Abstract
All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.
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Data availability
NSG data generated for this study are available at the Gene Expression Omnibus (GEO) under accession number GSE214019. The histone mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD043952. Source data are provided with this paper. All other reagents and material supporting the findings of this study are available from the corresponding author on reasonable request.
Code availability
The source code of Ceto pipeline and custom scripts used for analysing the next-generation sequencing data from this study is available at the GitHub site (https://github.com/ebartom/NGSbartom and https://github.com/ebartom/Shapiroetal2023).
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Acknowledgements
We thank H. Harris, M. Shang, C. Chen, Y. Chen, X. Jiang and K. Xu for their technical help, managing our mouse colony and genotyping the mice used in this study. We also thank C. J. Payne for his sound advice and providing initial aliquots of histone methylation antibodies to run preliminary experiments. We thank Y. Shah for providing us with reagents to perform the FPN overexpression studies. We thank E. Kiskinis and M. Gharib for generously providing us with hIPS neurons. We thank P. Burridge for providing us with hIPS cells for differentiation into CMs. We thank N. A. Lack for generously providing us the KDM3B plasmids. Metal analysis was performed at the Northwestern University Quantitative Bio-element Imaging Center generously supported by the National Research Resource for Quantitative Mapping in the Life Sciences (QE-Map) award number P41GM135018. Metabolomics services were performed by the Metabolomics Core Facility at Robert H. Lurie Comprehensive Cancer Center of Northwestern University. Proteomics services were performed by Y. Ah Goo, in conjunction with N. L. Kelleher at the Northwestern Proteomics Core Facility, generously supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center, instrumentation award (S10OD025194) from NIH Office of Director, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569. We thank M. J. Schipma, and B. Wray at the Northwestern University Sequencing Core and P. A. Ozark for their contribution to the ChIP–seq analyses. This work was supported through core services and support from the Northwestern University George M. O’Brien Kidney Research Core Center (NU GoKidney), an NIH/NIDDK-funded programme (P30 DK114857). This work was supported by the Northwestern University–Flow Cytometry Core Facility supported by Cancer Center Support Grant (NCI CA060553). Flow Cytometry Cell Sorting was performed on a BD FACSAria SORP system and BD FACSymphony S6 SORP system, purchased through the support of NIH 1S10OD011996-01 and 1S10OD026814-01. Additional funding for this work was provided through NRSA 5F30DK109608-02, and T32-DK007169 awarded to J.S.S., NHLBI HL127646, HL140973 and HL138982 awarded to H.A., U54CA193419 and R01GM038784 awarded to T.V.O., and the Spanish Ministry of Science, Innovation and Universities (MICINN) grant BIO2017-87828-C2-1-P and FEDER funds to S.P. Z.S.W. was supported in part by the National Institutes of Health Training Grant T32GM008449 through Northwestern University’s Biotechnology Training Program.
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Conceptualization: J.S.S., H.-C.C. and H.A.; methodology: J.S.S., H.-C.C., Z.Z., B.J.H., Z.S.W., H.C., E.T.B., S.P., S.-C.J., S.K.S.-T., L.L., I.B.-S., T.V.O., J.B. and P.K.; investigation: J.S.S., H.-C.C., Z.Z., Y.T., B.J.H., Z.S.W., H.C., J.A.G., E.T.B., A.D.J., Z.S., F.K.N., A.M., T.S., L.R.-A., A.M.R., M.T.M.-P., S.-C.J., S.K.S.-T. and G.L.; resources: S.P., L.L., D.B., G.L., I.B.-S., T.V.O., A.S., J.B., P.K. and H.A.; writing—original draft: J.S.S. and H.A.; writing—review and editing: all authors; visualization: J.S.S. and J.A.G.; supervision: H.A.; funding acquisition: J.S.S., S.P., T.V.O. and H.A.
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Extended data
Extended Data Fig. 1 Long-term ID inactivates mTORC1 in multiple cell types.
