Smc3 dosage regulates B cell transit through germinal centers and restricts their malignant transformation

Abstract

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation.

Extended Data Fig. 1. Smc3 knockout abrogates GC formation whereas Smc3 haploinsufficiency induces GC Hyperplasia.

(a) Box plots showing transcripts per million (TPM) of cohesin core subunits (Smc3, Smc1a, Stag1, Stag2 and Rad21) and regulator proteins (Nipbl, Wapl, Pds5a, Pds5b) in mouse and human (b). NB: naive B cells, CB: centroblasts, CC: centrocytes, PC: splenic plasma cells, TPC: tonsillar plasma cells, BMPC: bone marrow plasma cells. (c) Scheme depicting Smc3 conditional knockout model breeding strategy and (d) genotyping strategy. (e) Gating strategy used for flow cytometric analysis of GC B cells. (f) Genotyping of sorted GC B cells. Representative gel of one experiment out of four performed. (g) Representative flow cytometry plots showing total B cells (B220⁺ cells) and follicular B cells (B220⁺CD23^hiCD21^lo cells) and marginal zone B cell populations (B220⁺CD23^loCD21^hi cells) and quantitative data for one representative experiment out of 4 performed in Smc3^wt/wt (n=6), Smc3^wt/– (n=4) and Smc3^–/– (n=3) as shown (right). One-way ANoVA followed by Tukey test for multiple comparisons was used. (h-i) Quantification of GC B cells, defined as B220⁺Fas⁺CD38^–, 4 days (h, p=0.002) or 15 days (I, p=0.0434) after SRBC immunization. Two-tail unpaired t-test was used. (j) RT-qPCR comparing Smc3 mRNA expression in GC B cells (centroblasts, CB and centrocytes, CC), plasmablasts (PB) and plasma cells (PC) in Cγ1^wt/cre;Smc3^wt/wt and Cγ1^wt/cre;Smc3^wt/– mice as indicated. Two-tail unpaired t-test was used. CB, p=0.0039, CC: p=0.0005, PB: p=0.0041 and PC: p=0.0271. (k) Capillary immunoblot analyses comparing Smc3, Smc1a, Rad21 and Sag2 protein levels in centroblasts (CB), centrocytes (CC) and naïve B cells (NBC) from Cγ1^wt/cre;Smc3^wt/wt (n=3) and Cγ1^wt/cre;Smc3^wt/– (n=3) mice as indicated. Representative gel our of two experiments performed. Right panels show quantifications of the cohesin complex subunits using β-actin protein as loading control. Two-way ANoVA was used. **p<0.01. In (a) and (b) box plots show the median as center, first and third quartiles as the box hinges, and whiskers extend to the smallest and largest value no further than the 1.5 × interquartile range (IQR) away from the hinges. In (g), (h), (i), (j) and (k) data are presented as mean +/− SD.

Extended Data Fig. 2. Smc3 haploinsufficiency confers proliferative advantage to GC B cells without chromosomal instability.

(a) Cγ1^wt/cre;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Smc3^wt/– (n=5) were immunized with SRBCs to induce GC formation. One hour prior to euthanasia, mice were injected with 50 mg/kg i.v. 5-ethynyl-2’-deoxyuridine (EdU). (b) GC organoids were performed with splenocytes from Cγ1^wt/cre;Smc3^wt/wt and Cγ1^wt/cre;Smc3^wt/– mice. (c) On day 0 and day 4 after organoid plating, the presence of B cells and GC-like cells (B220⁺Fas⁺GL7⁺) was determined by flow cytometry. (d) Gating strategy followed to detect apoptotic active Caspase-3⁺ GC B cells.

Extended Data Fig. 3. GC hyperplasia induced by Smc3 haploinsufficiency is due to expansion of centrocytes.

