* * IgL locus (V - J ) λ λ

Transcription

1 Supplementary Figures and Figure Legend Supplementary Figure S1 A C F Vav-P-BCL2 doner mice Lymphoma free (%)1 Tumor Pathology Score Retroviral transduction Vav-P-BCL2 HPCs HPC + shrna VavP-Bcl2 HPCs GFP (n=1) Crebbp KD #3 (n=1) Irradiated recipent (WT) p= Time to disease onset (days) Spleen Kidney Lung vavp-bcl2/gfp vavp-bcl2/crebbp-kd vavp-bcl2/gfp vavp-bcl2/crebbp-kd vavp-bcl2/gfp vavp-bcl2/crebbp-kd E Lymphoma onset Crebbp KD GFP SSC B22 G B Crebbp relative mrna levels 73.2% 74.9% GFP B22 FL5-12 cells vector #1 #2 #3 sh-crebbp CD % 7.7% Crebbp KD * * IgL locus (V - J ) λ λ IgD 24.7% 33.6% H Mismatches % D 4.2% 5.97% 52.5% vector GFP sh-crebbp % 6.57% 8.68% IgM IgG1 GL7 Crebbp KD H3K27ac Total H3 Supplementary Figure S1. Crebbp deficiency results in accelerated germinal center derived B-

2 cell lymphoma development in mice. A, A schematic diagram showing the adoptive transfer model of FL. HPCs of the VavP-Bcl2 transgenic mouse were transduced with retroviral shrnas against either Crebbp or vector alone, and then reconstituted into lethally irradiated, syngenic, female mice. B, Bar plots showing the efficiency of shrnas against Crebbp measured by RT-qPCR in murine FL5-12 cells. C, Kaplan-Meier curve of C57BL/6 mice transplanted with VavP-Bcl2 HPCs transduced with MSCV-GFP retroviral vector alone (GFP, black, n=1), or containing a second shrnas against Crebbp (red, n=1). Statistical significance of survival difference was determined by the log-rank test between shcrebbp and vector alone. D, Western blots showing global H3K27ac and total histone H3 in B22+ enriched murine splenic tumour cells transduced with either Crebbp shrna or vector alone. E, Representative flow cytometry analysis for B22, CD19, IgD, IgM, IgG1 and GL7 of whole spleens from recipient mice in Figure 1A. F, Dot plots representing tumor pathology scores (see Methods) of the spleen, kidney and lung tissues from the VavP-Bcl2/GFP and the VavP-Bcl2/Crebbp KD animals. The mean and standard error of the mean were shown. G, Electrophoresis gel image showing the PCR analysis of the IgL locus of tumour cells isolated from spleens of recipient mice in Figure 1A. H, Dot plots showing the mismatch frequency of the VDJH locus of tumour cells isolated from spleens of recipient mice in Figure 1A. The mean and standard error of the mean were represented for each transplant group.

3 Supplementary Figure S2 A Ep3 relative mrna levels FL5-12 cells vector sh-ep3 B VavP-Bcl2 HPS Transplants 2 GFP n=31 Ep3 KD n=37 p= Time to disease onset (days) C Spleen H&E B22 CD3 Lymphoma Free (%) Ki67 GFP 5 μm 5 μm 5 μm 5 μm Ep3 KD H&E Kidney B22 H&E Lung B22 GFP 5 μm 5 μm 5 μm 5 μm Ep3 KD D Ep3 KD SSC 76.8% B22 68% 14.1% 42.3% 7.63% 8.34% B22 CD19 IgD IgM IgG1 GL7 E Ep3 KD IgL locus (V λ - J λ ) * F Mismatches % Ep3 KD

4 Supplementary Figure S2. Ep3 deficiency results in accelerated germinal center derived B- cell lymphoma development in mice. A, Bar plots showing the efficiency of shrna against Ep3 measured by RT-qPCR in murine FL5-12 cells. B, Kaplan-Meier curve of C57BL/6 mice transplanted with VavP-Bcl2 HPCs transduced with MSCV-GFP retroviral vector alone (GFP, black, n=31, same set of animals as in Figure 1A), or containing an shrnas against Ep3 (blue, n=37). Statistical significance of survival difference was determined by the log-rank test between shep3 and vector alone. C, H&E, B22, CD3, and Ki67 staining of spleen, kidney or lung tissues extracted from recipient mice upon sacrifice. Scale bars, 5 μm. The images from the VavP-Bcl2/GFP animals are the same shown in Figures 1D and 1E. D, Representative flow cytometry analysis for B22, CD19, IgD, IgM, IgG1 and GL7 of whole spleens from recipient mice. E, Electrophoresis gel image showing the PCR analysis of the IgL locus of tumour cells isolated from spleens of recipient mice. F, Dot plot showing the mismatch frequency of the VDJH locus of tumour cells isolated from spleens of recipient mice. The mean and standard error of the mean were represented for each transplant group.

