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BAHCC1 binds H3K27me3 via a conserved BAH module to mediate gene silencing and oncogenesis

Abstract

Trimethylated histone H3 lysine 27 (H3K27me3) regulates gene repression, cell-fate determination and differentiation. We report that a conserved bromo-adjacent homology (BAH) module of BAHCC1 (BAHCC1BAH) ‘recognizes’ H3K27me3 specifically and enforces silencing of H3K27me3-demarcated genes in mammalian cells. Biochemical, structural and integrated chromatin immunoprecipitation-sequencing-based analyses demonstrate that direct readout of H3K27me3 by BAHCC1 is achieved through a hydrophobic trimethyl-l-lysine-binding ‘cage’ formed by BAHCC1BAH, mediating colocalization of BAHCC1 and H3K27me3-marked genes. BAHCC1 is highly expressed in human acute leukemia and interacts with transcriptional corepressors. In leukemia, depletion of BAHCC1, or disruption of the BAHCC1BAH–H3K27me3 interaction, causes derepression of H3K27me3-targeted genes that are involved in tumor suppression and cell differentiation, leading to suppression of oncogenesis. In mice, introduction of a germline mutation at Bahcc1 to disrupt its H3K27me3 engagement causes partial postnatal lethality, supporting a role in development. This study identifies an H3K27me3-directed transduction pathway in mammals that relies on a conserved BAH ‘reader’.

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Fig. 1: BAHCC1 sustains acute leukemia cell growth in vitro and in vivo.
Fig. 2: BAHCC1BAH specifically ‘reads’ H3K27me3.
Fig. 3: Structural analysis of BAHCC1BAH reveals a unique H3K27me3 ‘reading’ pocket.
Fig. 4: Integrated RNA-seq and ChIP–seq analyses demonstrate a direct involvement of BAHCC1 in silencing of the H3K27me3-targeted genes.
Fig. 5: BAHCC1BAH-mediated binding of H3K27me3 is crucial for Polycomb target gene repression and oncogenesis.
Fig. 6: BAHCC1 interacts with corepressors, maintaining a hypoacetylated chromatin state at target genes.
Fig. 7: BAHCC1 and PRC1 corepress the H3K27me3-marked genes in cells of different lineage origin.

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Data availability

Next-generation sequencing data are deposited with the NCBI GEO under accession number GSE151578. Coordinates and structural factors for the mBAHCC1BAH–H3K27me3 complex are deposited in the PDB under accession code 6VIL. The mass spectrometry proteomics data are deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org/) via the PRIDE partner repository with the dataset identifier PXD021679. The publicly available gene expression profiling datasets used in the study for examination of BAHCC1 (also known as KIAA1447 or BAHD2) include the Cancer Cell Line Encyclopedia data80, the NCBI GEO datasets under accession numbers GSE1159, GSE24505, GSE13204, GSE33315, GSE7186, GSE28460 and GSE34861 and the dataset of the St. Jude Hospital ‘Pediatric ALL’ cohort (http://www.stjuderesearch.org/data/ALL3/). Source data are provided with this paper.

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Acknowledgements

We thank P. Ntziachristos, M. Brenner, C. Vakoc, J. Lu, P. Liu and M. Minden for graciously providing reagents and cell lines used in the study and the Wang laboratory members, T. Ptacek, H. Uryu and J. Simon for helpful discussions and technical support. We thank the UNC facilities, including Imaging Core, HTSF, Bioinformatics Core, Flow Cytometry Core, Tissue Culture Facility, Proteomics Core and Animal Studies Core for their professional assistance in this work. The core facilities affiliated with the UNC Cancer Center are supported in part by the UNC Lineberger Comprehensive Cancer Center Core Support Grant P30-CA016086. We thank staff members at the Advanced Light Source, Lawrence Berkeley National Laboratory for access to X-ray beamlines. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, the National Institute of General Medical Sciences and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under contract no. DE-AC02-05CH11231. This work was supported by National Institutes of Health grants (R01-CA215284 and R01-CA218600 to G.G.W.; R35GM119721 to J.S.; R35GM126900 to B.D.S.), a V Scholar Award (to G.G.W.), Kimmel Scholar Awards (to J.S. and G.G.W.), a University of California Cancer Research Coordinating Committee (UC CRCC) grant (CRR-20-634140 to J.S.), a Concern Foundation for Cancer Research grant (to G.G.W.), Gabrielle’s Angel Foundation for Cancer Research (to G.G.W.), Gilead Sciences Research Scholars Program in hematology/oncology (to G.G.W.) and When Everyone Survives Leukemia Research Foundation (to G.G.W.). G.G.W. is an American Cancer Society Research Scholar, an American Society of Hematology Scholar in basic science and a Leukemia and Lymphoma Society Scholar.