(A) Non-heme iron levels in HEK293T cells treated with 150 µM DFO. (n = 5 replicates, two-way unpaired t-test, mean ± s.e.m.). (B) mRNA levels of TTP and TFRC at indicated time points in HEK293T cells treated with 150 µM DFO. Internal control: POLR2A (n = 6 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (C) Immunoblot of mTORC1 activity in HEK293T cells treated with indicated concentrations of DFO. Representative image of two independent experiments (D) Immunoblot of mTORC1 activity in HEK293T cells treated with 150 µM DFO or 50 µM BPD for 3 hours. Representative image of two independent experiments. (E) mRNA levels of indicated genes 3 hours after addition of 150 µM DFO or 50 µM BPD. Internal control: POLR2A (n = 8 TFRC Control; n = 7 TFRC 150 µM DFO and 50 µM BPD; n = 4 REDD1; n = 8 TTP replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (F) Immunoblot of mTORC1 activity in response to 250 nM Torin1 at indicated time points. Representative image of two independent experiments. (G) Immunoblot of mTORC1 activity in primary murine hepatocytes treated with increasing concentrations of DFO. Representative image of two independent experiments (H) mRNA of Ttp and Tfrc in primary murine hepatocytes treated with indicated concentrations of DFO. Internal control: Polr2a (n = 3 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (I) Immunoblot of mTORC1 activity in hPS-CM treated with 150 µM DFO. Representative image of one experiment (J) Immunoblot of mTORC1 activity in hiPS-neurons treated with 150 µM DFO. Representative image of one experiment. (K) Immunoblot of HEK293T cells chelated for 18 hours followed by addition of ferric ammonium citrate (FAC) for indicated times. Representative image of two independent experiments. (L) BrdU incorporation in HEK293T cells treated with 150 µM DFO. Representative image of six independent samples. (M) Quantification of cellular proliferation by Hoescht staining 48 hours after treatment with DFO (n = 6 replicates, two-way unpaired t-test, mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels. Source numerical data and unprocessed blots are available in source data files.
Extended Data Fig. 2 Non-pharmacologic means of inducing ID inactivates mTORC1.
(A) Immunoblot of mTORC1 activity in HEK293T cells treated with high Tf-sat (66%) or low Tf-sat (6.6%) media for 18 hours. Representative image of two independent experiments. (B) Summary of immunoblot in panel A (n = 3 replicates, two-way unpaired t-test, mean ± s.e.m.). (C) Immunoblot of mTORC1 activity in HepG2 cells transfected with the FPN-GFP fusion protein and TET inducible rtTA3 plasmids in the presence and absence of 500 ng/ml doxycycline for 48 hours. Representative image of two independent experiments. (D) Summary of immunoblot in panel C (n = 3 replicates, two-way unpaired t-test, mean ± s.e.m.). (E) Fluorescent microscopy of cells transfected with FPN-GFP construct and treated with 500 ng/ml doxycycline for 24 hours demonstrating appropriate expression and localization of the FPN-GFP fusion protein. Representative image of three independent samples. (F) Immunoblot of puromycin incorporation in rtTA3/FPN-GFP stable HEK293T cells in the presence and absence of 500 ng/ml doxycycline for 48 hours. Representative image of one experiment. (G) mRNA expression of ER stress makers CHOP and BNIP3 in HepG2 cells transfected with rtTA3/FPN-GFP plasmids and treated with 500 ng/ml doxycycline for 48 hours. Internal control: 18S (n = 4 replicates, two-way unpaired t-test, mean ± s.e.m.). (H) Immunoblot of protein levels of the key components of the mTORC1 complex after 24 hours of 150 µM DFO in HEK293T cells. Representative image of two independent experiments. (I) Summary of results shown in panel A (n = 4 replicates, two-way unpaired t-test, mean ± s.e.m.). (J) Immunoblot of total and phosphorylated TSC2, AKT, ERK, GSK3β, and S6 proteins and total P53 at different time points after treatment with 150 µM of DFO in HEK293T cells. Representative image of one experiment. (K) Immunoblot of mTORC1 activity and mitochondrial function in HepG2 cells treated with DFO for 18 hours at the indicated doses. Representative image of one experiment. (L) Oxygen Consumption Rate (OCR) measured by the Seahorse Assay in HepG2 cells treated for 24 hours of DFO at the indicated doses (n = 10 replicates per group, mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels.
Extended Data Fig. 3 Leucine sensing is required for mTORC1 inactivation by ID.
