxidative phosphorylation proteins notably depleted in lung cancer, suggesting a link between nuclear metabolism and cell identity. Further, we explore the dynamic chromatin association of one-carbon folate enzymes, demonstrating their involvement in DNA damage and repair processes. Finally, we asked whether restricting metabolic enzymes to specific subcellular compartments rewires the transcriptome, thereby decoupling the observed transcriptional changes from mere metabolite diffusion. Our findings propose and validate novel non-canonical nuclear roles for several metabolic enzymes, providing new insights into the functional relationship between metabolism and chromatin regulation. This study underscores the hypothesis that the nucleus is populated by metabolic enzymes, offering new avenues for understanding how nuclear metabolism impacts chromatin function and cancer progression.

Introduction
Broad changes in the epigenetic and metabolic landscapes are both recognized hallmarks of cancer1–3. However, the interplay between these processes has remained elusive. Cancer transformation involves complex metabolic rewiring, which is essential for sustaining tumor growth and supporting metastasis. Traditionally, the connection between metabolism and epigenetics is mediated by metabolic substrates, such as acetyl-CoA, S-adenosylmethionine (SAM), or adenosine triphosphate (ATP), that facilitate epigenetic modifications4–7. These modifications, in turn, can induce transcriptional changes in specific metabolic enzymes, driving further metabolic rewiring. Classic examples, such as the c-Myc oncogene-BRD4 axis driving the transcription of folate metabolism enzymes8, or mutations in the enzymes Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) leading to aberrant genome-wide DNA and histone hypermethylation9, illustrate the strong, albeit indirect, connection between epigenetics and metabolism. Recent evidence, however, suggests a more direct interaction, particularly through the nuclear and chromatin translocation of metabolic enzymes.

Metabolic enzymes traditionally associated with cytoplasmic or organelle-specific metabolic reactions have also been shown to carry out distinct functions within the nucleus, phenomena often referred to as nuclear moonlighting functions10–13. These unexpected nuclear involvements often relate to chromatin dynamics and can occur independently of the enzyme’s catalytic properties. For example, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear localization is required to transcriptionally control apoptosis-related genes14, but also to facilitate the DNA damage repair by stabilizing RAD5115 and promoting the nuclear localization of Nicotinamide phosphoribosyltransferase (NAMPT)16. Nuclear fructose-1,6-bisphosphatase 1 (FBP1) interacts with the Polycomb Repressive Complex 2 (PRC2) to regulate levels of H3K27me317. Moreover, the nuclear localization of Pyruvate Kinase M2 (PKM2) is required for the phosphorylation of Histone H3 (T11), which consequently regulates histone acetylation10,11. In other cases, the canonical enzymatic activity of certain enzymes is essential for their nuclear function. For instance, Acyl-CoA synthetase short chain family member 2 (ACCS2) and ATP citrate lyase (ACLY) can both generate acetyl-CoA in the nucleus, which directly influences histone acetylation18–23. In a separate mechanism, the synthesis of nuclear ATP by the enzyme Nudix hydrolase 5 (NUDIX5) in breast cancer cells is required for estrogen-induced chromatin remodeling24. Additionally, the nuclear localization of Methylenetetrahydrofolate Dehydrogenase, Cyclohydrolase and Formyltetrahydrofolate Synthetase 1 (MTHFD1), a predominantly cytoplasmic folate metabolic enzyme, boosts the cancer-associated transcriptional landscape promoted by the epigenetic reader Bromodomain Containing 4 (BRD4)62. Finally, our most recent discoveries highlight a role for nuclear metabolic activities in the regulation of correct mitosis progression25, nuclear scavenging of reactive oxygen species (ROS)26, and the regulation of nuclear NAD+ availability during the DNA damage response27. This growing body of evidence suggests that nuclear metabolic enzyme activity is a regulated process essential for chromatin function and more widespread than previously thought. However, a thorough analysis of metabolic enzymes in the nucleus has not been performed, leaving this hypothesis unproven.

With the goal of comprehensively identifying novel moonlighting or non-canonical nuclear functions of metabolic enzymes, we optimized a subcellular fractionation protocol that enriches chromatin-bound proteins. We tested this protocol across a panel of tissue types for both cancer cell lines and healthy primary cells. In addition to canonical chromatin proteins, we identified metabolic enzymes that consistently localize to chromatin, many with tissue- and cancer-specific localization. For shortlisted metabolic enzymes, we validated their chromatin and nuclear localization in cell lines and patient-derived tissue samples and explored their functional relationships on chromatin. Our study provides a unique compendium of chromatin-associated metabolic activities that will serve as the foundation for the global analysis of chromatin and nuclear metabolism.

Results
The chromatome profile of ten different tissues
To achieve the characterization of chromatin-bound proteins across tissue types, we optimized a previously published chromatin enrichment protocol62, consisting of sequential cytoplasmic and nuclear lysis followed by sonication and benzonase-mediated chromatin digestion to extract the chromatin-bound proteome (the chromatome) (Fig. 1A). We applied this new chromatome protocol to ten tissue types (Fig. 1B), comparing cancer and healthy cells. In total, we processed and analyzed 44 cancer cell lines and ten healthy primary cell types in duplicates, which provided a comprehensive collection of samples. For each of those samples, the detergent concentrations and lysis duration were adjusted to obtain the best purification possible (Supplementary Data 1). A visual inspection of the fractionation protocol was conducted utilizing immunofluorescence to ascertain that non-nuclear proteins were systematically depleted at each step prior to nuclear lysis (Fig. 1C and Appendix 1). To accomplish this, samples were stained with an antibody directed against mitochondrial Ferredoxin 1 (FDX1), a protein that, according to subcellular fractionation data28, exclusively resides in the mitochondria (Fig. S1A) and that has not been previously identified as enriched in chromatome-MS experiments conducted by our group26,27. Moreover, at the end of the protocol, we performed Western blot of focal adhesion (Vinculin), mitochondrial (FDX1) and histone H3 (H3) markers to verify the purity of our final samples (Fig. 1D and Appendix 2).

Samples were acquired using data-independent acquisition mass spectrometry (DIA-MS) and, as expected, showed strong enrichment for nuclear and chromosome-related terms while being depleted of mitochondrial and cytoplasmic proteins (Fig. S1B, Source Data 1), further validating our protocol. We retrieved ~5100 proteins per sample, most quantified by multiple peptide-to-spectrum matches (PSMs), with high reproducibility in the number of retrieved proteins between biological replicates (Fig. 1E, Source Data 2). While most proteins were identified and quantified across the majority of samples (Fig. S1C, Source Data 3), some proteins were only sporadically detected (Supplementary Data 2).