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Contributions

H.F., J.L., Y.G., D.L., Z-.M.Z., W-.C.P., J.H.A., D.F.A., H.G., S.H., W-.Y.C., B.D.S. and L.C. performed experiments. Y-.H.T., W.G., Y.X. and Y.D. performed genomic data analysis under the direction of G.G.W. G.G.W. conceived the project. J.S. and G.G.W. organized and led the structural and functional aspects of the study, respectively. H.F., J.S. and G.G.W. prepared the manuscript with input from all authors.

Corresponding authors

Correspondence to Jikui Song or Gang Greg Wang.

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Competing interests

B.D.S. is a cofounder and SAB member of EpiCypher. The Wang laboratory received research funds from Deerfield Management/Pinnacle Hill. No potential conflicts ofinterest are disclosed by the other authors.

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Extended data

Extended Data Fig. 1 BAHCC1, a nuclear factor of unknown function, shows overexpression among acute leukemias.

a-g, Boxplots showing BAHCC1 expression among primary AMLs (based on GEO dataset GSE115959 in panel a and GSE24505 in b), hematological cancer lines according to Cancer Cell Line Encyclopedia dataset (c), and primary ALLs carrying genetic abnormality such as MLL rearrangement (MLL-r), BCR-ABL or E2A-PBX1 according to GEO datasets GSE33315 (d) and GSE13204 (e), the St Jude Hospital “Pediatric Acute Lymphoblastic Leukemia” cohort (f) or GSE34861 (g), relative to the indicated normal controls. The line indicates the mean and two-sided Wilcoxon test was used for calculating P value denoted on top of the panel. n, sample size. h-i, Schematic diagram (h) and PCR genotyping (i) of Flag Epitope Tag ChIP (pFETCh)-based strategy utilized to introduce a 3×Flag-P2A-NeoR+ cassette in-frame into the C-terminus of endogenous BAHCC1 gene. HOM1/2 (h), homology arm 1/2; E28/29, exons 28/29 of BAHCC1. Genotyping primers used for validating the cassette knockin are denoted in different colors. Agarose gel image (i) shows DNA ladder (M) and genotyping products using the indicated primers and genomic DNA of parental (Par) cells or those carrying heterozygous (Het) or homozygous (Homo) BAHCC1-3×Flag knockin (KI) alleles. j, Representative Sanger sequencing results showing correct recombination and cassette knockin in the produced JURKAT cells carrying homozygous BAHCC1-3×Flag KI alleles. k-l, Anti-Flag immunoblotting for endogenous BAHCC1-3×Flag protein, which was detected as 300kD in size (k) and readily depleted by BAHCC1-targeting shRNAs (sh988 or sh993; l), in JURKAT cells carrying the BAHCC1-3×Flag KI alleles. Parental (par) cells and those transduced with empty vector (EV) were used as control. m, Representative images of confocal immunofluorescence reveal the exclusive nuclear localization of endogenous BAHCC1 (left), relative to DNA staining (middle), in HeLa cells. Scale bar, 5 μm.