(A) Ttp mRNA in WT and Tsc2 KO MEFs treated with 150 µM DFO. Internal control: Snrk (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (B) Immunoblot of mTORC1 activity in WT and Tsc2 KO MEFs with indicated treatments. Representative image of three independent experiments. (C) REDD1 mRNA in HEK293T cells treated with siREDD1. Internal control: POL2RA (n = 6 replicates per condition, two-way unpaired t-test, mean ± s.e.m.). (D) TFRC mRNA in HEK293T cells treated with siREDD1 and 150 µM DFO. Internal control: POL2RA (n = 6 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (E) Immunoblot of mTORC1 and AMPK activity with indicated treatments. Representative image of one experiment (F) Ttp mRNA in WT and Ampkα1/2 dKO cells treated with 150 µM DFO. Internal control: Polr2a (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (G) Acridine orange staining in HEK293T cells treated with 150 µM DFO. Representative image of six independent samples. (H) Summary of the results in Panel G (n = 7 replicates control; n = 6 150 µM DFO, two-way unpaired t-test, mean ± s.e.m.). (I) Leucine levels in HEK293T cells treated with 150 µM DFO (n = 5 replicates control; n = 4 150 µM DFO two-way unpaired t-test, median ± quartiles). (J) SAM levels in HEK293T cells treated with 150 µM DFO (n = 5 replicates control; n = 4 150 µM DFO, two-way unpaired t-test, median ± quartiles). (K) Immunoblot of mTORC1 activity in WT and NPRL2 KO HEK293T cells with indicated treatments. Representative image of one experiment. (L) mTORC1 localization to lysosomes in NPRL2 KO cells treated with 150 µM DFO. Representative image of six independent samples. (M) Quantification of images in panel L. (n = 5 replicates control; n = 6 150 µM DFO, two-way unpaired t-test, mean ± s.e.m.) (N) Cell death using Hoescht and propidium iodide (PI) in WT and NPRL2 KO HEK293T cells treated with 150 µM DFO for indicated times. Representative image of six independent samples. (O) Quantification of images in panel N. (n = 6 replicates, two-way unpaired t-test, mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels.
Extended Data Fig. 4 ID increases H3K9 di-methylation independent of ATF4, the IRP system, and 2-HG.
(A) Immunoblot of H3K9me2 in hiPS-CM treated with 150 µM DFO. Representative image of one experiment. (B) Immunoblot of H3K9me2 in hiPS-Neurons treated with 150 µM DFO. Representative image of one experiment. (C) Immunoblot of H3K9me2 levels in MEFs with indicated treatments. Representative image of two independent experiments. (D, E) Summary of immunoblot in panel C (n = 3 replicates, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (F) Immunoblot of H3K9me2 in HepG2 cells expressing rtTA3/GFP-FPN with indicated treatments. Representative image of two independent experiments. (G) mRNA of indicated genes in WT and Arnt KO MEFs treated with 150 µM DFO. Internal control: 18S (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (H) Immunoblot of H3K9me2 levels in WT and Atf4 KO MEFs with indicated treatments. Representative image of one experiment. (I) Summary of immunoblot in panel H (n = 3 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (J) Immunoblot of H3K9me2 levels in WT and Irp1/2 KD/KO MEFs with indicated treatments. Representative image of two independent experiments. (K) Summary of immunoblot in panel J (n = 3 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (L) Immunoblot of IRP1 levels in Irp2 KO MEFs treated with indicated siRNA. Representative image of one experiment. (M) Summary of immunoblot in panel L (n = 3 replicates per condition, two-way unpaired t-test, mean ± s.e.m.). (N) mRNA of indicated genes in WT and Irp1/2 KD/KO MEFs treated with 150 µM DFO. Internal control: Snrk (n = 6 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (O) Ratio of 2-HG/succinate levels in HEK293T cells treated 150 µM DFO (n = 5 replicates control; n = 4 150 µM DFO, two-way unpaired t-test, median ± quartiles). (P) Immunoblot of H3K9me2 in HEK293T cells with indicated treatments. Representative image of one experiment. (Q) Immunoblot of H3K9me2 in HEK293T cells with indicated treatments. Representative image of one experiment. * indicates P value < 0.05 when noted for all panels.
Extended Data Fig. 5 ID alters occupancy of POLR2A at the promoters of genes involved in metabolic pathways.