(a) Mice were immunized with sheep red blood cells (SRBCs) to induce GC formation and euthanized 8 days after. (b) Gating strategy followed to identify DZ (B220⁺Fas⁺CD38^–CXCR4⁺CD86^–) and LZ (B220⁺Fas⁺CD38^–CXCR4^–CD86⁺) GC B cells. (c) Quantification of absolute numbers of DZ centroblasts (Cγ1^wt/cre;Smc3^wt/wt (n=7) and Cγ1^wt/cre;Smc3^wt/– (n=6) mice) or LZ centrocytes (Cγ1^wt/cre;Smc3^wt/wt (n=6) and Cγ1^wt/cre;Smc3^wt/– (n=6) mice). (d) Cγ1^wt/cre;Smc3^fl/fl were crossed to Rosa26^YFP/YFP to produce mice that express the yellow fluorescent protein when expressing the cre recombinase. (e) Gating strategy used in Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/– and Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/wt mice to identify YFP⁺ GC B cells. (f) Plot showing YFP⁺ GC B cells in the reporter Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/– (n=4) and Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/wt (n=9) mice. In (c) and (f) data are presented as mean +/− SD.

Extended Data Fig. 4. Smc3 haploinsufficient mice have impaired terminal differentiation

(a) Mice were immunized on day 0 with 100 μg NP-KLH:Alum 1:1 i.p., and received a boost on day 21 of 100 μg NP-CGG: Alum 1:1 i.p. Mice were bleed on days 0, 14, 26, 35 and 70, at which time were euthanized. (b) Representative flow cytometry gating strategy followed to determine the presence of bone marrow plasma cells (BMPC, CD138⁺B220^– cells) and long-lived BMPCs (NPcyt⁺IgG1cyt⁺CD138⁺B220^– cells). (c) Representative flow cytometry gating strategy followed to determine the percentage of IgM- and IgG1-expressing GC B cells. (d) Frequency of IgG1⁺, IgM⁺, or IgG1⁺/IgM⁺ GC B cells in 4, 8 or 15 day SRBC immunized Cγ1^wt/cre;Smc3^wt/wt (n=5) or Cγ1^wt/cre; Smc3^wt/– (n=5) mice. Two tail unpaired t-test. All differences are statistically non-significant. (e) Splenocytes from Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/– (n=5) mice were cultured in the presence of IL-4, LPS and anti-CD40 antibody and PB/PC followed by flow cytometry and (f) levels of active Cspase-3 were detected by flow cytometry using the presented gating strategy. (g) Active Caspase-3 levels in B cells, PBs and PCs from ex vivo cultures. (h-i) 8, 15 and 25-day immunized mice were sacrificed and splenocytes stained for GC markers. Nuclear staining of Irf4 was done afterwards. (h) Representative flow cytometry plots of IRF4⁺ GC B cells and (i) plots depicting the percentage of IRF4⁺ GC B cells in Cγ1^wt/cre;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Smc3^wt/– (n=5) mice (at every time point). (j) At each time point, mice were injected with 50 μg/kg EdU i.p. and sacrificed one hour later. Quantification of IRF4⁺ GC B cells in S-phase in the spleens of SRBC immunized mice for the indicated times in Cγ1^wt/cre;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Smc3^wt/– (n=5) mice at every time point. **p<0.01, ***p<0.001 and ****p<0.0001. In (d), (i) and (j) data are presented as mean +/− SD. (i) and (j) two-way ANoVA and Sidak multi-comparison test was used.

Extended Data Fig. 5. Smc3^wt/– centrocytes fail to upregulate GC exit genes and transition towards the plasma cell lineage.

(a) Gene set enrichment analysis (GSEA) plots for TET2_KO and shKMT2D_DOWN. (b) Sorting strategy used for single cell RNA-seq by BDRhapsody.

Extended Data Fig. 6. Smc3 haploinsufficiency disrupts intra-TAD interactions and GC exit gene looping