Extended Data Fig. 2 BAHCC1 promotes the in vitro growth, colony formation and cell cycle progression of acute leukemia cells.

a, RT-qPCR validating shRNA-mediated BAHCC1 knockdown (KD; with independent hairpins sh988 and sh993) in OCI-AML3, RS4;11 and K562 cells (n = 3 biologically independent samples). Data are presented as mean ± SD. b-f, Proliferation (left) after BAHCC1 KD (RT-qPCR shown in the right panel), relative to empty vector (shEV)-treated control, in acute leukemia lines, including AML (MV4;11, b) and T-ALL (CUTLL1, CCRF-CEM, ALL-SIL and MOLT4; c-f). n = 3 biologically independent experiments. Data are presented as mean ± SD. g, RT-qPCR of Bahcc1 (left) and summary of colony-forming unit (CFU) assay (right) post-KD of Bahcc1 (sh242075), relative to shEV, in mouse primary HSPCs (n=3 biologically independent experiments). Data are presented as mean ± SD. h, Dropout plot suggests that BAHCC1 regions targeted by the indicated sgRNAs are potentially crucial for JURKAT cell growth. Plotted at the bottom are sgRNAs showing significant dropout 10 days post-transduction of a viral BAHCC1 sgRNA pool, compared to day 1, among the same infected cells. Y-axis represents dropout beta-value, produced by the “Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout” module, with a cutoff of P value < 0.08. i, K562 cell proliferation (left) after dCas9-KRAB-mediated repression of BAHCC1 (RT-qPCR shown on the right; n=3 biologically independent experiments). j, Quantification of colonies formed by JURKAT cells post-KD of BAHCC1 compared to mock (n=2 biologically independent experiments). Data are presented as mean ± SD. k-l, Representative plate scan images (k) and quantification of colony formation (l) using OCI-AML3 cells post-KD of BAHCC1 relative to mock (n=2 biologically independent experiments). m, Cell cycle phase quantification using OCI-AML3 cells post-KD of BAHCC1 relative to mock (n=3 biologically independent experiments). ***, P < 0.001. Data are presented as mean ± SD. n, Measurement of apoptosis in JURKAT cells post-KD of BAHCC1 relative to mock.

Extended Data Fig. 3 BAHCC1 potentiates acute leukemia growth in vivo, a function that relies on an evolutionarily conserved BAH domain (BAHCC1BAH) as an H3K27me3/2-specific ‘reader’.

a, b, Bioluminescence imaging (a, two weeks post-transplantation) and Kaplan-Meier survival curve (b) of NSG mice xenografted with luciferase-labeled CUTLL1 cells post-transduction of empty vector or a BAHCC1-targeting shRNA. c, Averaged bioluminescence signals (collected from ventral view) of NSG mice xenografted with luciferase-labeled OCI-AML3 cells that harbor the CRISPR-Cas9-introduced disruption of BAHCC1BAH (ΔBAH), relative to WT (n=5 mice). Data are presented as mean ± SD. d, Plot showing the in vivo growth of K562 cell subcutaneous xenografts, after BAHCC1 KD or mock treatment. Data of tumor volume was shown as mean ± SD for each group (n=8). e, Images of K562 subcutaneous xenografts excised from the indicated cohort at the end of experiments. f, Domain architecture of the Homo sapiens, Mus musculus, Xenopus tropicalis and Danio rerio BAHCC1 proteins, revealing a conserved BAH domain (BAHCC1BAH). g, Alignment using the amino acid sequences of BAHCC1BAH from different species. Conserved residues are highlighted in red. The secondary structure is drawn based on the BAHCC1:H3K27me3 co-crystal structure, with the H3K27me3-caging and other histone-binding residues designated by red stars and black dots, respectively. h-i, Peptide pulldown using GST-BAHCC1BAH (h) or GST-ORC1BAH (i) protein and biotinylated histone H3.1 or H4 peptide carrying the indicated methylation (bottom). Unmodi, un-modified. j-p, ITC fitting curves of BAHCC1BAH with the indicated histone peptide. q, Summary of thermodynamic and curve fitting parameters for ITC assays using murine BAHCC1BAH recombinant protein and histone peptide. NDB, no detectable binding. *, N value set manually. #, mean and SD values derived from two independent measurements. For all the other titrations, ITC parameters were determined by curve fitting of single titration. **, due to behavior of CBX7CD and for a comparable measurement, ITC was performed at 25 °C instead of 7 °C for other measurements.

