Supplementary Information for The Crebbp acetyltransferase is a haploinsufficient tumor suppressor in B cell lymphoma Jiyuan Zhang1, Sofija Vlasevska1, Victoria A. Wells1, Sarah Nataraj1, Antony B. Holmes1, Romain Duval1, Stefanie N. Meyer1, Tongwei Mo1, Katia Basso1,2, Paul K Brindle3, Shafinaz Hussein4, Riccardo Dalla-Favera1,2,5,6,7 and Laura Pasqualucci1,2,7* 1 Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA 2 Department of Pathology and Cell Biology, Columbia University, New York, NY, 10032, USA 3 Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN 4 Department of Pathology and Laboratory Medicine, NorthWell Health, Staten Island University Hospital, Staten Island, New York, USA 5 Department of Genetics & Development, Columbia University, New York, NY, 10032, USA 6 Department of Microbiology & Immunology, Columbia University, New York, NY, 10032, USA 7 Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, 10032, USA *Correspondence: [email protected] SI Guide Supplementary Figure S1. CREBBP binds to GC-specific super-enhancers Supplementary Figure S2. Crebbp and EP300 expression in the B cell lineage Supplementary Figure S3. GSEA of Crebbp-conditional knock-out GC B cells and human DZ vs LZ B cells Supplementary Figure S4. Analysis of GC B cell responses in Crebbpfl/fl Cγ1-Cre and CD19-Cre cohorts Supplementary Figure S5. Analysis of plasma cell differentiation in Crebbpfl/fl Cγ1-Cre and CD19-Cre cohorts Supplementary Figure S6. Distribution pattern of CREBBP mutations in FL and de novo DLBCL Supplementary Figure S7. Analysis of Crebbp conditional knock-out tumor cohorts Supplementary Table S1. Overlap between CREBBP bound regions and predicted super- enhancers in GC B cells Supplementary Table S2. Genes differentially expressed in Crebbpfl/fl vs Crebbp+/+ GC B cells Supplementary Table S3. List of CREBBP “core target” genes (bound by CREBBP in human GC B cells and downregulated in Crebbpfl/fl Cγ1-Cre GC B cells) Supplementary Table S4. Pathway enrichment analysis of CREBBP "core target" genes Supplementary Table S5. Leading edge associated with GSEA of CREBBP core target genes in the rank of genes differentially expressed in DZ vs LZ GC B cells Supplementary Table S6. Pathway enrichment analysis of genes co-bound by CREBBP and BCL6 in human GC B cells Supplementary Table S7. V gene rearrangement analysis of B-cell lymphomas in the Crebbpfl/+/ Cγ1-Cre /VavP-BCL2 conditional knock-out mouse model Supplementary Table S8. List of antibodies used in FACS analysis 2 SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure S1. CREBBP binds to GC-specific super-enhancers. ChIP-seq plots of CREBBP and H3K27Ac binding at genes associated with GC-specific super-enhancers (SE), as predicted by the ROSE algorithm. Read density tracks of BCL6, H3K4me1, H3K4me3 and H3K27me3 ChIP-Seq enrichment are also shown. Supplementary Figure S2. CREBBP and EP300 expression in the B cell lineage. A, CREBBP and EP300 mRNA expression levels in naive, GC and memory B cells purified from reactive human tonsils, measured by RNA-seq (n = 3 donors each, representing 3 biological replicates; mean ± SD). Data were normalized to the total number of mapped reads in each sample and to the transcript size, and are expressed as reads per kilobase per million mapped reads (RPKM). B, CREBBP and EP300 protein expression in human naive, GC and memory B cells. BCL6 controls for the identity of the populations and tubulin serves as loading control. C, CREBBP and EP300 mRNA expression in GC dark zone (DZ) and light zone (LZ) B cells sorted from reactive human tonsils (left bars) or from the spleen of immunized mice (right bars), and analyzed by gene expression profiling using Affymetrix HG U133_plus 2 (human) or MG 430_2.0 (mouse) arrays (Accession No: GSE38697 and GSE38696, respectively). Data (MAS5- normalized) are expressed as linear absolute levels, and the probe ID is provided for each transcript (n = 3 donors/subset, representing biological replicates; mean ± SD). * P < 0.05, ** P < 0.01, Student’s t-test. Only statistically significant differences are indicated. D, CREBBP and EP300 mRNA expression levels during hematopoietic development in the mouse, as obtained from the Immgen.org database at https://www.immgen.org/. All data were generated on the Affymetrix Mouse Gene 1.0ST array. Supplementary Figure S3. GSEA of Crebbp-conditional knockout GC B cells and human GC DZ vs LZ cells. A, Enrichment plots of selected gene sets in the rank of genes differentially expressed between Crebbpfl/fl Cγ1-Cre (KO) and Crebbp+/+ Cγ1-Cre (WT) GC B cells. The preferential enrichment of previously identified FOXO1-bound genes in the Crebbp-proficient cells may reflect subtle changes imposed by Crebbp loss on transcription, which were not detected in the less sensitive, supervised gene expression analysis. B, Enrichment plots of the top 500 CREBBP-bound genes in the rank of