Given that we expected most chromatin proteins to be present across tissues in our chromatome analysis, we hypothesized that proteins with sparse presence in our dataset would likely be non-chromatin proteins. To demonstrate this, we predicted sequence-based nuclear localization signals (NLS) genome-wide and used them as a proxy for nuclear proteins (Fig. S1D, Source Data 4) due to their agreement with known nuclear proteins29,30. We found that proteins with few missing values were enriched for NLS-containing nuclear proteins, while proteins detected sparsely lacked NLS signals. (Fig. S1E, Source Data 5). After filtering out these sparsely observed proteins, we defined a “core chromatome” consisting of 3467 proteins which were consistently found on chromatin across the majority of samples analyzed (Supplementary Data 3).

To benchmark our optimized chromatome protocol and data analysis, we compared the results with other publicly available chromatome extraction methods31–33. Our chromatome outperformed these approaches (Fig. S1F), even though it is based on native chromatin extraction without the use of fixatives that could increase the presence of false-negative chromatin interactions, chemicals that would interfere with DNA properties and cell behavior, or expensive beads that would make it unaffordable to scale up32,33. When we classified the chromatin-associated proteins identified by our newly developed protocol according to their subcellular annotation, we observed that approximately 42% were not currently annotated as nuclear factors (Fig. S1F). However, the intersection of our data with the OpenCell resource34 led us to conclude that most of these non-nuclear factors identified on chromatin have at least one nuclear interactor (Fig. S1G), which provides further support for their nuclear localization. Finally, given the fact that metabolic enzymes often act in complexes to facilitate the execution of stepwise reactions, we explored the presence of protein complexes on the chromatome with hu.MAP2.035. As expected, we retrieved well-known chromatin complexes (Fig. 1F, Source Data 6) such as SWI/SNF and HDAC proteins as “core chromatome” proteins35,36. Interestingly, we also identified metabolic assemblies such as those involved in oxidative phosphorylation (OXPHOS).

Together, our annotated core chromatome reveals key chromatin regulators and metabolic complexes consistently associated with chromatin, strengthening the evidence for the nuclear localization of metabolic enzymes.

Metabolic enzymes on chromatin
To further identify functional pathways on chromatin, we explored ‘Biological Processes’ Gene Ontology (GO) terms enriched in the “core chromatome”. While we retrieved many well-characterized processes on chromatin, such as “DNA repair” and “DNA replication”, we also unexpectedly detected “ATP metabolic process”, a term mainly driven by proteins constituting the electron transport chain (ETC), such as members of the NADH:ubiquinone oxidoreductase (NDUF) supernumerary subunits (Fig. S2A, Source Data 7). In total, over 200 metabolic enzymes were identified within the chromatin environment, accounting for more than 7% of the proteins in the core chromatome (Fig. 2A and Supplementary Data 3), underscoring the broad integration of metabolic enzymes into nuclear functions. To investigate the evolutionary stages at which chromatin proteins emerged, we intersected the known localization of each “core chromatome” protein [either nuclear or non-nuclear according to the Human Protein Atlas (HPA)] with their evolutionary age (Fig. 2B, Source Data 8). We observed that most chromatin-bound proteins were already assigned a nuclear HPA annotation. However, many KEGG-related metabolic enzymes, excluding those with primarily epigenetic functions37, lacked such annotation. One example is NDUF subunit B11 (NDUFB11), which lacks a nuclear HPA label despite nuclear staining in HPA immunofluorescence images (Fig. 2B). This is similar to what is observed for enzymes such as Acyl-CoA Oxidase 1 (ACOX1) and Sirtuin 6 (SIRT6), which also show clear nuclear staining in HPA immunofluorescence images. However, unlike NDUFB11, both are annotated as nuclear by HPA. Interestingly, most enzymes have an evolutionary age pre-dating the eukaryotic branching event, regardless of their HPA annotation. This suggests that any nuclear function these proteins may perform on chromatin is highly likely to be a moonlighting function, as they did not co-evolve with the nuclear compartment.

To specifically check which metabolic pathways were present on chromatin, we performed an enrichment analysis on our “core chromatome” against KEGG pathways38,39. Among other pathways, OXPHOS was found to be the most complete pathway on chromatin, with more than 60% of subunits identified in our entire dataset (Fig. 2C and Fig. S2B, Source Data 9 and 10). OXPHOS was followed by lysine degradation, which includes lysine methyltransferase family members that are well established to be on chromatin, such as the histone lysine methyltransferase (KMTs), the nuclear receptor binding SET domain protein (NSDs) and the SET domain-containing proteins (SETDs). Following the identification of OXPHOS subunits on chromatin, we then examined the chromatin presence per complex for the entire ETC (Fig. 2D, Source Data 11). We observed a high presence of subunits across all complexes, with the exception of Complex II, for which less than 50% of its protein members were found on chromatin. This may suggest that nuclear-encoded components may have acquired additional, mitochondria-independent functions in the nucleus, possibly reflecting an evolutionary adaptation.

To explore tissue-specific pathways on chromatin, we annotated “core chromatome” proteins using KEGG pathways and clustered proteins based on their detection in each sample. We observed a strong tissue-based clustering of samples, with blood, lung, skin, brain and cervix showing high consistency across cell lines, while breast and pancreas demonstrated high heterogeneity in the chromatome across cell lines (Fig. S2C, Source Data 12). Interestingly, proteins belonging to the “Thermogenesis” pathway, were consistently detected across samples, except in lung, colon and pancreas samples, suggesting their near absence from the chromatin environment in these tissues. The term “Thermogenesis” consisted primarily of OXPHOS-related proteins, which are generally not expected in the nucleus. This unexpected finding prompted us to explore whether other metabolic enzymes might also be enriched in our dataset. While lung samples were depleted in OXPHOS proteins, some breast samples appeared to be depleted in pyrimidine and pyruvate metabolism proteins (Fig. 2E, Source Data 13), suggesting tissue specificity of chromatin localization of these enzymes.