(A) Fold change in POLR2A occupancy within predefined regions of the promoter and gene body to categorize genes defined by increased POLR2A binding, POLR2A loss, and promoter-pausing. (B) Gene-ontology (GO) terms of genes enriched in the increased POLR2A group after ID. (C) GO terms of genes that had decreased POLR2A after ID. (D) UCSC genome browser tracks for the LAT3 (top) and PAT1 (bottom) gene loci. POLR2A and H3K9me2 tracks from ChIP-seq analysis were loaded and represented as the difference in normalized reads between the DFO and control groups. Regions of H3K9me2 enrichment in the DFO group are underlined in red. Direction of transcription is indicated by a black arrow. Encode Histone (LAT3 and PAT1) and Genehancer (PAT1) browser tracks are displayed beneath and represent predicted enhancer regions which align with regions of increased H3K9me2 signal in response to DFO. Yellow bar, red and grey arrows indicate enhancer regions designated by Encode Histone and Genehancer browser tracks.
Extended Data Fig. 6 ID increases H3K9 di-methylation within the promoter of RPTOR and correlates with decreased RPTOR expression.
(A) H3K9me2 signal and POL2RA occupancy in the promoter for RPTOR from ChIP-Seq analysis. (B) ChIP-PCR of RPTOR in HEK293T cells. Cells treated with 150 µM DFO or 250 µM IOX1 for 12 hours and vehicle controls were followed by IP of lysates using an antibody against H3K9me2. IgG was used as a negative control for the IP (n = 2 replicates). (C) RPTOR mRNA levels at indicated time points after the addition of 150 µM DFO. Samples matched with Fig. 4I. Internal control: POLR2A (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (D) Immunoblot of RPTOR, mTOR and HIF1-α levels in HEK293T cells treated with 100 µg/ml cycloheximide (CHX) for indicated time points. HIF1-α, which is rapidly turned over under normoxic conditions via the actions of the EGLN and Von-Hippel Lindau (VHL) proteins, was used as a positive control. Representative image of one experiment. (E) Immunoblot of mTORC1 activity and complex member RAPTOR in A549 cells after treatment with 150 µM DFO for 48 hours. Representative image of two independent experiments. (F) Immunoblot of mTORC1 activity and complex member RAPTOR in patient-derived primary tumor cell cultures treated with 150 µM DFO. Representative image of one experiment. (G) Quantification of immunoblot in panel (F) (n = 3 replicates, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (H) Immunoblot of lysates from cells transfected with FLAG-RAP2A or FLAG-mTOR and treated with or without 150 µM DFO for 48 hours. Immunoprecipitation was performed with anti-FLAG antibody. RAP2A = negative control. (I) Summary of IP studies from panel (H) (n = 3 replicates, two-way unpaired t-test, mean ± s.e.m.). Representative image of two independent experiments (J) Immunoblot of mTORC1 activity in WT and NPRL2 KO HEK293T treated with and without 150 µM DFO for 48 hours. Representative image of one experiment. (K) Quantification of immunoblot in panel (J) (n = 3 replicates, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels.
Extended Data Fig. 7 The Jumonji-C KDM family inhibitor IOX1 mimics the actions of ID on mTORC1 activity.
(A) RT-PCR of AA transporters, RPTOR and TTP in HEK293T cells treated with various Jmj-C domain inhibitors for 12 hours. Internal control: POLR2A (n = 4 replicates per condition; except n = 3 TTP 1 mM DMOG, two-way unpaired t-test, mean ± s.e.m.). (B) Immunoblot of mTORC1 activity and H3K9me2 levels in A549 cells treated with 150 µM DFO or 250 µM IOX1 for 18hrs. Representative image of two independent experiments. (C) Immunoblot of indicated proteins in A549 cells treated with 250 µM IOX1 for 48 hours. Representative image of two independent experiments. (D) Immunoblot of cytosol and membrane fractions from HepG2 cells treated with 250 µM IOX1 for 12 hours. Representative image of one experiment. (E) Densitometry summary of data in Panel D (n = 3 replicates per condition, two-way unpaired t-test, mean ± s.e.m.). (F) 14C-leucine uptake into HEK293T cells treated with 150 µM DFO or 100 µM IOX1 for 18 hours (n = 5 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (G) 14C-leucine uptake into HeLa cells treated with 150 µM DFO or 100 µM IOX1 for 18 hours (n = 4 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). CPM = counts per minute. (H) Immunoblot of mTORC1 activity WT and NPRL2 KO HEK293T cells treated with 250 µM IOX1 for 18 hours. Representative image of two independent experiments (I) Quantification of immunoblot in panel H (n = 3 replicates per group, one-way ANOVA with Tukey’s post-hoc test. mean ± s.e.m.). * indicates P value < 0.05 when noted for all panels.
