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Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes

Nature Cell Biology volume 25, pages 258–272 (2023)Cite this article

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Abstract

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR–Cas9 screens, we identify specific roles for MTF2–PRC2.1, PCGF1–PRC1.1 and Menin–KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin–KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

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Fig. 1: Genome-scale CRISPR–Cas9 screens identify specific polycomb and KMT2 complex components regulating bivalent gene activation.
Fig. 2: PRC1.1 and PRC2.1 cooperate to restrict the activation of bivalent genes.
Fig. 3: Targeting Menin drives derepression of bivalent genes.
Fig. 4: Pharmacological inhibitors targeting the Menin–KMT2A/B interaction drive derepression of bivalent MHC-I genes in MHC-Ilow cancer cells and enhance T cell-mediated tumour killing.
Fig. 5: Targeting Menin alleviates polycomb-mediated repression of bivalent genes.
Fig. 6: Displacement of Menin from distant genomic loci activates bivalent gene expression.
Fig. 7: Opposing functions of KMT2A/B and Menin in the regulation of bivalent gene expression.
Fig. 8: Transcription factor binding bypasses the requirement of KMT2A/B for bivalent gene activation.

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Data availability

ChIP–seq, RNA-seq, CUT&Tag and CUT&RUN data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE181829. ChIP–seq data from the hESC H9 line were used from GEO accession nos GSE96336 and GSE96353, EZH2-null H9 hESC RNAs-seq data were from GEO accession no. GSE76626 and human induced pluripotent stem cell line iPS-20b ChIP–seq data from GEO accession nos GSM772844 and GSM772847. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank the Peter MacCallum Cancer Centre Molecular Genomics Core and the flow cytometry facility. We thank the following funders for fellowship, scholarship and grant support: Snow Medical Research Foundation Fellowship (M.L.B. and M.E.-M.), Cancer Research UK Clinician Scientist Fellowship C53779/A20097 and NHMRC Investigator Grant 1196598 (M.L.B.), Sir Edward Dunlop Fellowship, Cancer Council of Victoria, NHMRC Investigator Grant 1196749 and Howard Hughes Medical Institute International Research Scholarship 55008729 (M.A.D.), CSL Centenary Fellowship and NHMRC Investigator Grant 1196755 (S.-J.D.), Peter and Julie Alston Centenary fellowship (K.D.S.), Wellcome Trust Principal Research Fellowship 101835/Z/13/Z (P.J.L.), Peter MacCallum Postgraduate Scholarship (C.E.S.), NHMRC Postgraduate Scholarship (K.L.C.), Maddie Riewoldt’s Vision 064728 (Y.-C.C.), Victorian Cancer Agency (E.Y.N.L.), VCA Mid-Career Fellowship MCRF19033 (D.J.G.), CSL Centenary Fellowship (S.-J.D.) and NHMRC grants 1164054 and 2010275 (M.L.B.), 1085015 and 1106444 (M.A.D.), and 1128984 (M.A.D. and S.-J.D.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Schematics in Fig. 1a,d, Fig. 4b and Extended Data Fig. 10a were created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Marian L. Burr, Mark A. Dawson.

Authors and Affiliations

  1. Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

    Christina E. Sparbier, Andrea Gillespie, Juliana Gomez, Nishi Kumari, Ali Motazedian, Kah Lok Chan, Charles C. Bell, Omer Gilan, Yih-Chih Chan, Melanie A. Eckersley-Maslin, Sarah-Jane Dawson, Enid Y. N. Lam, Marian L. Burr & Mark A. Dawson

  2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia

    Christina E. Sparbier, Ali Motazedian, Kah Lok Chan, Charles C. Bell, Yih-Chih Chan, Melanie A. Eckersley-Maslin, Sarah-Jane Dawson, Enid Y. N. Lam, Marian L. Burr & Mark A. Dawson