When looking at the protein abundance of the “core chromatome”, we observed a variation in the degree of chromatin enrichment, as quantified by total histone protein intensity across samples (Fig. S2D, Source Data 14). This variability is likely attributable to subtle, cell line-specific differences affecting the efficiency of cell lysis and chromatin enrichment. To enable comparison across samples for downstream analysis, we regressed all protein abundances against each sample’s relative chromatin enrichment (Fig. S2E, Source Data 15). As expected, nuclear proteins showed positive regression coefficients, while non-nuclear proteins displayed negative ones. This pattern is exemplified by the nuclear protein H3-3 and the primarily non-nuclear protein LAMP2, which showed opposite trends in relation to chromatin enrichment, reflecting their relative chromatin associations40 (Fig. S2F). The effect of the relative chromatin enrichment per sample can be observed where the primary subcellular localization of a protein (e.g., chromatin or endoplasmic reticulum), is a large driving force on the quantified protein abundance (Fig. S2G, Source Data 16). This regression-based normalization removed the compartment-specific separation within our dataset (Fig. S2H17), with the largest differences on normalized chromatome abundances being present between blood, lung and skin cell lines, suggesting tissue-specific chromatin proteomic profiles (Fig. S2I, Source Data 18). When investigating proteins more enriched on chromatin in blood-derived cell lines compared to skin-derived ones, the GO term “Regulation of DNA replication” emerged as one of the top enriched categories in the blood samples. This enrichment is primarily driven by components of the minichromosome maintenance (MCM) complex, which was recently reported to be particularly abundant in blood cell lines41 (Fig. S2J). Further confirming our model, regression coefficients of each protein, representing relative protein proportion on chromatin, strongly agreed with hyperLOPIT subcellular annotations29 and the presence of NLS motifs for nuclear proteins (Fig. S2K, L, Source Data 19 and 20).

We noticed blood, skin, lung, and pancreas tissues to display more homogeneity regarding the abundance of chromatin-bound proteins, with the pancreatic cancer cell lines showing similarity to their respective non-cancerous tissue (Fig. 2F, Source Data 21). Focusing on metabolic pathways, we observed that enzymes on chromatin vary in a tissue-specific manner (Fig. 2G, Source Data 22). For instance, several samples, including those of skin, brain and breast, showed high levels of chromatin-bound OXPHOS subunits compared to lung samples, while blood samples exhibited higher chromatin levels of purine metabolism proteins (Fig. 2G and Fig. S3A, B). To systematically identify metabolic pathways differentially present between tissues, we compared the abundance of enzymes across KEGG pathways in tissues for which we analyzed more than five samples (Supplementary Data 4). When evaluating differentially present pathways between these tissues, we found OXPHOS to be preferentially more abundant in skin, colon and breast samples, while steroid biosynthesis was more abundant in colon and breast samples (Fig. 2H, Source Data 23). Interestingly, only OXPHOS-related subunits encoded by nuclear DNA were identified on chromatin, while none of the mitochondrially encoded subunits survived the regression workflow described above (Data 23). To explore whether the increased chromatin association of OXPHOS components is consistent across subunits, we compared the average chromatin abundance of ubiquinol-cytochrome c oxidoreductase complex (Complex III) subunits between lung and breast cancer cell lines, which confirmed that all detected subunits were consistently more enriched on chromatin in breast compared to lung (Fig. 2I, Source Data 24). Leveraging CCLE proteomics and transcriptomics data, we ascertained that the higher or lower chromatin presence of OXPHOS enzymes on chromatin is not a consequence of differential expression of these enzymes across cell types (Fig. S3C). To validate our MS data, we performed chromatin extraction followed by Western blot analysis in lung (Hcc44 and H460) and breast (MDA-MB-231 and MDA-MB-468) cancer cell lines. The results confirmed a higher presence of OXPHOS enzymes on chromatin in breast cancer cell lines than in lung cancer cell lines. Notably, this difference was not mirrored in their cytosolic abundance, suggesting tissue-specific, active recruitment of OXPHOS subunits to chromatin (Fig. S3D).

We next attempted to computationally explore the mechanisms driving enzyme nuclear localization. Although most of these enzymes are smaller than the ~80 kDa size threshold permitted by nuclear pore diffusion42,43, and thus would not require active transport, only a small fraction of their cellular pool was found on chromatin (Fig. S3E, Source Data 25). An exception was observed for enzymes with NLS signals and enzymatic functions, such as KMTs, which are canonically nuclear and showed high chromatin association. In contrast, enzymes specific to membrane-bound compartments, such as alpha-1,3-mannosyltransferase (ALG3) and the catalytic subunit STT3B of the oligosaccharyltransferase complex, were only minimally detected on chromatin. Finally, the presence of enzymes larger than 80kDa that lack an NLS signal on chromatin suggests the involvement of alternative active mechanisms mediating their nuclear localization. This evidence, together with the uneven distribution of small enzymes between the nucleus and the rest of the cell, rules out the possibility that the nuclear localization of metabolic enzymes is controlled solely by passive diffusion.

To further explore the mechanisms of nuclear localization and retention, we analyzed the interactome of OXPHOS enzymes using the OpenCell database34. Nearly all OXPHOS enzymes were found to interact with nuclear factors, with COX5A, a subunit of the cytochrome c oxidase complex (Complex IV), exhibiting the highest number of interactors. Among these, we identified FKBP Prolyl Isomerase 5 (FKBP5) and Lamin B1 (LMNB1) as key interactors of COX5A (Fig. S3F). FKBP5 acts as a co-chaperone in complex with Heat Shock Protein 90 (HSP90), regulating the nuclear trafficking of target proteins44. LMNB1 plays a crucial role in maintaining chromatin integrity, nuclear structure and the localization of nuclear proteins45. These interactions may thus provide insights into the nuclear shuttling and retention mechanisms of COX5A.

Taken together, our findings suggest that the nuclear localization of metabolic enzymes is more widespread than previously appreciated, with a notable enrichment of OXPHOS proteins. This raises the possibility that these enzymes may perform yet unidentified, moonlighting functions within the nuclear environment.

Nuclear localization of OXPHOS subunits in human breast and lung tissue
To corroborate the presence of metabolic enzymes in human cells, we focused on OXPHOS subunits and their differential localization in breast and lung cancer. We first performed high-throughput immunofluorescence analysis on breast and lung cancer cell lines to determine whether the differential localization previously observed in the chromatin compartment was also reflected in their nuclear levels. Our findings demonstrated a significant nuclear enrichment of OXPHOS subunits CYC1 (Complex III), COX4 (Complex IV), and ATP5A1 (Complex V) in breast cancer cell lines compared to lung cancer cell lines (Fig. 3A–C, Source Data 26). Notably, and particularly for COX4 and ATP5A1, the observed differences could not be attributed to variations in overall protein expression between cell lines, as their cytoplasmic abundance did not consistently mirror the nuclear trends (Fig. S4A–C). Interestingly, the three OXPHOS subunits exhibited distinct nuclear localization patterns, with COX4 showing prominent accumulation in nucleolus-like regions, particularly in breast cancer cells. Given that cell proliferation rates, cell cycle phase distribution, and phase duration vary across cell lines, and that some metabolic enzymes exhibit phase-specific nuclear localization25–27, we explored whether OXPHOS subunit localization is cell cycle-dependent. To address this, we classified cells into G1, S, and G2/M phases by combining nuclear size measurements with Hoechst intensity profiles from immunofluorescence imaging, as previously performed46,47. This allowed us to quantify nuclear OXPHOS subunit levels in a cell cycle–resolved manner. Our results showed that CYC1 nuclear (Fig. S4D) and cytoplasmic (Fig. S4G) abundance increased during cell cycle progression. However, cell cycle did not alter the nuclear (Fig. S4E, F) nor cytoplasmic (Fig. S4H, I) abundance of COX4 or ATP5A1. This suggests the existence of a distinct regulatory mechanism governing their nuclear localization, independent of cell cycle dynamics. To cross-validate the differential presence of OXPHOS subunits in the nucleus across different cell lines, we leveraged the HPA image dataset. The use of different antibodies and unrelated cell lines in the HPA database provided independent support for our observations. Consistent with our findings, nuclear staining of CYC1, COX4, and ATP5A1 was evident across cell lines, although its intensity and subnuclear distribution varied. The nucleolar localization of COX4 was also confirmed (Fig. 3D–F).