5 Supplementary Figure S3 A kda shscr shcrebbp-1 shcrebbp-2 B kda shscr shcrebbp-1 shcrebbp CREBBP 15 H3K27ac 5 Tubulin 15 Total H3 C Others Enhancer Promoter 76.9% 19.% Supplementary Figure S3. A, Western blots showing the knockdown efficiency of shrnas against human CREBBP in human DLBCL MD91 cells. B, Western blots showing H3K27ac reduction in MD91 cells transduced with shrnas against human CREBBP. C, Stacked bar plot showing the genome-wide distribution of CREBBP ChIPseq peaks in OCI-Ly7 cells.

6 Supplementary Figure S4 A B Enrichment Score Log 2 FC Enhancer H3K27ac Loss > 25% in murine lymphomas NES=-1.24, NES= FDR q= FDR q= Crebbp -4 KD vs GFP -2 shcrebbp vs Scr -4 Supplementary Figure S4. A, GSEA enrichment plots showing correlation of genes with >25% loss of H3K27ac at enhancers in VavP-Bcl2/shCrebbp tumours with ranked expression change between B22+ lymphoma cells from VavP-Bcl2/shCrebbp tumours (n=3) and VavP-Bcl2/GFP tumours (n=3). NES, normalized enrichment score. B, GSEA of enrichment plots showing correlation of genes with >25% loss of H3K27ac reads at their enhancers in CREBBP KD MD91 cells with ranked expression change between CREBBP KD and Scr MD91 cells. Enrichment Score Log 2 FC Enhancer H3K27ac Loss > 25% in human DLBCL

7 Supplementary Figure S5 A H3K4me2+ H3K4me3- H3K27ac+ peaks (N=5219) Common peaks (N=179) NBC (N=7577) B Normalized H3K27ac read density NB Specific Specific NBC NBC (kb) (kb) Normalized H3K27ac read density C NB-H3K27ac -H3K27ac NB-GEP -GEP D NB Specific Active Enhancer IRF4 S1PR1 NB H3K27ac NB RNAseq NB H3K27ac NB RNAseq chr6: IRF4 chr1: S1PR1 CB Specific Active Enhancer (kb) Reads Density AICDA BCL6 z scores chr3: NB 15 H3K27ac 15 NB 15 RNAseq 15 BCL6 chr12: NB 3 H3K27ac 3 NB 15 RNAseq 15 AICDA Supplementary Figure S5. A, Venn diagram showing the overlap of H3K4me2+H3K4me3- H3K27ac+ (active enhancer) peaks between human tonsilar IgD+ Naïve B cells (NBs) and CD77+

8 germinal center B cells (s). B, Normalized H3K27ac read density profiles of the top NB specific (n=575, left panel) and specific (n=592, right panel) active enhancer peaks (FDR<.1) in either NB (red) or (blue) cells. C, Left panel: Read density heatmap of normalized H3K27ac at enhancer loci (interval ± 3kb from H3K27ac peak center) identified in either human tonsilar NB or cells. The data represents average of two biological replicates for each cell type. Right panel: heatmap represents the z-scores of the expression value (RPKM) characterized by RNA-seq of the top NB specific and specific H3K27ac associated genes in NB (n=7) and (n=6) cells. D, UCSC read-density tracks of normalized H3K27ac ChIP-seq and RNAseq reads at four representative gene loci. Shaded areas highlight the regions showed change of histone marks between NB and cells.

9 Supplementary Figure S6 RNAseq B-cell enhancer genes Enhancer K27ac loss genes in VavP-sh Crebbp Enhancer activity loss genes in human Enhancer K27ac loss genes in human DLBCL B-cell biological functions Plasma cell genes CD4 induced genes in lymphoma IRF4 induced genes in lymphoma Antigen presentation and MHC Class II B-cell transcription factor targets BCL6/SMRT enhancer target genes in sibcl6 up-regulated genes in DLBCL EP3 targets in DLBCL CREBBP targets in DLBCL H. DLBCL M. Crebbp KD Lymphomas -log 1 (BH adjusted p-value) > Top repressed genes Supplementary Figure S6. Pathway analysis of down-regulated genes in top 5 differentially expressed genes between murine VavP-Bcl2/shCrebbp tumours and VavP-Bcl2/GFP tumours (left panel), or between CREBBP knockdown and scramble control human DLBLC MD91 cells (right panel). Heatmap represents the BH-adjusted p value of each geneset tested.