  3. The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia

    Juliana Gomez, Sarah Popp & Marian L. Burr

  4. Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Victoria, Australia

    Kah Lok Chan & Mark A. Dawson

  5. Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia

    Omer Gilan

  6. Department of Molecular Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia

    Daniel J. Gough

  7. Centre for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria, Australia

    Daniel J. Gough

  8. Department of Anatomy and Physiology, University of Melbourne, Melbourne, Victoria, Australia

    Melanie A. Eckersley-Maslin

  9. Centre for Cancer Research, University of Melbourne, Melbourne, Victoria, Australia

    Sarah-Jane Dawson & Mark A. Dawson

  10. Cambridge Institute of Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, Cambridge, UK

    Paul J. Lehner

  11. ACRF Cancer Biology and Stem Cells Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Kate D. Sutherland

  12. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

    Kate D. Sutherland

  13. Section of Hematology, Oncology and Bone Marrow Transplant, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Patricia Ernst

  14. Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Patricia Ernst

  15. Syndax Pharmaceuticals, Waltham, MA, USA

    Gerard M. McGeehan

  16. Department of Anatomical Pathology, ACT Pathology, Canberra Health Services, Canberra, Australian Capital Territory, Australia

    Marian L. Burr

Authors
  1. Christina E. Sparbier
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  2. Andrea Gillespie
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  3. Juliana Gomez
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  4. Nishi Kumari
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  5. Ali Motazedian
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  6. Kah Lok Chan
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  7. Charles C. Bell
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  8. Omer Gilan
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  9. Yih-Chih Chan
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  10. Sarah Popp
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  11. Daniel J. Gough
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  12. Melanie A. Eckersley-Maslin
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  13. Sarah-Jane Dawson
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  14. Paul J. Lehner
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  15. Kate D. Sutherland
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  16. Patricia Ernst
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  17. Gerard M. McGeehan
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  18. Enid Y. N. Lam
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  19. Marian L. Burr
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  20. Mark A. Dawson
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Contributions

M.L.B. and M.A.D. conceived, designed and supervised the research and wrote the manuscript. C.E.S. designed the research, conducted experiments, analysed data and helped write the manuscript. J.G., N.K., K.L.C., A.M., C.C.B., O.G. and S.P. conducted experiments, analysed data and provided expertise. C.E.S. conducted the CRISPR screens. A.G. and E.Y.N.L. led the analysis of the genomic data and CRISPR screens with contributions from Y.-C.C. K.D.S., D.J.G., M.A.E.-M., S.-J.D., P.J.L., P.E. and G.M.M. provided critical expertise and/or reagents and contributed to manuscript preparation.

Corresponding authors

Correspondence to Marian L. Burr or Mark A. Dawson.

Ethics declarations

Competing interests

M.A.D. has been a member of advisory boards for GSK, CTX CRC, Storm Therapeutics, Celgene and Cambridge Epigenetix. The Dawson Laboratory is a recipient of grant funding through the emerging science fund administered through Pfizer. S.J.D. has been a member of advisory boards for Adela and Inivata. P.E. owns Amgen stocks (less than 5% value of the company) and has undertaken previous consulting for Servier (less than $10,000). G.M.M. is employed by Syndax Pharmaceuticals. The remaining authors declare no competing interests.

Peer review

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Nature Cell Biology thanks Yali Dou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 MHC-I genes harbour bivalent H3K4me3 and H3K27me3 modifications.

a, Genomic snapshots of MHC-I genes showing H3K4me3 and H3K27me3 CUT&Tag in K-562 and ChIP–seq in neuroblastoma KELLY cell lines. The K-562 tracks are also shown in the control cells in Fig. 2h and H3K27me3 control cells in Fig. 6f. b,c, Cell surface MHC-I in K-562 (left) and KELLY (right) cells following treatment with EPZ-011989 and (c) ± 10 ng ml−1 IFN-γ (48 h K-562; 24 h KELLY). d, Genomic snapshots of MHC-I genes showing ChIP–seq for H3K4me3, H3K27me3 and H3K27ac in KELLY cells treated with ethanol (control) or EPZ-011989 ± IFN-γ. e,f, ChIP reChIP–seq of single H3K27me3, single H3K4me3 and reChIP (H3K27me3 and H3K4me3) in K-562 cells. e, Genomic snapshots of bivalent MHC-I genes. f, Heatmaps show bivalent genes −3 kb TSS/+3 kb TES, with genomic regions ordered by H3K27me3 read density in the single H3K27me3 ChIP sample. b,c, Representative plots from three experiments (Supplementary Fig. 3).