(a) Genome wide chromosomal interactivity was mapped by in situ Hi-C in Cγ1^wt/cre;Smc3^wt/wt (n=2) or Cγ1^wt/cre;Smc3^wt/– (n=2) centrocytes (B220⁺Fas⁺CD38^–CXCR4^–CD86⁺) from 8 day SRBC immunized mice. (b) Cartoon depicting the definition of intra- and inter-TAD connectivity. (c) Log₂ fold change of inter- and intra-TAD interactions in Smc3 haploinsufficient versus wild-type centrocytes. p-value expresses significant difference between inter- and intra-TAD interactions. Wilcoxon rank-sum test was used, p<2.2×10^–16. (d) TAD boundary strength for Smc3 haploinsufficient centrocytes versus wild-type centrocytes by Hi-C. Wilcoxon rank-sum test was used, p<2.2×10^–16. (e) z-score of promoter short-range interactivity change plotted for genes which are downregulated, non-changing or upregulated in centrocyte RNA-seq of Smc3 haploinsufficient cells versus control. Wilcoxon rank-sum test was used, Down vs NS p<1.6×10^–7, NS vs Up p=0.055 and Down vs Up p=0.00024. (f) Gene set enrichment of compartment changes in Smc3 haploinsufficient vs wild-type centrocytes. Compartment C-scores are calculated in 100 kb intervals and annotated to genes based on the location of their TSS within these bins. Genes are ranked by Δ c-score and analyzed by GSEA. (g) Gene set enrichment of enhancer-promoter interactivity changes in Smc3 haploinsufficient vs wild-type centrocytes. Enhancers are annotated to the nearest TSS (as explained in Methods), normalized contacts between a gene and all its enhancers are summed to get an aggregate promoter-enhancer looping score. Genes are ranked by Δ promoter-enhancer score between Smc3 haploinsufficient and wild-type mice and analyzed by GSEA. In (c) and (e) box plots show the median as center, first and third quartiles as the box hinges, and whiskers extend to the smallest and largest value no further than the 1.5 × interquartile range (IQR) away from the hinges.

Extended Data Fig. 7. Smc3 dosage does not impair B cell differentiation and is only required for GC exit.

(a) Scheme depicting Smc3 conditional knockout model breeding strategy. (b) Early B cell differentiation stages and markers used for identification. (c) Representative flow cytometry plots of splenic CD3⁺ T cells, B220⁺ B cells and naïve B cells in CD19^wt/cre;Smc3^wt/wt (n=4), C19^wt/cre;Smc3^wt/– (n=7) and C19^wt/cre;Smc3^–/– (n=4) and (d) quantification of ratio between splenic B220⁺/CD3⁺ cells in Cd19^wt/cre;Smc3^wt/wt (n=4), Cd19^wt/cre;Smc3^wt/– (n=7) and Cd19^wt/cre;Smc3^–/– (n=4) (****p<0.0001) or (e) naïve B cells in Cd19^wt/cre;Smc3^wt/wt (n=5) and Cd19^wt/cre;Smc3^wt/– (n=9). (f) Quantification of the percentage of bone marrow pre-BI cells, immature B cells, and splenic follicular B cells in Cd19^wt/cre;Smc3^wt/wt (n=4) and Cd19^wt/cre;Smc3^wt/– (n=5). NS: non-significant differences. In (d), (e), and (f) data are presented as mean +/− SD. In (d) and (e) one-way ANoVA and Tukey multi-comparison test was used, and two-way unpaired t-test in (f).

Extended Data Fig. 8. Haploinsufficiency of Smc3 cooperates with Bcl6 to induce lymphoma.

(a) Bone marrow transplantation experimental design. Bone marrow cells were isolated from Cγ1^wt/cre;Smc3^wt/wt, Cγ1^wt/cre;Smc3^wt/–, IμBcl6;Cγ1^wt/cre;Smc3^wt/w and IμBcl6;Cγ1^wt/cre;Smc3^wt/– CD45.2 donors, and transplanted through tail vein injection into lethally irradiated CD45.1 mice. After engraftment, mice were immunized with SRBCs every 3 weeks for the duration of the experiment. Mice were euthanized when presented with signs of disease. (b) Representative flow cytometry plots performed 300 days after engraftment showing that all B220⁺ are CD45.2⁺ and CD45.1^–, demonstrating that all B cells in these mice come from the donor and not from the recipient. (c) Representative allele occurrence was showed by performing PCR in DNA from tumors from IμBcl6;Cγ1^wt/cre;Smc3^wt/w and IμBcl6;Cγ1^wt/cre;Smc3^wt/– mice. One representative gel is shown out of 2 experiments performed.

Figure 1.. Smc3 knockout abrogates GC formation whereas Smc3 haploinsufficiency induces GC Hyperplasia.