Extended Data Fig. 8 H3K9 di-methyl ChIP-seq signal in iron chelated samples correlate with changes after loss of KDM3A and KDM3B.
(A) Heatmap of the log2FC in H3K9me2 signal between DFO treatment and control plotted against KDM3A and KDM3B occupancy. K-means cluster =3. (B) Browser tracks with KDM3B and H3K9me2 track data from HCT116 cells treated with control shRNA (shC) or shRNA targeting KDM3A and KDM3B (sh3A3B). Track data for KDM3A was downloaded from the GEO database accession GSE127624. Track data for KDM3B and H3K9me2 from HCT116 cells were downloaded from the GEO database accession GSE71885. (C) Hierarchical clustering and correlation analysis between indicated samples using deepTools plotCorrelation. Values indicate the Pearson correlation between the sample listed in the corresponding row and column. (D) Immunoblot showing deletion of KDM4B and KDM4C, using CRISPR-Cas technology in HEK293T cells. Representative image of one experiment. (E) Immunoblot of mTORC1 activity and H3K9me2 in Cas9 (WT) and KDM3A, KDM4B or KDM4C KO HEK293T cells in the presence and absence of 150 µM DFO for 18 hours. Representative image of one experiment. (F) Immunoblot of mTORC1 activity and H3K9me2 in HEK293T cells with overexpression of HA-tagged KDM4A, KDM4B or combination of the two proteins treated with 150 µM DFO for 18 hours. Representative image of one experiment.
Extended Data Fig. 9 KDM3B KO cells do not repress LAT3/PAT1 expression or mTORC1 activity, and have increased cell death during ID.
(A) Immunoblot of mTORC1 activity and H3K9me2 in Cas9 (WT) and sg.3B c.7 (KDM3B KO) TSC2 KO HeLa cells in the presence and absence of 150 µM DFO for 18 hours. Representative image of one experiment. (B) Summary of immunoblot in panel A (n = 3 replicates per group, one-way ANOVA with Tukey’s post-hoc test. Mean ± s.e.m.). (C) Assessment of mTORC1 on the lysosome in KDM3B KO HepG2 cells treated with 150 µM DFO for 18 hours. Representative image of five independent samples. (D) Quantification of images in panel C. (n = 5 replicates per condition, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (E) Immunoblot of LAT3 protein in KDM3B KO HepG2 cells treated with DFO for 18 hours. Representative image of one experiment. (F) Immunoblot of PAT1 protein in KDM3B KO HepG2 cells treated with DFO for 18 hours. Representative image of one experiment. (G) Fluorescent microscopy of cell death using Hoescht and propidium iodide (PI) double staining in KDM3B KO HEK293T cells treated with 150 µM DFO for 0, 24, 36 and 60 hours. Representative image of six independent samples. (H) Quantification of images in panel G. (n = 6 replicates, one-way ANOVA and Tukey’s post-hoc test, mean ± s.e.m.). (I) SDS-PAGE gel of purified N-terminal FLAG-tagged WT KDM6A and KDM6AMT/ED mutant expressed using a baculoviral overexpression system. Representative image of three independent experiments. (J, K) Enzyme kinetics of WT KDM6A (J) and KDM6AMT/ED (K) in the presence of increasing concentrations of iron. (L) Enzyme kinetics of KDM3B in the presence of increasing concentrations of R-2HG. dpm = disintegration per minute. (M) Immunoblot of mTORC1 activity in HEK293T cells after treatment with 150 µM DFO or increasing concentrations of octyl-R-2HG. Representative image of one experiment. * indicates P value < 0.05 when noted for all panels.
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Source data table for extended data figures.
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Shapiro, J.S., Chang, HC., Tatekoshi, Y. et al. Iron drives anabolic metabolism through active histone demethylation and mTORC1. Nat Cell Biol 25, 1478–1494 (2023). https://doi.org/10.1038/s41556-023-01225-6
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DOI: https://doi.org/10.1038/s41556-023-01225-6