Source data

Extended Data Fig. 2 Genome-wide CRISPR–Cas9 screen identifies regulators of MHC-I expression.

a, Cell surface MHC-I, pan-HLA-A,B,C (top)- and HLA-B (bottom)-specific antibodies in K-562 Cas9 cells treated with the indicated IFN-γ doses for 24 h. b, K-562 cells stably expressing Cas9 were mutagenized by infection with a pooled lentiviral sgRNA library and treated with 1 ng ml−1 IFN-γ for 24 h prior to FACS sorting. Rare MHC-I high cells were enriched by two successive rounds of FACS sorting for mCherry+ (containing sgRNA vector) MHC-I+ cells. FACS dot plots and histograms show MHC-I expression in unsorted, post sort 1 and post sort 2 in K-562 Cas9 cells transduced with the CRIPSR sgRNA library and sorted with either pan-HLA-A,B,C (top)- or HLA-B (bottom)-specific antibodies. c, Table depicting correlation between CRISPR gene-effect scores (Fig. 1e) for top-20 shared EZH2 and EED co-dependent genes calculated from combined CRISPR survival screens in 990 cancer cell lines in Cancer Dependency Map (https://depmap.org/portal/)31,32. Table indicates Pearson’s correlation coefficients. d,e, Immunoblots of K-562 Cas9 cells transduced with control and MTF2 (d) or AEBP2 (e) sgRNA. f, H3K4me3 and H3K27me3 CUT&Tag. Genomic snapshots of bivalent MHC-I genes in K-562 cells transduced with control, MTF2 and AEBP2 sgRNA. The H3K4me3 control tracks are the same control tracks in Fig. 7c. g, Cell surface MHC-I in K-562 Cas9 cells transduced with control or BAHD1-specific sgRNAs and treated with 10 ng ml−1 IFN-γ for 48 h. Representative plots from three experiments (Supplementary Fig. 3). h, Knockout scores of individual sgRNA targeting BAHD1 measured using Synthego Performance Analysis, Interference of CRISPR editing (ICE) Analysis.

Source data

Extended Data Fig. 3 Loss of PRC1 drives derepression of bivalent genes.

a, Immunoblot of K-562 Cas9, PCGF1-KO and EED-KO cells ± 10 ng ml−1 IFN-γ (40 h). b,c, Cell surface MHC-I in K-562 Cas9 cells transduced with either control or PCGF1 sgRNA. c, Mean percentage of MHC-I expression from three experiments, indicated by points. Unpaired two-tailed Student’s t-test, P = 0.0295. d, qRT-PCR for MHC-I genes in K-562 Cas9 cells transduced with control or PCGF1 sgRNA. Bars indicate mean ± s.d. of technical triplicates from a representative experiment. e, Cell surface MHC-I in EED-KO cells transduced with control or MTF2 sgRNA. Representative plot from three experiments (Supplementary Fig. 3). f, Immunoblot of K-562 Cas9 and EED-KO cells transduced with control and PCGF1 sgRNA. g,h, Cell surface MHC-I in K-562 Cas9 cells transduced with RING1A and/or RING1B sgRNA, following treatment with 10 ng ml−1 IFN-γ for 36 h. h, Bars show mean fold change in MFI from 3–5 experiments, indicated by points. Unpaired two-tailed Student’s t-test, P values are indicated. i, Immunoblot of K-562 Cas9 cells transduced with the indicated sgRNA. j, Genomic snapshots of bivalent MHC-I genes showing H3K4me3, H3K27me3 and H2AK119Ub CUT&Tag in K-562 Cas9 (control), EED-KO and PCGF1-KO cells. The H3K4me3 and H3K27me3 control tracks are the same control tracks in Fig. 6f. k, H2AK119Ub CUT&Tag in K-562 cells transduced with control or MTF2 sgRNA. Heatmaps show bivalent genes −3kb TSS/+3 kb TES. Genomic regions are ordered by H2AK119Ub read density in the control sample.