(a-f) Cγ1^wt/cre;Smc3^wt/wt (n=6), Cγ1^wt/cre;Smc3^wt/– (n=5) and Cγ1^wt/cre;Smc3^–/– (n=3) were immunized with sheep red blood cells (SRBCs) to induce GC formation. (a) Immunohistochemistry (IHC) of GC using peanut-agglutinin (PNA), the proliferation marker Ki67, the B cell marker B220 or hematoxylin and eosin (H&E) in representative tissue sections in Smc3^wt/wt, Smc3^wt/– and Smc3^–/– mice as indicated. For pictures in the first, third, fifth and seventh column bar measure is 500 μm, and for pictures in the second, fourth, sixth and eighth column bar measure is 100 μm. (b) Quantitative data of the number of GC per spleen area from IHC images and (c) quantitative data of the GC area from IHC images. (d) Representative flow cytometry plots of GC B cells, defined as B220⁺Fas⁺CD38^– (top row) or B220⁺Fas⁺GL7⁺ (lower row) in Smc3^wt/wt, Smc3^wt/– and Smc3^–/– mice. (e-f) Quantitative GC B cell data for one representative experiment out of 4 performed as shown. **p<0.01, ***p<0.001 and ****p<0.0001. In (b), (c), (e) and (f) data are presented as mean +/− SD. In all four cases, one-way ANoVA and Tukey multi-comparison test was used.

Figure 2.. Smc3 haploinsufficiency confers proliferative advantage to GC B cells without chromosomal instability.

(a-b) Cγ1^wt/cre;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Smc3^wt/– (n=5) were immunized with SRBCs to induce GC formation. One hour prior to euthanasia, mice were injected with 50 mg/kg i.v. 5-ethynyl-2’-deoxyuridine (EdU). (a) Representative flow cytometry plots of cell cycle distribution of GC B cells and (b) quantitative data for one representative experiment out of 2 performed as shown, *p=0.0492. (c) Four days after plating, organoids were incubated 30 min in the presence of 3 μg/ml 5-bromo-2’-deoxyuridine (BrdU) and analyzed by flow cytometry for the presence of GC-like cells (B220⁺Fas⁺GL7⁺). Percentage of cells in S-phase of the cell cycle was analyzed by BrdU staining. (d) Quantification of the frequency of GC-like cells, ***p<0.0001 and (e) S-phase GC-like cells in Smc3^wt/fl (n=3) and Smc3^wt/– (n=3), **p=0.0042. (f) Genotyping for the detection of Smc3^wt, Smc3^fl and Smc3^– alleles from organoids in figure (c). Genotyping was routinely performed to confirm excision of the Smc3 allele. (g) Quantification of the percentage of Caspase-3⁺ GC B cells in Cγ1^wt/cre;Smc3^wt/wt (n=8) and Cγ1^wt/cre;Smc3^wt/– (n=8), p=0.7079. In (b), (d), (e) and (g) data are presented as mean +/− SD. In all four cases, two tail unpaired t-test was used.

Figure 3.. GC hyperplasia induced by Smc3 haploinsufficiency is due to expansion of centrocytes.

(a-e) Mice were immunized with sheep red blood cells (SRBCs) to induce GC formation and euthanized 8 days after. (a) Representative flow cytometry plots of dark zone (DZ) and light zone (LZ) GC B cells, defined as B220⁺Fas⁺CD38^–CXCR4⁺CD86^– (DZ) or B220⁺Fas⁺CD38^–CXCR4^–CD86⁺ (LZ) in Cγ1^wt/cre;Smc3^wt/wt and Cγ1^wt/cre;Smc3^wt/– mice. (b) LZ-to-DZ ratio calculated for Cγ1^wt/cre;Smc3^wt/wt (n=17) and Cγ1^wt/cre;Smc3^wt/– mice (n=11). **p<0.0036. (c) Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/wt (n=3) and Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/– (n=3) mice were injected with 2.5 μg anti-CD35-BV421 at day 7 after immunization, and sacrificed at day 8. Spleens were fixed in 4% paraformaldehyde overnight and cut at 70 μm in a vibratome, mounted and imaged in a Zeiss LSM 880 Airyscan High Resolution Detector confocal microscope. For pictures in the first to third column bar measure is 250 μm, and for pictures in the fourth to sixth column bar measure is 100 μm. (d) LZ to DZ area was calculated using Image J as CD35-BV421⁺YFP surface area / YFP surface area. **P<0.0014. In (b) and (d) data are presented as mean +/− SD. In both cases, two tail unpaired t-test was used.