To validate these findings in human breast and lung tissue, both healthy and cancerous, we analyzed commercially available tissue microarrays (TMAs) comprising tumor tissue, adjacent tissue (AT), and normal adjacent tissue (NAT). The breast TMA (Supplementary Data 5) included 20 cases of invasive ductal carcinoma (primarily grade 2), two atypical medullary carcinomas, one mucinous adenocarcinoma, and one intraductal carcinoma, together with 24 unmatched AT and 24 unmatched NAT samples. The lung TMA (Supplementary Data 6) included...

Methods
To achieve the characterization of chromatin-bound proteins across tissue types, we optimized a previously published chromatin enrichment protocol62, consisting of sequential cytoplasmic and nuclear lysis followed by sonication and benzonase-mediated chromatin digestion to extract the chromatin-bound proteome (the chromatome) (Fig. 1A). We applied this new chromatome protocol to ten tissue types (Fig. 1B), comparing cancer and healthy cells. In total, we processed and analyzed 44 cancer cell lines and ten healthy primary cell types in duplicates, which provided a comprehensive collection of samples. For each of those samples, the detergent concentrations and lysis duration were adjusted to obtain the best purification possible (Supplementary Data 1). A visual inspection of the fractionation protocol was conducted utilizing immunofluorescence to ascertain that non-nuclear proteins were systematically depleted at each step prior to nuclear lysis (Fig. 1C and Appendix 1). To accomplish this, samples were stained with an antibody directed against mitochondrial Ferredoxin 1 (FDX1), a protein that, according to subcellular fractionation data28, exclusively resides in the mitochondria (Fig. S1A) and that has not been previously identified as enriched in chromatome-MS experiments conducted by our group26,27. Moreover, at the end of the protocol, we performed Western blot of focal adhesion (Vinculin), mitochondrial (FDX1) and histone H3 (H3) markers to verify the purity of our final samples (Fig. 1D and Appendix 2).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript explores metabolic enzymes functioning within the nucleus. The study uses chromatin profiling technique to\nmap metabolic enzyme localization in various cancer cell lines and healthy tissues. It uncovers tissue- and cancer-specific\nnuclear localization patterns to highlight their role on the chromatin.\nAlthough Authors searched metabolic enzymes localized on the chromatin, there is no further confirmation nor validation of\ntheir finding, function, or physiological/pathologcal relevances. It is stongly advised that the authors perform ChIPseq and\ncell-based assays to validate their findings.", "reviewer_2": "Kourtis S et al present a comprehensive study of the function of metabolic enzymes involved in nuclear metabolism in both\nhealthy and cancer cells. The authors optimized the chromatin enrichment protocol and identified metabolic enzymes that\nlocalize to chromatin with tissue- and cancer-specific patterns, suggesting a broader role for metabolic enzymes in\nchromatin-related processes, such as DNA repair and transcriptional regulation. These findings suggest that nuclear\nmetabolism may reflect cellular states such as carcinogenesis, differentiation, and aging, and provide new avenues for\nstudying the functional dynamics of metabolic enzymes in the nucleus.\nI have some concerns about some of the claims made by the authors in the current manuscript.\n1. The authors describe a complex subcellular fractionation protocol to isolate chromatin-bound proteins. However, FDX1\nexpression could still be detected by immunofluorescence in the clean nuclear part of some cells. Moreover, there is little\ndiscussion of the reproducibility of this optimized method, and comparisons with alternative fractionation methods.\n2. The authors used several bioinformatics tools to analyze the proteomics data, but without sufficient experimental\nconfirmation. In addition, predictions of protein localization and function are based on existing databases, which may not\nfully capture the complexity of the nuclear proteome. Validation of bioinformatics predictions with experimental data, by\nusing immunoprecipitation or chromatin immunoprecipitation (ChIP) assays, could provide more robust conclusions.\n3. The authors found that the majority of metabolic enzymes do not have NLSs on chromatin, but the authors do not explore\nin depth the mechanisms by which these enzymes are translocated to the nucleus and retained on chromatin. The\ndiscussion is mostly speculative. Further investigations to identify NLS or interacting partners that facilitate their chromatin\nassociation should be explored.\n4. The study includes a wide range of tissue types and cell lines, both cancerous and non-cancerous. However, the\nheterogeneity of the samples that could introduce variability makes it difficult to interpret the results. In addition, some tumor\ntypes only contain 1-2 cell lines, which may not fully represent the heterogeneity of certain tissues or cancer types. Thus,\nmore focused analysis on a smaller, more homogeneous set of samples will reduce variability and help to better understand\ntissue-specific nuclear metabolism.\n5. The study mentions tissue-specific and cancer-specific enzymes on chromatin, but this is based on a small number of\nsamples. A wider range of samples on specific tumor or tissue types should be included to confirm the conclusion.", "reviewer_3": "This manuscript presents a proteomics resource defining chromatin-associated metabolism-related proteins across human\nhealthy samples and cancer cells. The strategy used to prepare chromatin fractions is well done, and the datasets generated\nwill be valuable to the scientific community. The study makes the interesting and notable point that many metabolism-\nassociated proteins are found in chromatin across many sample types. The primary utility of this type of study in my view is\nin making the datasets easily accessible and usable by other researchers, and improvements are needed in this regard.\n1. I found it difficult to mine the data to understand which specific proteins are regulated in each setting, which is a major\naspect of the utility of the study. Some suggestions:\na. Table 3 lists the “core-chromatome” chromatin-associated proteins detected across samples. In addition to the Uniprot\ndesignations, it would be much appreciated to also include the protein names so that one can more readily query the table.\nb. The link to the datasets listed under Data Availability did not work for me. I appreciate that the authors provided an\nupdated link during the review process, which should replace the prior link.\nc. Could the various Data files also be updated to include as much information as possible about each dataset so that the\ndata can be readily understood? For example, add titles to explain what is presented in each dataset, indicate what the\nnumerical values mean (e.g., z scores, etc), and so on. Basically, make the data user-friendly. They could also consider\nmaking a web-based portal to query the datasets, though I wouldn’t require this.\nd. Fig 2E and F. With the current presentation, I find it difficult to glean useful information, since the tissue samples don’t\nnecessarily cluster together. If the point is to understand how pathways are regulated within each type of sample, it might be\nmore useful to present replicate tissue samples next to one another and cancer samples from each tissue next to the\ncorresponding normal tissue samples. That way the reader could at least understand the overall patterns within each tissue.