Source data

Extended Data Fig. 4 Depletion of Menin or LEDGF enhances basal and IFN-γ-induced bivalent MHC-I gene expression.

a,b, Cell surface MHC-I in K-562 Cas9 cells transduced with control, MEN1 or PSIP1 sgRNA. b, Bars show mean percentage of MHC-I expression from three experiments, indicated by points. Unpaired two-tailed Student’s t-test, significant changes are indicated, P = 0.0356. c, qRT-PCR for MHC-I genes in K-562 Cas9 cells transduced with control or MEN1 sgRNA. Bars indicate mean ± s.d. of technical triplicates from a representative experiment. d, Immunoblot of K-562 Cas9, MEN1-KO and PSIP1-KO cells ± 10 ng ml−1 IFN-γ for 40 h. e, Cell surface MHC-I in K-562 Cas9 cells transduced with control or the indicated sgRNA targeting MEN1. f,g, Immunoblots of K-562 Cas9 cells transduced with control sgRNA or sgRNA targeting MEN1 (f), MEN1-KO cells ± MEN1 cDNA (g). h,i, JunD is not required for enhanced MHC-I expression following MEN1 KO. K-562 Cas9 and MEN1-KO cells transduced with control or JunD sgRNA and analysed by flow cytometry following treatment with 10 ng ml−1 IFN-γ for 48 h (h) and immunoblot (i). h, Representative plots from three experiments (Supplementary Fig. 3).

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Extended Data Fig. 5 Pharmacological targeting of Menin–KMT2A/B and PRC2 similarly augment IFN-γ-induced MHC-I expression in MHC-Ilow cancers and enhance T cell-mediated killing.

a, qRT-PCR analysis of K-562 cells treated ± 500 nM VTP50469. Bars indicate the mean ± s.d. of technical triplicates. b, MI-503, a chemically distinct inhibitor of the Menin–MLL interaction, also enhanced IFN-γ induced MHC-I expression. Cell surface MHC-I in K-562 Cas9 cells pre-treated with 500 nM MI-503 and 10 ng ml−1 IFN-γ (48 h). Representative plot from three experiments (Supplementary Fig. 3). c, Cell surface MHC-I in cells treated with DMSO or 3 µM EPZ-011989 and 10 ng ml−1 IFN-γ (24 h SCLC, 40 h KELLY), (VTP50469 treatment: Fig. 4a). Representative plots from independent experiments (n = 2 SCLC, n = 3 KELLY (Supplementary Fig. 3)). d, Cell surface MHC-I expression in SCLC cells treated with DMSO, 1 µM VTP50469 or 3 µM EPZ-011989 and 10 ng ml−1 IFN-γ for 24 h. Representative plots from two experiments (Supplementary Fig. 3). e, Scatter plot indicating MEN1 and EED CERES gene perturbation effects for neuroblastoma cell lines evaluated in combined CRISPR screens in DepMap (DepMap 21Q2 Public+Score, CERES (https://depmap.org/portal/)31,32. f, Flow cytometry analysis of RP-48-OVA cells pre-treated with DMSO or 1 µM VTP50469 and 10 ng ml−1 murine IFN-γ (24 h) prior to co-culture with OVA antigen-specific OT-I T cells at the indicated effector:target (E:T) ratios. Bars indicate mean percent remaining mCherry+ (RP-48-OVA) cells compared with no T cell control from three independent replicates, indicated by points. Unpaired two-tailed Student’s t-tests compared with the respective DMSO controls. Significant changes are indicated. g, Cytometric Beads Array (CBA) assay for mIFN-γ following 24 h co-culture of RP-48-OVA cells pre-treated with DMSO or 1 µM VTP50469 and 10 ng ml−1 murine IFN-γ (24 h) prior to co-culture with OVA antigen-specific OT-I T cells at a 2:1 (E:T) ratio. Bars show mean expression from 2–3 independent replicates, indicated by points. Unpaired two-tailed Student’s t-test, P = 0.01. h, Cell surface MHC-I in SPC-545-OVA cells pre-treated with DMSO, 1 µM VTP50469 and/or 3 µM EPZ-011989, and 1 ng ml−1 murine IFN-γ (24 h). Representative plot from two experiments (Supplementary Fig. 3). i, CBA assay for mIFN-γ and TNF following 4 d of co-culture of pre-treated SPC-545-OVA cells (DMSO, 1 µM VTP50469 and/or 3 µM EPZ-011989 and 2 h 20 ng ml−1 mIFN-γ) with OVA antigen-specific OT-I T cells at a 2:1 (E:T) ratio. Bars show mean expression from three independent replicates, indicated by points. Unpaired two-tailed Student’s t-test compared with the respective DMSO + mIFN-γ controls. Significant changes are indicated.