Extended Data Fig. 4 Characterization of BAHCC1BAH binding to H3K27me3 through their co-crystal structure and structure-based mutagenesis.

a, Crystal structures of the eight color-coded murine BAHCC1BAH molecules in one asymmetric unit, with chain ID labeled. Four of the BAHCC1BAH molecules (chain B, C, E and G) are complexed with H3K27me3 peptides (chain J, I, L and M, respectively.). The BAHCC1BAH:H3K27me3 complex with the best model-to-map fit (Chain C and I) was selected for structural analysis. b, Electrostatic surface view of murine BAHCC1BAH bound to the H3K27me3 peptide (yellow sticks). The Fo-Fc omit map for the H3K27me3 peptide, contoured at 1.5 sigma level, was shown as mesh in magenta. c-h, ITC binding curves of the H3K27me3 peptide with murine BAHCC1BAH recombinant protein carrying an indicated mutation, either Y2537A (c), W2558G (d), Y2560A (e), E2564A (f), H2583A (g) or D2585A (h). i, CRISPR-cas9-mediated genomic editing to introduce homozygous point mutation of W2554G (left two panels) or Y2533A (right) into the BAHCC1 gene in JURKAT cells. Shown are Sanger sequencing results using cDNA (middle) or genomic DNA (left and right) as template. See also Extended Data Fig. 3g for murine and human BAHCC1 amino acid numerations.

Extended Data Fig. 5 Transcriptome profiling reveals the gene-expression programs regulated by BAHCC1 in JURKAT cells.

a, Venn diagrams show overlapping for upregulated (left) or downregulated (right) transcripts in JURKAT cells post-transduction of a BAHCC1-targeting shRNA, relative to empty vector-treated control (EV), as revealed by RNA-seq (n=3 biologically independent samples). The threshold for differentially expressed genes (DEGs) is an adjusted p (q) value of less than 0.05 and fold-change (FC) of over 1.50 for transcripts with baseMean value of more than 10. b, c, Ingenuity Pathway Analysis (IPA; b) and Gene Ontology (GO; c) analyses with the genes found to be commonly upregulated post-KD of BAHCC1 by the two independent shRNAs, relative to mock, in JURKAT cells. d, Summary of GSEA using RNA-seq profiles of JURKAT cells post-KD of BAHCC1 by sh988 (left) or sh993 (right), relative to EV. Representative genesets are categorized into differentiation, Polycomb (PRC2 and PRC1) or cell death-related or others. e-f, GSEA demonstrates that BAHCC1 KD is correlated with derepression of genesets associated with myogenesis (e) and erythroid differentiation (f). g-l, GSEA showing BAHCC1 KD correlated with reactivation of genesets suppressed by PRC1 (g) or PRC2 (h-k) and the H3K27me3-bound genes (l). m, Platform for Integrative Analysis of Omics data (Piano) analysis results using RNA-seq profiles of JURKAT cells post-KD of BAHCC1 by sh988 (left) or sh993 (right), relative to EV.

Extended Data Fig. 6 BAHCC1 co-localizes with H3K27me3, mediating optimal repression of Polycomb target genes in multiple examined leukemia cells.