\nIt should also be made clear in the legend what is “Relative abundance”. Is this a z score?\n2. More validation of the patterns in chromatin-associated proteins identified by proteomics should be conducted, using IF\nand/or biochemical fractionation and western blotting. It would be nice to see careful validation of examples of proteins\nshown to be chromatin-associated across sample types and those that are chromatin-associated in specific sample types.\nFor example, validating the OXPHOS proteins in skin or brain vs lung cells would be appropriate.\nMinor\n- Fig 2A. Legend needs info on IF data. Is it from HPA? Or their own data?\n- The 300mM of salt in the nuclear extraction buffer allows the investigators to be rigorous in defining chromatin-associated\nproteins- but they might be losing some proteins that are less strongly bound, which many metabolic enzymes may be. This\ncaveat could be mentioned in the discussion."}, "author_response": "Sara Sdelci, Ph.D.\nCentre for Genomic Regulation (CRG)\nUniversity Pompeu Fabra (UPF)\nBarcelona Institute of Science and Technology\nJune 16, 2025\nNCOMMS-24-42424-T\nComprehensive Chromatome Profiling Reveals Hundreds of Metabolic Enzymes\nin the Nucleus\nResponse to reviewers:\nWe sincerely thank the reviewers for their thoughtful and constructive feedback. Their\ninsightful comments not only helped us clarify several aspects of the manuscript but also\nprompted us to perform additional analyses that extended beyond the original scope of\nour study. In addressing their suggestions, we were able to validate key findings and\nfurther substantiate the biological relevance of our results. We believe these revisions\nhave significantly strengthened the manuscript and helped shape it into a more\ncomprehensive and compelling scientific contribution.\nReviewer #1 (Remarks to the Author):\nThe manuscript explores metabolic enzymes functioning within the nucleus. The study\nuses chromatin profiling technique to map metabolic enzyme localization in various\ncancer cell lines and healthy tissues. It uncovers tissue- and cancer-specific nuclear\nlocalization patterns to highlight their role on the chromatin.\nAlthough Authors searched metabolic enzymes localized on the chromatin, there is no\nfurther confirmation nor validation of their finding, function, or physiological/pathologcal\nrelevances. It is stongly advised that the authors perform ChIPseq and cell-based assays\nto validate their findings.\nWe sincerely thank the reviewer for this constructive and insightful comment.\nThe primary aim of our study was to provide a resource for the scientific community by\ncharting the chromatin-bound metabolic proteome across different biological cells and\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\ntissue types. For this reason, we initially focused on generating and reporting a robust\ndataset without delving into mechanistic or functional analyses. However, inspired by the\nreviewer’s helpful suggestion, we have now extended the study in several directions to\noffer a deeper level of validation and initial functional insight:\n- Validation of nuclear localization: We performed Western blot analyses on chromatin\nfractions from cell lines (Figure S3D and S7E), as well as quantitative high-throughput\nimmunofluorescence (HT-IF) in both cultured cells (Figure 3 and Figure S4 and human\ntissues (healthy and cancer; Figure 4 and Figure S5-6). These experiments confirm the\nnuclear localization of key metabolic enzymes and validate the reproducibility and\nrobustness of our chromatome-MS profiling approach.\n- Tissue-specific distribution in cancer vs. healthy cells: Using image-based analyses\non healthy and cancerous lung and breast tissue sections, we examined the nuclear\ndistribution of selected metabolic enzymes, focusing on the OXPHOS complex. Our\nanalysis distinguishes between epithelial and stromal compartments and reveals\nconsistent differences in nuclear localization between healthy and cancer tissues, as well\nas across cell types—suggesting a possible role in defining cell identity (Figure 4 and\nFigure S5-6).\n- Dynamic nuclear relocalization during DNA damage response: To explore the\npotential functional implications, we examined the chromatin association of folate\nmetabolism and de novo purine synthesis enzymes following DNA damage (irradiation).\nWe observed dynamic nuclear relocalization patterns correlating with DNA damage\nresponse kinetics (Figure 6 and Figure S8). These results align with our recent work on\nthe nuclear function of IMPDH2—a key enzyme in purine synthesis—which modulates\nPARP1 activity during DNA damage repair (Espinar et al., Nat. Commun. 2024,\nhttps://doi.org/10.1038/s41467-024-53877-z).\nWhile we acknowledge that numerous avenues remain to be explored, including more\nmechanistic investigations such as ChIP-seq, we believe that the validation experiments\nand targeted analyses now included in the revised manuscript provide solid support for\nthe conclusions drawn. We opted not to perform ChIP-seq in this context, as many\nmetabolic enzymes may not directly bind DNA, and the approach poses technical\nchallenges, particularly for proteins with weak or transient chromatin interactions and\nlimited ChIP-seq grade antibody availability. Given that the primary goal of this work is\nto provide a resource highlighting the widespread nuclear localization of metabolic\nenzymes, we are confident that the additional data and analyses now presented\nsubstantially strengthen our contribution and will serve as a foundation for future studies\nin the field.\nReviewer #2 (Remarks to the Author):\nKourtis S et al present a comprehensive study of the function of metabolic enzymes\ninvolved in nuclear metabolism in both healthy and cancer cells. The authors optimized\nthe chromatin enrichment protocol and identified metabolic enzymes that localize to\nchromatin with tissue- and cancer-specific patterns, suggesting a broader role for\nmetabolic enzymes in chromatin-related processes, such as DNA repair and\ntranscriptional regulation. These findings suggest that nuclear metabolism may reflect\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\ncellular states such as carcinogenesis, differentiation, and aging, and provide new\navenues for studying the functional dynamics of metabolic enzymes in the nucleus.\nWe sincerely thank the reviewer for their positive assessment of our manuscript and for\nrecognizing the robustness and comprehensive scope of our approach.\nI have some concerns about some of the claims made by the authors in the current\nmanuscript.\n1.The authors describe a complex subcellular fractionation protocol to isolate chromatin-\nbound proteins. However, FDX1 expression could still be detected by\nimmunofluorescence in the clean nuclear part of some cells. Moreover, there is little\ndiscussion of the reproducibility of this optimized method, and comparisons with\nalternative fractionation methods.\nThe lack of FDX1 detection by immunofluorescence at the “Clean Nuclei” stage does not\nindicate a failure of the protocol. After the sucrose gradient step—resulting in the so-\ncalled clean nuclei—nuclei are subjected to three consecutive washes, followed by lysis\nto extract chromatin, digest DNA, and isolate chromatin-bound proteins. As shown in the\nrevised manuscript, we intentionally used FDX1 as an example to demonstrate its\nprogressive loss throughout the extraction procedure, until the clean nucleai stage where\nIF is still feasible (Figure 1C and Appendix 1). Western blot analysis of protein extract of\nthe final chromatome preparation confirms no contamination of FDX1, as expected from\na highly purified chromatin-bound fraction (Figure 1D, Figure S3D; Figure S7E).