genes differentially expressed between GC DZ and LZ cells (left panel). The analysis was performed separately with the top 500 genes bound by CREBBP at promoter regions (middle panel) or at TSS-distal, putative enhancer regions (right panel). Bound genes were identified as described in Methods and were ranked based on increasing P value. Supplementary Figure S4. Analysis of GC B cell responses in Crebbpfl/fl Cγ1-Cre and CD19- Cre cohorts. A, Immunofluorescence staining of CREBBP and EP300 in representative spleen sections from SRBC immunized Cγ1-Cre mice. PNA (green) identifies the GC area (outlined). B, Representative flow cytometric analysis of splenic B220+ cells from 12-16 week-old Crebbp+/+ (WT), Crebbpfl/+ (HET), and Crebbpfl/fl (KO) Cγ1-Cre mice analyzed 10 days after SRBC immunization. GC B cells are identified as CD95+PNAhi cells, and numbers in each panel indicate the percentage in the gate. C, Percentage of GC B cells in mice from the indicated genotypes, analyzed at 3 months of age, 10 days after SRBC immunization. D, Representative contour plots of GC B cell DZ (CXCR4hiCD86lo) and LZ (CXCR4loCD86hi) populations in Cγ1- 3 Cre mice of the indicated genotypes, analyzed 10 days after SRBC immunization according to an established gating strategy (1-3); cells are gated on the B220+CD95+PNAhi population, and numbers in each panel indicate the percentage in the DZ and LZ gates. E, Western blot analysis of Crebbp and Ep300 in sorted GC B cells from Crebbp+/+ (WT), Crebbpfl/+ (HET), and Crebbpfl/fl (KO) CD19-Cre mice. The relative intensity of the bands is quantified below the image after normalization for the loading control, with the wild type band arbitrarily set as 1. Bcl6 serves as control for the identity of the population. F, Representative flow cytometric analysis of splenic B220+ cells from 12-16 week-old mice of the indicated genotypes in the CD19-Cre cohort, analyzed 10 days after SRBC immunization. G, Percentage of GC B cells in mice from the indicated genotypes, analyzed at 3 months of age, 10 days after SRBC immunization. H, Analysis of DZ and LZ GC B cells in the CD19-Cre cohort. In all panels, ns denotes not significant, as calculated by one-way ANOVA. Supplementary Figure S5. Analysis of plasma cell differentiation in Crebbpfl/fl Cγ1-Cre and CD19 cohorts. A, Representative flow cytometric analysis of splenic plasma cells and surface IgG1+ B cells in Crebbp+/+ (WT), Crebbpfl/+ (HET), and Crebbpfl/fl (KO) Cγ1-Cre mice. Numbers denote the percentage of cells within the gate, relative to total lymphocytes. B, Percentage (top) and absolute number (bottom) of bone marrow (BM) plasma cells in 12-16 week-old mice of the indicated genotypes, analyzed 10 days after SRBC immunization. Each symbol represents one mouse, and the horizontal bar denotes the mean. C, Quantification of frequencies (top) and absolute numbers (bottom) of splenic plasma cells, BM plasma cells, and IgG1+ B cells in the CD19-Cre cohort, illustrated as in B (cumulative data from at least two independent experiments performed on 3-4 mice each, 10 days after SRBC immunization; data for BM plasma cells are also provided at day 7 post-secondary immunization in the far right panels). As reported, Crebbpfl/fl CD19-Cre mice show reduced percentages and numbers of B220+ cells (not shown) (4), impacting on the number of all mature B cell subsets. * P < 0.05, *** P < 0.005, one-way ANOVA. D, Antibody titers in the serum of 12-16 week-old Crebbp- floxed CD19-Cre mice, measured before (top) and 14 days after (bottom) immunization with the T-cell specific NP-KLH hapten. IgG3 and IgA titers were only obtained from 5 of the 6, Crebbp KO animals, due to insufficient amounts of serum. * P < 0.05, ** P < 0.01, Student’s t-test. Only statistically significant differences are highlighted in the figure. Supplementary Figure S6. Distribution pattern of CREBBP mutations in FL and de novo DLBCL. A, Percentage of CREBBP mutated samples showing monoallelic (red) vs biallelic (grey) mutations in DLBCL and FL/transformed FL (tFL). The total number of cases analyzed is given below the pie. B, Overall percentage of CREBBP/EP300 mutated cases harboring concurrent chromosomal translocations of BCL2 in FL, GCB-DLBCL and ABC-DLBCL. Data in the left panel refer to CREBBP mutated/deleted cases, while data in the right panel also include EP300 mutated cases. C, Distribution of CREBBP mutations, EP300 mutations and BCL2 chromosomal translocations in FL, GCB-DLBCL and ABC-DLBCL samples. In the heatmap, each column represents one case, and color codes denote the presence (red) or absence (white) of the genetic lesion. Tx, translocation; M, mutation; D, deletion. Only cases with available information for all indicated lesions were included in the analysis displayed in panels B and C. DLBCL data integrate published studies by our group, while data on FL cases derive from Morin et al., Nature 2011.