a, Summary of read counts of Flag ChIP-seq using JURKAT cells carrying 3×Flag-KI-alleles of BAHCC1, either wildtype or Y2533A-mutated. b, Correlation analysis of replicated Flag-BAHCC1 ChIP-seq data in JURKAT cells. c, Venn diagram showing overlapping of called BAHCC1 and H3K27me3 peaks in JURKAT cells. d, Heatmap showing the called BAHCC1 peaks (Flag; two replicated experiments) that overlap (top) or does not overlap (bottom) with H3K27me3 in JURKAT cells after normalization to input and sequencing depth. Color bar, log2(ChIP/Input). e, Heatmap showing BAHCC1 and H3K27me3 ChIP-seq peaks across ±3 kb from all genes in JURKAT cells. TSS, transcription start site; TES, transcription end site. f, Flag-tagged BAHCC1 (blue) and H3K27me3 (red) ChIP-seq profiles at classic Polycomb/H3K27me3-targeted loci (SMAD7, KLF9, GATA2 and the HOXB cluster) in JURKAT cells. g, H3K27me3 and H3 immunoblots using JURKAT and CUTLL1 cells post-treatment with UNC1999. h, i, Venn diagrams show significant overlapping for DEGs, either all DEGs (h) or those directly bound by H3K27me3 (i), that were found upregulated post-KD of BAHCC1 relative to mock (black) or post-treatment with UNC1999 relative to DMSO (red) in JURKAT cells. j, EZH2, SUZ12 and H3 immunoblotting in various used T-ALL cell lines. k-n, RT-qPCR of the indicated H3K27me3-targeted gene post-KD of BAHCC1 (red), relative to mock (black), in T-ALL cells including JURKAT (k), CUTLL1 (l), CCRF-CEM (m) and ALL-SIL (n). Data of three independent experiments are plotted as mean ± SD after normalization to GAPDH and to mock-treated cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant. o-p, RT-qPCR of the indicated H3K27me3-targeted gene after UNC1999 treatment, compared to mock, in JURKAT (o) and CUTLL1 (p) cells. Data of three independent experiments are plotted as mean ± SD after normalization to GAPDH and to mock-treated cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant.

Extended Data Fig. 7 The H3K27me3-binding-defective mutation of BAHCC1BAH decreases overall occupancy of BAHCC1 at H3K27me3-bound genes, leading to their concurrent derepression.

a, Immunoblots of BAHCC1, Tubulin and histone H3 using chromatin-bound and soluble fractions of 293 cells post-treatment with DMSO or UNC1999. b, Flag ChIP-qPCR to examine binding of endogenous 3×Flag-BAHCC1 to TSS of the indicated gene in JURKAT cells post-treatment with UNC1999 versus DMSO. Data of three independent experiments are plotted as mean ± SD after normalization to those of input and to DMSO-treated. **P < 0.01; ***P < 0.001. c, Heatmap of Flag-BAHCC1 ChIP-seq signals across all genes in JURKAT cells carrying either WT or the Y2533A homozygous mutation of 3×Flag-BAHCC1 (endogenous). d, Flag ChIP-qPCR to examine the endogenous Flag-BAHCC1 binding to TSS of the indicated gene in JURKAT cells that carry either WT or the W2554G homozygous mutation of BAHCC1. Data of three independent experiments are plotted as mean ± SD after normalization to those of input and to WT. *P < 0.05; **P < 0.01; ***P < 0.001. e, Immunoblots of BAHCC1, Tubulin and H3 using chromatin-bound and soluble fractions of JURKAT cells carrying WT or the W2554G homozygous mutation of BAHCC1BAH. f, Summary of GSEA using RNA-seq profiles of JURKAT cells carrying an indicated H3K27me3-binding-defective mutation of BAHCC1BAH and their WT counterpart cells. Representative genesets showing the most significant correlations to Y2533A (left) or W2554G (right) homozygous mutation of BAHCC1BAH, relative to WT, are categorized into Polycomb-related, differentiation-related or others. g-o, GSEA reveals that, relative to WT, the H3K27me3-binding-defective mutation of BAHCC1BAH is positively correlated to derepression of gene signatures related to PRC1 (g), SUZ12 (h, j, k), EZH2 (i, l, n), EED (m) or myogenesis (o). p, Immunoblotting of EZH2, SUZ12, H3K27me3 and H3 in the indicated cell lines with WT or the H3K27me3-binding-defective mutation of BAHCC1BAH.