\nAdditionally, FDX1 was not detected in our mass spectrometry results (Data 2), and this\nis consistent with previous chromatin MS data we have published, where FDX1 was\nnever enriched:\nhttps://doi.org/10.1038/s41588-019-0413-z;\nhttps://doi.org/10.1038/s41467-024-53877-z;\nhttps://doi.org/10.15252/msb.202211267;\nhttps://doi.org/10.1101/2024.12.20.629614)\nRegarding benchmarking, we agree with the reviewer that this was initially missing. We\nhave now included a benchmarking analysis in the revised manuscript, which shows that\nour chromatome extraction protocol largely outperforms other recently published\nprotocols (Figure S1F-G)\n2. The authors used several bioinformatics tools to analyze the proteomics data, but\nwithout sufficient experimental confirmation. In addition, predictions of protein\nlocalization and function are based on existing databases, which may not fully capture\nthe complexity of the nuclear proteome. Validation of bioinformatics predictions with\nexperimental data, by using immunoprecipitation or chromatin immunoprecipitation\n(ChIP) assays, could provide more robust conclusions.\nWe sincerely thank the reviewer for their thoughtful and encouraging feedback. Our study\nwas designed as a resource-building effort to chart the chromatin-bound metabolic\nproteome across a broad spectrum of biological systems. In line with this objective, we\ninitially focused on generating a high-quality dataset with extensive coverage and\nreproducibility, rather than on dissecting specific molecular mechanisms.\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\nIn response to the reviewer’s helpful suggestions, we have substantially expanded the\nrevised manuscript to incorporate additional layers of validation and functional insight.\nWe performed Western blot analyses on chromatin-enriched fractions (Figure S3D and\nS7E), complemented by high-throughput immunofluorescence (HT-IF) in both cultured\ncell lines (Figure 3 and Figure S4) and human tissue samples from healthy and\ncancerous origins (Figure 4 and Figures S5–S6). These experiments confirmed the\nnuclear localization of selected metabolic enzymes and reinforced the robustness of our\nchromatome-MS profiling pipeline.\nNotably, we observed tissue-specific differences in nuclear localization patterns,\nparticularly for OXPHOS enzymes such as COX4. COX4 exhibited striking nucleolar\nenrichment in breast cancer samples—both in cell lines and in patient-derived tissues,\nbut not in lung cancer or healthy breast tissue. This unexpected subnuclear localization\nwas independently supported by publicly available immunofluorescence images from the\nHuman Protein Atlas (HPA), further underscoring the physiological relevance of our\nfindings. Our image-based analysis also revealed consistent differences in enzyme\nlocalization between epithelial and stromal compartments, as well as between healthy\nand malignant tissues, suggesting possible roles in tissue identity and disease-specific\nchromatin regulation. To further explore functional relevance, we examined nuclear\nrelocalization of folate and purine biosynthesis enzymes following ionizing radiation.\nThese enzymes exhibited dynamic nuclear enrichment that paralleled DNA damage\nresponse kinetics (Figure 6 and Figure S8), pointing to context-dependent regulatory\nfunctions. These findings build upon and complement our previous work identifying\nnuclear IMPDH2 as a modulator of PARP1 activity during DNA repair (Espinar et al., Nat.\nCommun. 2024, https://doi.org/10.1038/s41467-024-53877-z).\nTaken together, the newly included experimental validations and targeted analyses\nsignificantly enhance the depth and interpretability of our dataset, and we hope they will\nprovide a strong foundation for future investigations into the nuclear roles of metabolic\nenzymes.\n3. The authors found that the majority of metabolic enzymes do not have NLSs on\nchromatin, but the authors do not explore in depth the mechanisms by which these\nenzymes are translocated to the nucleus and retained on chromatin. The discussion is\nmostly speculative. Further investigations to identify NLS or interacting partners that\nfacilitate their chromatin association should be explored.\nWe thank the reviewer for this insightful and constructive suggestion. While it would not\nbe technically feasible to perform compartment-specific immunoprecipitation for the\nmore than 200 metabolic enzymes identified on chromatin, we have taken this\nopportunity to explore potential mechanisms that may underlie their nuclear localization.\nTo this end, we leveraged publicly available protein interaction data and incorporated a\nnew analysis using the OpenCell database. This allowed us to identify putative binding\npartners that could facilitate nuclear import. For example, we found that COX5A interacts\nwith FKBP5 and LMNB1, two proteins with known roles in nuclear transport and\nchromatin organization, suggesting a plausible shuttling/retaining mechanism (Figure\nS3E). We have included this analysis in the revised manuscript and expanded the\ndiscussion accordingly to offer additional insight into how metabolic enzymes lacking\ncanonical nuclear localization signals might gain access to the chromatin compartment.\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\n4. The study includes a wide range of tissue types and cell lines, both cancerous and\nnon-cancerous. However, the heterogeneity of the samples that could introduce\nvariability makes it difficult to interpret the results. In addition, some tumor types only\ncontain 1-2 cell lines, which may not fully represent the heterogeneity of certain tissues\nor cancer types. Thus, more focused analysis on a smaller, more homogeneous set of\nsamples will reduce variability and help to better understand tissue-specific nuclear\nmetabolism.\n5. The study mentions tissue-specific and cancer-specific enzymes on chromatin, but\nthis is based on a small number of samples. A wider range of samples on specific tumor\nor tissue types should be included to confirm the conclusion.\nResponse to point 4 and 5\nWe thank the reviewer for this thoughtful comment. Our initial rationale for including a\nbroad range of cell types was to define a robust “core chromatome”, a set of proteins\nconsistently associated with chromatin across diverse biological contexts (Table 1),\nirrespective of cell line or tissue origin. This inclusive approach allowed us to confidently\nidentify a group of metabolic enzymes reliably present on chromatin (Table 3).\nThat said, we fully acknowledge that this design is not optimal for capturing condition-\nspecific or tissue-specific differences in nuclear metabolism, particularly when comparing\nhealthy versus cancer states. In response to this valuable suggestion, we have now\nconducted a focused analysis of OXPHOS-related enzymes by comparing breast and\nlung cancer cell lines. Notably, we observed that breast cancer cell lines display\nsignificantly higher nuclear enrichment of OXPHOS enzymes (Figure 3), despite showing\ncomparable expression and cytoplasmic protein levels to lung cancer cell lines (Figure\nS3 C- D and S4).