10 Supplementary Figure S7 -log 1 (BH adjusted p-value) > Biological processes Pr Proliferation signature genes in DLBCL Upregulated in plasma cells IRF4 induced genes in lymphoma IL1 induced genes in lymphoma Downregulated genes upon Glutamin starvation B-cell transcription factor targets Pr BCL6 promoter bound genes in BCL6 promoter bound genes in DLBCL BCL6/SMRT enhancer target geens in DLBCL BCL6/SMRT enhancer target genes in Upregulated genes upon sibcl6 in DLBCL EP3 promoter bound genes in DLBCL EP3 enhancer target genes in DLBCL PU.1 promoter bound gene in PU.1 enhancer target genes in CTCF enhancer target genes in IKZF1 promoter bound genes in IKZF1 enhancer bound genes in Supplementary Figure S7. Pathway analysis of genes with > 25% reduction of H3K27ac reads at promoters in murine VavP-Bcl2/shCrebbp tumors. Heatmap represents the BH-adjusted p value of each geneset tested.

11 Supplementary Figure S8 BCL6 () SMRT () H3K27ac (NBC) H3K27ac () chr16:1,95,-11,2, chr5:149,781,-149,83, H3K27ac (Scr) H3K27ac (shcrebbp) CIITA CD74 Supplementary Figure S8. UCSC read-density tracks of normalized BCL6 (purple) and SMRT (orange) ChIP-seq reads in human tonsilar s, H3K27ac ChIP-seq reads in human tonsilar NBCs (red) and s (blue), and H3K27ac ChIP-seq in control scramble (Scr, green) and CREBBP KD (shcrebbp, turquoise) MD91 cells at the human CIITA and CD74 loci. BCL6 and SMRT peaks determined by MACS2 are indicated by grey bars under the read density track. Shaded areas highlight the enhancers that were bound by BCL6 and SMRT, and showed loss of H3K27ac in CREBBP KD cells.

12 Supplementary Figure S9 A Percent FL FL DLBCL Cohort 1 (Exome) (N=26) Cohort 2 (Targeted) (N=78) Cohort 3 (Targeted) (N=347) WT Mut D Numbers DLBCL, Cohort 3 vs ABC, p=1.42e-5 Mutated WT ABC Not Classified B TAZ KIX Bromo HAT ZZ TAZ CBD Nonsense Missense a.a a.a HAT a.a a.a C FL Sample 5 FL Sample 15 FL Sample 25 Tumor p.r148c/c.4222g>a Germline Supplementary Figure S9. A, Stacked bar plots showing the frequencies of CREBBP somatic

13 mutations in two cohorts of FL and one cohort of DLBCL primary patient samples. B, Schematic diagram representing the CREBBP somatic mutations identified in two cohorts of FLs. C, Sanger sequencing validation of CREBBP somatic mutation p.r148c/c.4222g>a in three FL samples from cohort 1. D, Bar plots showing the number of samples harbouring CREBBP mutations by DLBCL subtypes (cohort 3). Statistical significance was determined by Fisher Exact Test.

14 Supplementary Figure S1 A Cohort 1 TOP1 TOP5 TOP1 31% 31% 33% 69% 68% 67% B Enrichement Score (ES) Enrichment Plot, C1-DOWN NES=-1.82, FDR= Enrichment Plot, C1-UP NES=1.27, FDR=.38 Cohort 2 61% 39% 59% 41% 53% 47% Ranked List Metric 1..5 C3-UP C3-DOWN Rank in Ordered List C3-UP C3-DOWN Rank in Ordered List Cohort 3 55% 45% 6% 4% 62% 38% Enrichement Score (ES) Enrichment Plot, C2-DOWN NES=-2.3, FDR= Enrichment Plot, C2-UP NES=2., FDR= UP DOWN Ranked List Metric 1..5 C3-UP C3-DOWN Rank in Ordered List C3-UP C3-DOWN Rank in Ordered List Supplementary Figure S1. A, The proportions of the Up (up-regulated) and Down (down-regulated) genes in the top 1, 5, and 1 most differentially expressed genes of the respective cohorts profiled in Figure 3. B, GSEA enrichment plots showing the top down-regulated genes in CREBBP MUT patients in cohort 1 and 2 are significantly enriched in the top down-regulated genes in CREBBP MUT patients of cohort 3 (left panels); and the top up-regulated genes in CREBBP MUT patients in cohort 1 and 2 are significantly enriched in the top up-regulated genes in CREBBP MUT patients of cohort 3 (right panels).

15 Supplementary Fig. S11 A GL FAS NS-DADwt NS-DADmut B GC B cells (GL7 + FAS + ) %B22 + DAPI NS-DADwt p=.8 NS-DADmut Supplementary Figure S11. A, Representative flow cytometry analysis for FAS and GL7 of whole spleens from NS-DAD wild type (wt) or mutant (mut) animals. B, Dot plots showing the percentage of GC B cells (GL7 + FAS + ) in total live B cells (B22 + DAPI - ) in spleens of either NS-DADwt animals (n=8) or NS-DADmut animals (n=7). Mean and standard error of the mean (S.E.M) are shown. Statistical analysis was performed using student t-test.

