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

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COOPERATES WITH SWI/SNF COMPLEXES TO FACILITATE TRANSCRIPTIONAL PROGRESSION Article Open access 24 August 2024 GENOMIC PROFILING OF THE TRANSCRIPTION FACTOR ZFP148 AND ITS IMPACT ON THE P53

PATHWAY Article Open access 25 August 2020 BAHCC1 BINDS H3K27ME3 VIA A CONSERVED BAH MODULE TO MEDIATE GENE SILENCING AND ONCOGENESIS Article 02 November 2020 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

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https://doi.org/10.1101/845529 (2019). Download references 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 * These authors contributed equally: Marian L. Burr, Mark A. Dawson. AUTHORS

AND AFFILIATIONS * 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 * 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 * The John Curtin School of Medical Research, The Australian National University, Canberra,

Australian Capital Territory, Australia Juliana Gomez, Sarah Popp & Marian L. Burr * Department of Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital,

Melbourne, Victoria, Australia Kah Lok Chan & Mark A. Dawson * Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia Omer Gilan * Department of

Molecular Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria, Australia Daniel J. Gough * Centre for Cancer Research, Hudson

Institute of Medical Research, Clayton, Victoria, Australia Daniel J. Gough * Department of Anatomy and Physiology, University of Melbourne, Melbourne, Victoria, Australia Melanie A.

Eckersley-Maslin * Centre for Cancer Research, University of Melbourne, Melbourne, Victoria, Australia Sarah-Jane Dawson & Mark A. Dawson * Cambridge Institute of Therapeutic Immunology

and Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, Cambridge, UK Paul J. Lehner * ACRF Cancer Biology and Stem Cells Division, Walter and Eliza Hall

Institute of Medical Research, Parkville, Victoria, Australia Kate D. Sutherland * Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia Kate D.

Sutherland * Section of Hematology, Oncology and Bone Marrow Transplant, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Patricia Ernst *

Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Patricia Ernst * Syndax Pharmaceuticals, Waltham, MA, USA Gerard M. McGeehan * Department of Anatomical

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publications You can also search for this author inPubMed Google Scholar * Kate D. Sutherland View author publications You can also search for this author inPubMed Google Scholar * Patricia

Ernst View author publications You can also search for this author inPubMed Google Scholar * Gerard M. McGeehan View author publications You can also search for this author inPubMed Google

Scholar * Enid Y. N. Lam View author publications You can also search for this author inPubMed Google Scholar * Marian L. Burr View author publications You can also search for this author

inPubMed Google Scholar * Mark A. Dawson View author publications You can also search for this author inPubMed Google Scholar 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 PEER REVIEW INFORMATION _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. ADDITIONAL INFORMATION 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). Source data 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 (_P_adj < 0.05 and fold change > 2) after CRISPR deletion of _MEN1_, _PSIP1_ or _EED_. C, Venn diagrams depicting overlap in genes up- and downregulated

(_P_adj < 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

(_P_adj < 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. Source data

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. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–3. 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. SOURCE DATA SOURCE DATA FIG. 1 Source data. SOURCE DATA

FIG. 2 Source data. SOURCE DATA FIG. 2 Unprocessed western blots. SOURCE DATA FIG. 3 Source data. SOURCE DATA FIG. 3 Unprocessed western blots. SOURCE DATA FIG. 4 Source data. SOURCE DATA

FIG. 5 Source data. SOURCE DATA FIG. 5 Unprocessed western blots. SOURCE DATA FIG. 6 Source data. SOURCE DATA FIG. 7 Source data. SOURCE DATA FIG. 8 Source data. SOURCE DATA FIG. 8

Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 1 Source data. SOURCE DATA EXTENDED DATA FIG. 2 Source data. SOURCE DATA EXTENDED DATA FIG. 2 Unprocessed western blots. SOURCE DATA

EXTENDED DATA FIG. 3 Source data. SOURCE DATA EXTENDED DATA FIG. 3 Unprocessed western blot. SOURCE DATA EXTENDED DATA FIG. 4 Source data. SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed

western blots. SOURCE DATA EXTENDED DATA FIG. 5 Source data. SOURCE DATA EXTENDED DATA FIG. 6 Source data. SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed western blots. SOURCE DATA EXTENDED

DATA FIG. 7 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 9 Source data. SOURCE DATA EXTENDED DATA FIG. 9 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 10 Unprocessed

western blots. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 30 September 2021 * Accepted: 15 November 2022 *

Published: 12 January 2023 * Issue Date: February 2023 * DOI: https://doi.org/10.1038/s41556-022-01056-x SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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