Figure 4.. Smc3 haploinsufficiency impairs plasma cell differentiation in vivo.

(a) Mice were immunized on day 0 with 100 μg NP-KLH:Alum 1:1 i.p., and received a boost on day 21 of 100 μg NP-CGG:Alum 1:1 i.p. Mice were bleed on days 0, 14, 26, 35 and 70, at which time were euthanized. ELISA of anti-NP₍₈₎ or NP₍₃₀₎ immunoglobulin isotypes (IgG1, IgG2b, IgG3 and Igλ) in serum of 14, 26, 35 and 70 day NP-KLH/NP-CGG-immunized Cγ1^wt/cre;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Smc3^wt/– (n=5) mice. All comparisons versus Cγ1^wt/cre;Smc3^wt/wt. ns: no significant statistical differences, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Two-way ANoVA and Sidak multi-comparison test was used. (b) ELISpot analyses and quantification of NP₍₈₎ and NP₍₃₀₎ specific IgG1 in bone marrow cells isolated from Cγ1^wt/cre;Smc3^wt/wt and Cγ1^wt/cre;Smc3^wt/–, as indicated. Two-way ANoVA and Sidak multi-comparison test was used. *p<0.05. (c) Quantification of bone marrow plasma cell (BMPC) from 70 day-immunized Cγ1^wt/cre;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Smc3^wt/– (n=4) mice. *p=0.033. Two tail unpaired t-test was used. (d) Quantification of IgG1⁺ cytoplasmatic NP⁺ cytoplasmatic BMPC from 70 day-immunized Cγ1^wt/cre;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Smc3^wt/– (n=4) mice. *p=0.0156. Two tail unpaired t-test was used. (e) Splenocytes from Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/wt (n=5) and Cγ1^wt/cre;Rosa26^YFP;Smc3^wt/– (n=5) mice were cultured in the presence of IL-4, LPS and anti-CD40 antibody and plasmablast (PB) and plasma cell (PC) occurrence was assessed by flow cytometry. Day 1 through day 7 flow cytometry plots are shown in (e) and PB (B220⁺CD138⁺) and PC (B220^–CD138⁺) quantification is shown on panel (f). Multiple t-test with Holm-Sidak correction for multiple comparison was used. Adjusted p-values for PB at days 5, 6 and 7: 0.000253, 0.000090 and 0.004262, and for PC at days 6 and 7: 0.000076 and <0.000001, respectively. (g) mRNA levels were determined by RT-qPCR performed in total RNA from B220⁺CD138⁺ PB from ex vivo cultures. Two-way ANoVA and Sidak multi-comparison test was used. **p<0.01, ***p<0.001, ****p<0.0001. In (a), (b), (c), (d), (f) and (g) data are presented as mean +/− SD.

Figure 5.. Smc3^wt/– centrocytes fail to upregulate GC exit genes and transition towards the plasma cell lineage.

(a) Supervised hierarchical clustering analysis of centrocyte RNA-sequencing. Heatmap shows differentially expressed genes in Cγ1^wt/cre;Smc3^wt/– (n=3) versus Cγ1^wt/cre;Smc3^wt/wt (n=3). (b) Gene set enrichment analysis (GSEA) for the differentially expressed gene in Cγ1^wt/cre;Smc3^wt/– (n=3) versus Cγ1^wt/cre;Smc3^wt/wt (n=3). (c) Gene expression of representative genes involved in proliferation or tumor suppressor genes. (d-f) UMAP representation of single-cell RNA-seq by BDRhapsody Immune Response Targeted Panel (mouse) + custom mouse panel (Supplementary Table 2). (d) LZ, DZ, PC and recycling signatures from RNA-seq analysis were projected into UMAP plots. (e) Single cells were identified as belonging to one of the following clusters, according to the gene signature expression: light zone (LZ) dark zone (DZ), T cells, or plasmablasts (PB). (f) Expression levels of germinal center (Aicda, Bcl6), plasma cell (Xbp1), centrocyte genes (Cd86, Cd86), T cell (Cd4, Trac), proliferation (Mki67) and recycling genes (Myc) were projected into UMAP plots. (g) Slingshot analysis was performed in GC B cells and PB. (h-i) Cells were projected in a PCA plot and pseudotimes were derived for the different populations in the GC. (j) DZ, LZ and PB density plotted along the differentiation pseudo-time and (k) difference in pseudo-time in Smc3 haploinsufficient minus wild-type cells are shown.