\nTo further support this finding, we extended our investigation to human tissue samples,\nboth healthy and cancerous breast and lung tissues, examining OXPHOS nuclear\nlocalization across epithelial and stromal compartments (Figure 4 and S5-6). While the\nfunctional significance of OXPHOS enzymes in the nucleus remains to be fully\nelucidated, our data suggest that tissue type and disease state may influence their\nchromatin association, underscoring the potential biological relevance of this\nobservation.\nReviewer #2 (Remarks on code availability):\nThe web page is displayed as not found.\nWe sincerely apologize for the inconvenience. Although we had communicated the\ncorrect link to the editor during the revision process, we understand that it may not have\nbeen received on time. We have now provided a revised version of the link, which we\nhope will work as intended.\nReviewer #3 (Remarks to the Author):\nThis manuscript presents a proteomics resource defining chromatin-associated\nmetabolism-related proteins across human healthy samples and cancer cells. The\nstrategy used to prepare chromatin fractions is well done, and the datasets generated\nwill be valuable to the scientific community. The study makes the interesting and notable\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\npoint that many metabolism-associated proteins are found in chromatin across many\nsample types. The primary utility of this type of study in my view is in making the datasets\neasily accessible and usable by other researchers, and improvements are needed in this\nregard.\n1. I found it difficult to mine the data to understand which specific proteins are regulated\nin each setting, which is a major aspect of the utility of the study. Some suggestions:\na. Table 3 lists the “core-chromatome” chromatin-associated proteins detected across\nsamples. In addition to the Uniprot designations, it would be much appreciated to also\ninclude the protein names so that one can more readily query the table.\nb.The link to the datasets listed under Data Availability did not work for me. I appreciate\nthat the authors provided an updated link during the review process, which should\nreplace the prior link.\nc. Could the various Data files also be updated to include as much information as possible about each dataset so that the\ndata can be readily understood? For example, add titles to explain what is presented in each dataset, indicate what the numerical values\nmean (e.g., z scores, etc), and so on. Basically, make the data user-friendly. They could\nalso consider making a web-based portal to query the datasets, though I wouldn’t require\nthis.\nd.Fig 2E and F. With the current presentation, I find it difficult to glean useful information,\nsince the tissue samples don’t necessarily cluster together. If the point is to understand\nhow pathways are regulated within each type of sample, it might be more useful to\npresent replicate tissue samples next to one another and cancer samples from each\ntissue next to the corresponding normal tissue samples. That way the reader could at\nleast understand the overall patterns within each tissue. It should also be made clear in\nthe legend what is “Relative abundance”. Is this a z score?\nWe sincerely thank the reviewer for their thorough and very helpful suggestions. We have\nnow incorporated this information into our tables and datasets, and we hope these\nrevisions make the content more accessible and easier to interpret. Making our work\nunderstandable to a broad scientific audience is one of our key goals, and we are truly\ngrateful for the reviewer’s insightful comments that helped us move closer to that\nobjective. Moreover, while we did not modify Figure 2E, we reorganized the heatmap in\nFigure 2F to cluster samples by tissue type, to highlight the emergence of tissue-specific\npatterns of chromatin-associated metabolic pathways across different samples.\n2. More validation of the patterns in chromatin-associated proteins identified by\nproteomics should be conducted, using IF and/or biochemical fractionation and western\nblotting. It would be nice to see careful validation of examples of proteins shown to be\nchromatin-associated across sample types and those that are chromatin-associated in\nspecific sample types. For example, validating the OXPHOS proteins in skin or brain vs\nlung cells would be appropriate.\nWe sincerely thank the reviewer for their thoughtful and encouraging feedback. As the\nreviewer noted, our study was conceived primarily as a resource-generating effort,\naiming to comprehensively chart the chromatin-bound metabolic proteome across a wide\nrange of healthy and cancerous human cell types and tissues. To ensure this resource\nwould be broadly useful and reliable, our initial focus was on generating a robust, high-\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\ncoverage dataset with strong reproducibility, rather than pursuing detailed mechanistic\ndissection for individual enzymes.\nIn response to the reviewer’s suggestions, and in line with their emphasis on utility and\ninterpretability, we have substantially expanded the revised manuscript to include\nadditional validation and context-specific analysis. We performed Western blotting on\nchromatin-enriched fractions (Figure S3D and S7E) and conducted high-throughput\nimmunofluorescence (HT-IF) in both cell lines (Figure 3 and Figure S4) and human tissue\nsamples spanning healthy and malignant conditions (Figure 4 and Figures S5–S6).\nThese experiments confirm the nuclear localization of selected metabolic enzymes and\nreinforce the reliability of the proteomic profiles presented.\nNotably, we identified tissue-specific differences in chromatin localization patterns—for\nexample, COX4 displayed striking nucleolar enrichment in breast cancer samples (both\ncell lines and patient-derived tissues), a pattern not observed in lung cancer or healthy\nbreast tissue. This subnuclear localization was independently supported by\nimmunofluorescence data from the Human Protein Atlas (HPA), further validating the\nphysiological relevance of our findings. In addition, our spatial analysis revealed distinct\nlocalization profiles between epithelial and stromal compartments, as well as between\nnormal and tumor tissues, suggesting potential roles in tissue-specific chromatin\nregulation or disease-associated nuclear remodeling.\nTo explore functional relevance, we also analyzed the nuclear dynamics of folate and\npurine biosynthesis enzymes following ionizing radiation. These enzymes showed time-\ndependent nuclear relocalization that paralleled DNA damage response kinetics (Figure\n6 and Figure S8), consistent with regulated chromatin engagement under stress. These\nfindings complement our recent work demonstrating the nuclear function of IMPDH2 in\nmodulating PARP1 activity during DNA repair (Espinar et al., Nat. Commun.\n2024, https://doi.org/10.1038/s41467-024-53877-z).\nAltogether, the additional experimental validations and targeted functional insights now\nincluded in the revised manuscript significantly enhance the depth, robustness, and\ninterpretability of the resource. We hope this strengthened dataset will prove valuable to\nthe community and provide a solid foundation for future investigations into the nuclear\nroles of metabolic enzymes.\nMinor\n- Fig 2A. Legend needs info on IF data. Is it from HPA? Or their own data?\nThank you for pointing out this inaccuracy. We have now corrected it accordingly.