Source data

Extended Data Fig. 6 Targeting Menin drives expression of bivalent genes independently of IFN and NF-κB signalling.

a,b, Immunoblot in K-562 EED-KO cells depleted of MEN1 and PSIP1 (a) or PCGF1 (b) and then transduced with the indicated sgRNA. c, Immunoblot in K-562 Cas9 and EED-KO cells transduced with the indicated sgRNA and treated ± 10 ng ml−1 IFN-γ for 48 h. d–h, K-562 EED-KO cells depleted of MEN1, PSIP1 or PCGF1 and transduced with the indicated sgRNA, analysed by flow cytometry (d,f), and immunoblot (e,g,h). i, Immunoblot of K-562 Cas9 and EED-KO cells transduced with the indicated sgRNA and treated ± 20 ng ml−1 TNF-α for 48 h. j, Cell surface MHC-I expression in K-562 EED-KO cells transduced with control or PCGF1 sgRNA and treated ± 25 ng ml−1 IFN-γ for 24 h. d,f,j, Representative plots from three experiments (Supplementary Fig. 3).

Source data

Extended Data Fig. 7 Loss of Menin alleviates repression of bivalent genes.

a, Volcano plot showing log2FC gene expression from RNA-seq data in K-562 cells expressing MEN1 sgRNA compared with control sgRNA. Selected MHC class I genes are labelled. Two-sided Wald test; P values adjusted for multiple testing. b, Venn diagram depicting overlap in genes downregulated (Padj < 0.05 and fold change > 2) after CRISPR deletion of MEN1, PSIP1 or EED. c, Venn diagrams depicting overlap in genes up- and downregulated (Padj < 0.05 and fold change > 2) after CRISPR deletion of MEN1 or PSIP1, or 500 nM VTP50469 treatment. d, Pharmacological inhibition of Menin–KMT2A/B induces genome-wide displacement of Menin from chromatin. Menin ChIP–seq in K-562 cells treated for 48 h with DMSO or 1 µM VTP50469. Average profile plots (top) and heatmaps (bottom) of Menin-occupied sites −3kb TSS/+3 kb TES. Genomic regions are ordered by Menin occupancy in the control sample. e,f, Immunoblots of K-562 Cas9 (control), MEN1-KO, PSIP1-KO and PCGF1-KO cells. g, Genomic snapshots of MHC-I genes from SUZ12 ChIP–seq data in K-562 Cas9 control and MEN1-KO cells. h, Genomic snapshots of H3K4me3, SUZ12 ChIP–seq and H3K27me3 CUT&Tag in K-562 Cas9 control and MEN1-KO cells.