Figure 6.. Smc3 haploinsufficiency disrupts intra-TAD interactions and GC exit gene looping.

(a) (a) Normalized chromosomal contacts for chromosome 1 of combined Cγ1^wt/cre;Smc3^wt/wt and Cγ1^wt/cre;Smc3^wt/– centrocytes. (b) Insulation contact maps example for chr19 showing insulation for different cell transitions: wild-type centroblasts (CB) minus wild-type naïve B cells (NBC), wild-type centrocytes (CC) minus wild-type CB, Smc3-haploinsufficient CC minus wild-type CC, and wild-type PC minus wild-type CC. x-axis shows chromosomal location, y-axis shows different insulation square sizes. (c) log₂ fold change plot showing intra-TAD vs inter-TAD connectivity. Wilcoxon rank-sum test was used, intra TAD connectivity p<2.2×10^–16 and inter TAD connectivity p<2.2×10^–16. (d) Connectivity maps for a representative region of chr19 showing overall connectivity for Smc3 haploinsufficient and wild-type CC and the difference between them. (e) Meta-TAD analysis summarizing results for all TADs stretched to the same size. Plots show the overall connectivity for centrocyte wild-type (upper), Smc3 haploinsufficient (middle) and log₂ fold change (lower). (f) Difference in genome-wide contacts by distance of centrocytes Smc3 haploinsufficient normalized over wild-type. Wilcoxon rank-sum test was used, <50 kb p<2.2×10^–16, 50–250 kb p<2.2×10^–16. (g) Proximal connectivity changes by region type (gene promoter or enhancer). Wilcoxon rank-sum test was used, enhancer vs promoter p<1.2×10^–32 and enhancers or promoters versus baseline p=0. (h) GSEA enrichment analysis for Smc3 haploinsufficient gene expression signature is enriched among genes that lose interactions by Hi-C. (i) Proximal connectivity difference for enhancers in Smc3 haploinsufficient versus wild-type centrocytes, normalized to mean background difference (top) and tornado plot showing ~12,000 individual enhancers. For each 5 kb bin the aggregate total proximal connectivity was taken by summing any contact within 100 kb of a bin. Delta proximal connectivity in Cγ1^wt/cre;Smc3^wt/– versus Cγ1^wt/cre;Smc3^wt/wt was calculated across each bin, and normalized to the background change by shifting the mean to zero. These differences were anchored around putative enhancers from H3K27ac peaks within centrocytes to show overall connectivity loss of centrocyte enhancers. (j) virtual 3C analysis of the Hi-C data for representative genes (Dusp2, Dusp4 and Zeb2) that lose promoter-enhancer connectivity in Smc3 haploinsufficient centrocytes. In (f) and (g) box plots show the median as center, first and third quartiles as the box hinges, and whiskers extend to the smallest and largest value no further than the 1.5 × interquartile range (IQR) away from the hinges.

Figure 7.. Smc3 dosage does not impair B cell differentiation and is only required for GC exit.