\n-The 300mM of salt in the nuclear extraction buffer allows the investigators to be rigorous\nin defining chromatin-associated proteins- but they might be losing some proteins that\nare less strongly bound, which many metabolic enzymes may be. This caveat could be\nmentioned in the discussion.\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\nWe thank the reviewer for raising this important point, which has also been a topic of\nextensive internal discussion within our lab. As the reviewer correctly noted, the chosen\nsalt concentration was selected to enrich for relatively strong and stable chromatin\ninteractions. While we acknowledge that this approach may exclude some more transient\nor weaker associations, potentially leading to the loss of certain metabolic enzymes, it is\nalso true that our protocol successfully retrieved over 200 metabolic enzymes,\nsuggesting that a substantial number of these proteins are robustly associated with\nchromatin.\nWe agree that the protocol could be further optimized, not only by adjusting salt\nconcentrations, but also by incorporating additional components, such as RNase\ntreatment, to distinguish RNA-dependent from RNA-independent chromatin interactions.\nHowever, we believe that our current methodology provides a comprehensive and\nreproducible starting point for the systematic study of nuclear metabolism, establishing\na valuable resource for the field. In response to the reviewer’s suggestion, we have\nadded these insights to the Discussion.\nReviewer #3 (Remarks on code availability):\nThis is not my skill set. Hopefully you had other reviewers who could review the code.\nC/ Dr. Aiguader, 88\n08003 Barcelona\nT +34 316 01 00\nwww.crg.es\nWe thank Reviewers 2 and 3 for their positive evaluation of our work and for recommending\nits acceptance.\nWe have carefully considered the comments raised by Reviewer 1 and have revised the\nmanuscript accordingly. We hope that the additional clarifications and analyses provided\nwill satisfactorily address the concerns and demonstrate the robustness of our findings."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Although the revised manuscript contains more detailed resource and includes additional cellular validation for selected\ncandidate enzymes, it still fall short of providing comprehensive experimental validation to substantiate their notions.\nThe study would be significantly strengthened by extending validation beyond localization and include molecular, cellular,\nand functional assessments of key metabolic enzymes. Specifically, rather than focusing solely on the spatial profiling of a\nsubset of candidates, the authors should demonstrate the functional consequences and biological significance of nuclear\nversus cytoplasmic localization for these enzymes.\nALso, the authors have previously published work (PMID: 39532854 and PMID: 39532843) that functionally validates the\nnuclear roles of certain metabolic enzymes. However, the integration of these prior findings and conceptually advancing the\ncurrent manuscript is limited and insufficient.", "reviewer_2": "In the revised manuscript the authors carefully addressed the raised questions and concerns. The\nmanuscript contains now all information and can be accepted for publication.", "reviewer_3": "The authors have done a good job in addressing my comments. One additional minor point is that the colors used for blood,\nbreast, and liver are very close shades of green and I couldn’t tell which was which in the figures. I recommend choosing\ncolors more distinguishable to the eye. Otherwise, this is a valuable resource for the scientific community and I recommend\nacceptance."}, "author_response": "Reviewer #1 (Remarks to the Author):\nAlthough the revised manuscript contains more detailed resource and includes additional\ncellular validation for selected candidate enzymes, it still fall short of providing\ncomprehensive experimental validation to substantiate their notions.\nThe study would be significantly strengthened by extending validation beyond localization\nand include molecular, cellular, and functional assessments of key metabolic enzymes.\nSpecifically, rather than focusing solely on the spatial profiling of a subset of candidates, the\nauthors should demonstrate the functional consequences and biological significance of\nnuclear versus cytoplasmic localization for these enzymes.\nALso, the authors have previously published work (PMID: 39532854 and PMID: 39532843)\nthat functionally validates the nuclear roles of certain metabolic enzymes. However, the\nintegration of these prior findings and conceptually advancing the current manuscript is\nlimited and insufficient.\nWe thank the reviewer for their constructive comments. While we fully agree that\nunderstanding the molecular and functional roles of nuclear metabolic enzymes is of great\nbiological interest, a comprehensive mechanistic dissection of each individual enzyme\nnecessarily exceeds the scope of this study. The goal of this work is to provide the first\nsystematic atlas of chromatin-associated metabolic enzymes, revealing that more than 200\nsuch enzymes localize to chromatin, a finding that substantially broadens our understanding\nof nuclear metabolism, previously limited to a few dozen examples. This resource\nestablishes the conceptual and experimental framework upon which targeted mechanistic\nstudies can now be built.\nWe emphasize that this manuscript already goes beyond a purely resource-oriented study\nby including functional and mechanistic validation of selected enzymes. Following the\nreviewer’s suggestion, we have now further extended our analyses by focusing on IMPDH2,\none of the key chromatin-associated metabolic enzymes identified in our atlas. Using\nIMPDH2 knockout cells reconstituted with wild-type, nuclear-enriched (NLS), or nuclear-\nexcluded (NES) forms, and including guanosine supplementation to distinguish enzymatic\nactivity from metabolite availability, we found that NLS and NES variants drive opposite\ntranscriptional programs compared to wild-type IMPDH2. These results reveal distinct\npathway alterations, including BRCA1 degradation and ERK1/2 signaling, demonstrating that\nenzyme compartmentalization has clear transcriptional and functional consequences.\nTogether with our previously published mechanistic studies on nuclear enzymes (PMID:\n37259925, 37937685, 39532854 and 39532843), these new data provide strong evidence\nthat enzyme compartmentalization is functionally relevant. A comprehensive mechanistic\ndissection of each of the >200 enzymes identified here would represent an independent line\nof research, better suited for future focused studies that can now directly build upon the\natlas established in this work.\nReviewer #2 (Remarks to the Author):\nIn the revised manuscript the authors carefully addressed the raised questions and concerns.\nThe\nmanuscript contains now all information and can be accepted for publication.\nWe sincerely thank the reviewer for their positive appreciation of our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors have done a good job in addressing my comments. One additional minor point\nis that the colors used for blood, breast, and liver are very close shades of green and I\ncouldn’t tell which was which in the figures. I recommend choosing colors more\ndistinguishable to the eye. Otherwise, this is a valuable resource for the scientific community\nand I recommend acceptance.\nWe sincerely thank the reviewer for their positive appreciation of our manuscript."}]

Decision letter