Source data

Extended Data Fig. 8 Targeting Menin potentiates bivalent gene derepression in human pluripotent stem cells.

a, RNA-seq in H9 hESCs treated with DMSO, 1 µM VTP50469 and/or 3 µM EPZ-011989 for 5 d. Heatmap includes bivalent genes significantly up- or downregulated in combination Menin/EZH2 inhibitor-treated cells compared with DMSO control (Padj < 0.05 and log2FC >1 or <−1). b,c, RNA-seq in wild-type (WT), EZH2-null (EZH2−/−) and EZH2-complemented EZH2-null (EZH2−/− + EZH2) H9 hESCs (GEO: GSE76626)60. b, Boxplots include the top upregulated bivalent genes in combination with Menin + EZH2 inhibitor-treated H9 hESCs (log2FC > 4 compared with the DMSO control) and depict median log2FC in expression in EZH2-null or EZH2-complemented H9 hESCs compared with the wild-type control60. Whiskers represent the minimum and maximum, the box represents the interquartile range and the centre line represents the median. c, Heatmap shows log2FC in expression of selected germ layer-specific genes in either EZH2-null or EZH2-complemented H9 hESCs compared with the wild-type control60. d, Heatmap shows log2FC in expression of selected germ layer-specific genes in H9 hESCs treated with 1 µM VTP50469 and/or 3 µM EPZ-011989 compared with the DMSO control. e,f, ChIP–seq of H9 hESCs. Genomic snapshots showing data from KMT2A (e), and KMT2A, H3K4me3 (GEO: GSE96336) and H3K27me3 (GEO: GSE96353)84 (f).

Extended Data Fig. 9 KMT2A/B is required for basal MHC-I expression.

a, Cell surface MHC-I in K-562 Cas9 cells transduced with KMT2A or KMT2B sgRNA compared with control sgRNA and treated with 10 ng ml−1 IFN-γ for 48 h. Bars show mean percentage of MHC-I expression from three experiments, indicated by points. Unpaired two-tailed Student’s t-test compared with control sgRNA. Significant changes are indicated; P < 0.0001. b,c, Immunoblots in K-562 Cas9 and KMT2B-KO cells (b), and KMT2A-KO ± KMT2B-KO cells (c). d, Cell surface MHC-I in K-562 KMT2B + PCGF1-KO cells transduced with the indicated sgRNA and treated for 5 d with DMSO, 1 µM VTP50469 or 3 µM EPZ-011989. Representative plot from three experiments (Supplementary Fig. 3). e, Genomic snapshots of H3K4me3 CUT&Tag in K-562 Cas9 and KMT2A/B-KO cells treated ± EPZ-011989. The EZH2i-treated (no IFN-γ) track is also shown in Fig. 8g. f, Immunoblots in K-562 Cas9, MEN1-KO and KMT2A-KO cells. g–i, Genomic snapshots of K-562 Cas9 and MEN1-KO cells (g,h) H3K4me3 ChIP–seq and KMT2A CUT&RUN (i). The H3K4me3 tracks are also shown in Extended Data Fig. 7h.

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Extended Data Fig. 10 KMT2A/B is dispensable for MHC enhanceosome-driven activation.

a, Schematic overview of cis-regulatory elements in the MHC-I promoter. NLRC5 forms an enhanceosome with the RFX (regulatory factor X) complex, made up of RFX5, RFXANK and RFAXP (RFX-associated ankyrin-containing protein); CREB (cAMP-responsive-element-binding); and NFY (nuclear transcription factor Y), which bind the SXY-molecule to activate transcription of MHC-I. b, Immunoblot of K-562 Cas9 cells transduced with control and RFX5 sgRNA. c, IFN-γ time course in K-562 Cas9 and the indicated KO cells treated with 3 µM EPZ-011989 and 25 ng ml−1 IFN-γ for the indicated time periods. d, Immunoblot of K-562 Cas9 and KMT2A/B-KO cells transduced with control, SETD1A and/or SETD1B sgRNA.

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Supplementary information

Supplementary Information

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Reporting Summary

Peer Review File

Supplementary Tables 1–7

Supplementary Tables 1–7. Supplementary Tables 1–3. CRISPR screen results. Related to Fig. 1. Supplementary Table 4. Gene lists for RNA-seq data. Related to Fig. 6 and Extended Data Fig. 7. Supplementary Table 5. Gene list intersection of CRISPR screen and RNA-seq results. Supplementary Tables 6 and 7. Primer sequences.

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Sparbier, C.E., Gillespie, A., Gomez, J. et al. Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes. Nat Cell Biol 25, 258–272 (2023). https://doi.org/10.1038/s41556-022-01056-x

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