Extended Data Fig. 8 The H3K27me3-binding-defective mutation of BAHCC1BAH slowed growth of acute leukemia in vitro and in vivo.

a, b, RT-qPCR of H3K27me3-marked genes in JURKAT cells harboring Y2533A (a) or W2554G (b) homozygous mutation of BAHCC1BAH, relative to WT (n=3 independent experiments). Data are plotted as mean ± SD after normalization to GAPDH and to WT. *P < 0.05; **P < 0.01; ***P < 0.001. c, d, Sanger sequencing (c) and Flag immunoblotting (d) using CUTLL1 cells with either WT or Y2533A homozygous mutation of endogenous 3×Flag-BAHCC1. e, RT-qPCR of cMyc in JURKAT and CUTLL1 cells with the indicated BAHCC1BAH homozygous mutation, relative to WT (n=3 independent experiments). Data are plotted as mean ± SD after normalization to GAPDH and to WT. n.s., not significant. f, Wright-Giemsa staining of JURKAT cells with WT or the Y2533A homozygous mutation of BAHCC1BAH (n=2 independent lines). Scale bar, 10 μm. g, h, Proliferation of JURKAT (g) or OCI-AML3 cells (h) carrying homozygous mutation of BAHCC1BAH relative to WT (n=3 biologically independent experiments). Data are presented as mean ± SD. i, Quantification of colonies formed by JURKAT cells carrying WT or Y2533A homozygous mutation of BAHCC1 (n=2 biologically independent experiments). Data are presented as mean values ± SD. j, Quantification of cell cycle progression of CUTLL1 cells carrying WT or Y2533A homozygous mutation of BAHCC1 (n=3 biologically independent experiments). Data are presented as mean ± SD. **P < 0.01; ***P < 0.001. k-l, Averaged bioluminescence signals collected from ventral (k) or dorsal view (l) of NSG mice (n=5) xenografted with luciferase-labeled JURKAT cells that carry WT or the Y2533A homozygous mutation (two independent lines) of BAHCC1BAH. Data are presented as mean ± SD. *P < 0.05; ***P < 0.001. m, Bioluminescence images (two weeks post-xenograft) of NSG mice xenografted with luciferase-labeled CUTLL1 cells that carry WT or the Y2533A homozygous mutation of BAHCC1BAH. n, Kaplan-Meier curve showing event-free survival of NSG mice xenografted with luciferase-labeled JURKAT cells that carry WT or the W2554G homozygous mutation of BAHCC1BAH. n, cohort size. o, Summary for top hits of the BAHCC1-interacting proteins identified by Flag pulldown and mass spectrometry analyses with HeLa cells carrying BAHCC1-3×Flag KI alleles. Parental HeLa cells were used as control for Flag pulldown (n=2 biologically independent experiments). Rep, replicate. p, CoIP for endogenous Flag-BAHCC1 and PRC1 in JURKAT cells.

Extended Data Fig. 9 BAHCC1 interacts with corepressors SAP30BP and HDAC1, maintaining a hypoacetylated state at target genes.