(a-h) Cd19^wt/cre;Smc3^wt/wt, Cd19^wt/cre;Smc3^wt/– and Cd19^wt/cre;Smc3^–/– were immunized with SRBCs to induce GC formation. (a) IHC of GC using PNA, Ki67, B220 or H&E in representative tissue sections in Smc3^wt/wt, Smc3^wt/– and Smc3^–/– mice as indicated. For pictures in the first, third and fifth column bar measure is 500 μm, and for pictures in the second and fourth column bar measure is 200 μm. One representative experiment is shown out of 3 performed. (b) Quantitative data of the number of GC per spleen area from IHC images of Cd19^wt/cre;Smc3^wt/wt (n=5) and Cd19^wt/cre;Smc3^wt/– (n=5) and (c) quantitative data of the GC area from IHC images. **p<0.01. (d) Representative flow cytometry plots of GC B cells, defined as B220⁺Fas⁺CD38^– or B220⁺Fas⁺GL7⁺ in Cd19^wt/cre;Smc3^wt/wt and Cd19^wt/cre;Smc3^wt/– mice. (e) Quantitative GC B cell data for one representative experiment with Cd19^wt/cre;Smc3^wt/wt (n=4) and Cd19^wt/cre;Smc3^wt/– (n=7) out of 3 performed as shown. ****p<0.0001. (f) LZ-to-DZ ratio calculated for Cd19^wt/cre;Smc3^wt/wt (n=8) and Cd19^wt/cre;Smc3^wt/– mice (n=6). ****p<0.0001. (g) Cd19^wt/cre;Smc3^wt/wt (n=8) and Cd19^wt/cre;Smc3^wt/– mice (n=6) were immunized with SRBCs to induce GC formation. One h prior to euthanasia, mice were injected with 50 mg/kg i.v. EdU. Quantitative data for one representative experiment out of 2 performed as shown (right). **p<0.01. (h) Apoptosis levels were determined by detection of Annexin V⁺ in gated GC B cells in Cd19^wt/cre;Smc3^wt/wt (n=4) and Cd19^wt/cre;Smc3^wt/– (n=5). (i) Phylogeny trees from compartments scores for plasma cell lines (THB-7 and MPC 11 OUAr), and FACS sorted naive B cells, centroblasts and centrocyte Hi-C. In (b), (c), (e), (f), (g) and (h) data are presented as mean +/− SD. In all cases, two tail unpaired t-test was used.

Figure 8.. Smc3 deficiency accelerates malignant transformation of GC B cells and is linked to inferior outcome of DLBCL patients.

(a) Spleen/body weight ratio of IμBcl6;Cγ1^wt/cre;Smc3^wt/– (Bcl6/Smc3) mice compared to Cγ1^wt/cre;Smc3^wt/wt (control), Cγ1^wt/cre;Smc3^wt/– (Smc3) or IμBcl6;Cγ1^wt/cre;Smc3^wt/wt (Bcl6) mice. Box plots show the median as center, first and third quartiles as the box hinges, and whiskers extend to the smallest and largest value no further than the 1.5 × interquartile range (IQR) away from the hinges. **p<0.01. (b-e) Representative images of histological analyses in (b) spleen, (c) mesenteric lymph node, (d) lung and (e) liver. Representative images from one mouse out of >30 per group. Hematoxylin and eosin staining (H&E) and B220 immunohistochemistry is shown in samples from control, Smc3, Bcl6 and Bcl6/Smc3 mice. In (b) an (c), for pictures in the first and third row bar measure is 1 mm, and for pictures in the second and fourth row bar measure is 250 μm. In (d) and (e), bar measure is 1 mm. (f) Percentage of tumor bearing mice 300 days after bone marrow transplantation in the different cohorts of mice as indicated (n=5 per group). (g) Kaplan-Meier analysis showing percentage of mice surviving lymphoma-free in Bcl6/Smc3 mice compared to control, Smc3 and Bcl6 cohorts. Log-rank (Mantel-Cox) test was used. p-values shown are resulting from pairwise comparison of any two groups. Control vs Smc3: p=0.1053, Control vs Bcl6: p<0.0001 and Bcl6/Smc3 vs Bcl6: p<0.0001. (h) Proportion of mice that developed B cell lymphoma was calculated at day 500 after bone marrow transplant for animals in all four groups. Binomial test was used to calculate p-values. (i) Univariable Cox regression analysis and (j) multivariable Cox regression analysis were performed in two DLBCL cohorts (cohort 1: n=322 patients, and cohort 2: n=757 patients). In both cases, Smc3 expression levels were used as a continuous variable. Multivariable analysis was adjusted by age, sex and DLBCL subtype of the patient. In (i) and (j) error bars represent 95% confidence intervals of hazard radio.