16 Supplementary Figure S12 A WB OCI-Ly7 ishluc ishhdac3-1 ishhdac3-2 MD91 ishluc ishhdac3-1 ishhdac HDAC3 ACTIN B efluor 67 MFI 5 ishluc ishhdac3-1 ishhdac3-2 OCI-Ly7 MD91 OCI-Ly18 OZ RIVA WT MUT C D OCI-Ly7 MD91 OCI-Ly18 OZ RIVA Tunnel ishluc Scr shhdac3 ishhdac3-1 ishhdac OCI-Ly7 FARAGE 5 μm 5 μm 5 μm 5 μm DAPI Annexin V Supplementary Figure S12. A, Western blots showing the knockdown efficiency of shrnas against human HDAC3 in DLBCL cell lines OCI-Ly7 and MD91 12 hr after induction of shrna expression. The amount of HDAC3 was quantified by using ImageJ, and the ratio of shrna knockdown samples and the control shluc sample was indicated below the blots. B, Quantification of the eflore67 signals in Figure 4J. Bar plots represent the mean and S.E.M. of the mean fluorescence intensity (MFI) of the eflore67 measured at 12 hr after the initial pulse. C, Flow cytometry zebra plots

17 showing the apoptotic cell population (Annexin V +, DAPI -, bottom right quadrant) in cells transduced with inducible shrnas against human HDAC3. Cell staining and flow cytometry analysis were performed 12 hr after induction of shrna expression. Cells were gated on the GFP+ population indicating the expression of shrnas. D, Representative images of tunnel staining of OCI-Ly7 or FARAGE xenograft tumours with either scramble or HDAC3 knockdown.

18 Supplementary Figure 13 Erichment (fold, over input) sint+ip HDAC3 sibcl6+ip HDAC3 sint+ip BCL6 sibcl6+ip BCL6 CD 74 DOA Supplementary Figure S13. BCL6 and HDAC3 ChIP-qPCR at enhancers of CD74 or HLA-DOA (primer sequences please check Supplementary Table S12) in OCI-Ly1 cells transfected with either non-target sirna (sint) or BCL6 sirna (sibcl6).

19 Supplementary Figure 14 A. B. Exp2 HLA-DR (log A.U.) MD91 # # * * Ctrli HDAC3i Ctrli HDAC3i shcrebbp-1 shcrebbp-2 Exp2 shscr Ctrli shscr HDAC3i shcrebbp-1 Ctrli shcrebbp-1 HDAC3i MD91 OCI-Ly18 HLA-DR (log A.U.) OCI-Ly18 # # * * Ctrli HDAC3i Ctrli HDAC3i shcrebbp-1 shcrebbp-2 shscr Ctrli shscr HDAC3i shcrebbp-2 Ctrli shcrebbp-2 HDAC3i HLA-DR C. Exp2 T-cell Proliferation (relative to shscr+ctrli) MD91 * * Ctrli HDAC3i Ctrli HDAC3i shcrebbp-1 shcrebbp-2 T-cell Proliferation (relative to shscr+ctrli) OCI-Ly18 * * Ctrli HDAC3i Ctrli HDAC3i shcrebbp-1 shcrebbp-2

20 Supplementary Figure S14. A, Quantification of HLA-DR measured by flow cytometry in shcrebbp or scramble transduced lymphoma cells MD91 and OCI-Ly18 in a second biological replicate experiment. Cells were treated with either a selective HDAC3 inhibitor (HDAC3i) or a control compound (Ctrli). Cells were transduced with shrnas for 3 days and then treated with compounds for 96hr. The data was represented as mean ± S.D. Statistical significance was determined by Student s T-test. * indicates significant difference between control compound-treated scamble or shcrebbp transduced cells (p<.5). # indicates significant difference between control compound and selective HDAC3 inhibitor treated cells (p<.5). B, Representative flow cytometry histograms showing cell surface level of MHC II molecule HLA-DR that were quantified in A. C, Bar plots showing the relative proliferation of T-cells stimulated by shcrebbp or scramble transduced lymphoma cells treated with either a selective HDAC3 inhibitor (HDAC3i) or a control compound (Ctrli) in a second biological replicate experiment. The data indicate relative folds of T cell proliferation, represented as ratio of fluorescence (56nm/59nm) from each treatments to that from scramble shrna transduced cells treated with control compound (set as 1, dotted lines). The data was represented as mean ± S.D. Statistical significance was determined by Student s T-test.