a, ChIP-seq profile of histone acetylation at the BAHCC1 target gene IGFBP4 in JURKAT cells carrying WT or the W2554G homozygous mutation of BAHCC1BAH. b, ChIP-qPCR of histone acetylation at the cMyc promoter in JURKAT cells that carry WT or the Y2533A homozygous mutation of BAHCC1 (n=3 biologically independent samples). Data are presented as mean ± SD. c, d, RT-qPCR of SAP30BP or HDAC1 after the dCAS9-KRAB/sgRNA-mediated repression of SAP30BP (c) or transduction of HDAC1-targeting shRNAs (d), relative to mock, in JURKAT cells (n=3 biologically independent samples). Data are presented as mean ±± SD. e, f, RT-qPCR of BAHCC1-targeted genes post-treatment of JURKAT cells with 5 µM of CI-994 (e) or 0.25 µM of Entinostat (f) for 2 days (n=3 biologically independent samples). Data are presented as mean values ± SD. **P < 0.01; ***P < 0.001; n.s., not significant. g, JURKAT cell proliferation post-treatment with 0.25 µM of Entinostat relative to mock (n=3 biologically independent experiments). Data are presented as mean ± SD. * P < 0.05; **P < 0.01; ***P < 0.001. h, Sanger sequencing to confirm the mouse genotype, either WT (left) or Y2537A-mutated (right) Bahcc1 allele. i, Averaged body weight of mouse pups with either WT (left, n=31) or the Y2537A homozygous alleles (right, n=26) of Bahcc1, at day 1 post-birth (P1). Data are presented as mean ± SD. ****P < 0.0001. j, Wright-Giemsa staining of E2A-PBX1-transformed murine leukemia cells, established with HSPCs from mice carrying either WT or the Y2537A homozygous mutation of BAHCC1BAH. Scale bar, 10 μM. k, l, CFU quantification (k) and representative plate images (l) using the indicated E2A-PBX1-transformed murine leukemia cells (duplicate per group; data presented as mean ± SD). m, ChIP-qPCR detects Bahcc1 binding to TSS of H3K27me3-marked genes relative to a negative locus, beta-actin (left), in the WT E2A-PBX1-transformed murine leukemia cells (n=3 independent experiments; mean ± SD after normalization to input). n, H3K27me3 ChIP-seq profile of the indicated gene in the WT E2A-PBX1-transformed leukemia cells. o, GSEA reveals that, relative to WT, the Y2537A mutation of Bahcc1BAH is positively correlated to derepression of Polycomb- or HDAC-related gene signature.

Extended Data Fig. 10 BAHCC1 and PRC1 can corepress H3K27me3-targeted genes in different cell lineages, suggesting a generalized functionality of BAHCC1.

a, RT-qPCR of the indicated gene post-depletion of RING1B by independent shRNAs, relative to mock, in JURKAT cells (n=3 biologically independent samples). Data are presented as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001. b, ChIP-seq profiles of H3K27me3 and H2Aub at BAHCC1- and PRC1-corepressed genes in 293 cells. c, RT-qPCR of BAHCC1, RING1A and RING1B in 293 cells transfected with siRNA of control (siCtrl), RING1A alone (siRING1A), RING1B alone (siRING1B), BAHCC1 alone (siBAHCC1), RING1A plus RING1B (siRING1A+B), or all three genes (si-3×). Data of three independent experiments are plotted as mean ± SD after normalization to GAPDH and to siCtrl-treated. *P < 0.05; **P < 0.01; n.s., not significant. d, e, RT-qPCR of BAHCC1 (d) and the indicated target gene (e) two and four days (D2 and D4) after BAHCC1 KD (sh988), compared to mock, in 293 cells (n=3 independent experiments). Data are plotted as mean ± SD after normalization to GAPDH and to shEV. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. f, g, H3 acetylation (f) and H3K27me3 (g) ChIP-qPCR for the indicated gene promoter at different timepoints after shRNA-mediated BAHCC1 KD in 293 cells (n=3 independent experiments). Data are plotted as mean ± SD after normalization to shEV cells. **P < 0.01; ***P < 0.001; n.s., not significant. h, H3K27me3 ChIP-seq profile at BAHCC1 targets in JURKAT cells carrying WT or the W2554G homozygous mutation of BAHCC1. i, Intermolecular interactions between CBX7CD (PDB 4X3K) and H3K27me3 peptide (yellow; left), along with the surface view of the H3K27me3-binding groove (right). The side chains of interacting residues are shown in stick representation and hydrogen bonds in dashed lines. j, Ribbon representation of ORC1BAH (pink) bound to H4K20me2 peptide (yellow sticks) (PDB 4DOV). The H4K20me2-binding residues of ORC1BAH are labeled. The hydrogen bonding interactions are shown as dashed lines.

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Supplementary Data 1

Full wwPDB X-ray structure validation report

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Unprocessed western blots and/or gels.

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Fan, H., Lu, J., Guo, Y. et al. BAHCC1 binds H3K27me3 via a conserved BAH module to mediate gene silencing and oncogenesis. Nat Genet 52, 1384–1396 (2020). https://doi.org/10.1038/s41588-020-00729-3

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