MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis

Domain: earth_science
Published: 2024-08-21
Editorial outcome: major_revision

Abstract
The myocyte enhancer factor 2B (MEF2B) transcription factor is frequently mutated in germinal center (GC)-derived B cell lymphomas. Its ammino (N)-terminal mutations drive lymphomagenesis by escaping interaction with transcriptional repressors, while the function of carboxy (C)-terminal mutations remains to be elucidated. Here, we show that MEF2B C-tail is physiologically phosphorylated at specific residues and phosphorylation at serine (S) 324 is impaired by lymphoma-associated mutations. Lack of phosphorylation at S324 enhances the interaction of MEF2B with the SWI/SNF chromatin remodeling complex, leading to higher transcriptional activity. In addition, these mutants show an increased protein stability due to impaired interaction with the CUL3/KLHL12 ubiquitin complex. Mice expressing a phosphorylation-deficient lymphoma-associated MEF2B mutant display GC enlargement and develop GC-derived lymphomas, when crossed with Bcl2 transgenic mice. These results unveil converging mechanisms of action for a diverse spectrum of MEF2B mutations, all leading to its dysregulation and GC B cell lymphomagenesis.

Introduction
Diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), are the most common forms of mature B cell malignancies1. Although the majority of patients (about 60%) are curable with current R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) immunochemotherapy, the remaining are not cured and about ~20% of DLBCL patients are subject to early relapses with poor outcomes2,3. DLBCL and FL originate from the germinal centers (GCs), histological structures that form in the secondary lymphoid organs and where B cells undergo multiple rounds of immunoglobulin (Ig) gene editing, in the dark zone (DZ), followed by affinity-driven selection, in the light zone (LZ), in order to generate memory B cells and antibody secreting plasma cells4,5.

In the last two decades, numerous efforts have contributed to dissecting the heterogeneity of GC-derived lymphomas and the complex networks that are hijacked during malignant transformation. Two major DLBCL subtypes were identified based on gene expression profiling: the Germinal Center B cell-like (GCB) DLBCL, displaying transcriptional similarities to normal GCB cells; and the Activated B Cell-like (ABC) DLBCL, expressing genes that are induced by B cell receptor (BCR) signaling and activation and associated with a more unfavorable prognosis6,7. The analysis of mutations affecting the DLBCL coding genome has allowed a genetic classification showing several subgroups of prognostic significance8–14. Recently, the identification in DLBCL of a pervasive mutational mechanism, which targets the non-coding regulatory domains affecting the transcriptional regulation of multiple genes, including established oncogenes (such as BCL6, BCL2, and CXCR4), has added a further layer of complexity15.

Among the genes that are genetically targeted during lymphomagenesis the transcription factor MEF2B is somatically mutated in 11% of FL and 10% of DLBCL, with a prevalence in the EZB-DLBCL genetic subgroup8–12,14,16. MEF2B belongs to the MEF2 (myocyte enhancer factor 2) family of transcription factors that includes three additional members MEF2A, MEF2C, and MEF2D. MEF2 family members were initially identified as transcriptional modulators of myocyte differentiation and muscle development17–19. The N-terminus portion of MEF2 family members includes highly similar MADS and MEF2 domains that are involved in DNA binding and protein dimerization, while the C-terminus is divergent among MEF2 proteins and it is believed to play a role in transcriptional activation19–21. In mature B cells, MEF2B is exclusively expressed at the GC stage with two isoforms, isoform A (isoA) and isoform B (isoB), which differ in their C-terminal regions from amino acid (aa) 257 to the end22,23. MEF2B contributes to GC physiology by promoting BCL6 expression and regulating an array of genes involved in cell cycle, DNA replication and repair, apoptosis, differentiation, and GCB cell confinement22–24. In vivo Mef2b deletion in GC B cells reduces GC formation and deletion of all Mef2 family members completely abrogates GC formation, indicating a functional redundancy between MEF2B and other MEF2 members23.

By analyzing lymphoma-associated MEF2B mutations, we previously identified a group of highly recurrent MEF2B N-terminal mutations which lead to escape from multiple layers of negative regulation by impairing interaction with the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex and class IIa histone deacetylases (HDACs)22,23. The most frequent lymphoma-associated N-terminal MEF2B mutant (D83V)8,10,13,22,25 shows hypomorphic features, including reduced DNA binding and protein stability, yet it acts dominantly due to its deregulated constitutive activity23,25,26. Mice conditionally expressing Mef2bD83V in GCB cells displayed enlarged GCs due to LZ expansion and, when crossed with Bcl2 transgenic mice, to mimic a common genetic lesion co-occurring with MEF2B mutations in human lymphoma, developed tumors recapitulating the phenotypic features of human FL and DLBCL23.

The C-terminus half of MEF2B is also targeted by genetic alterations, accounting for one-third of MEF2B-mutated cases and including nonsense, frameshift, and missense mutations that are scattered and rarely recurrent. The C-terminal mutations seem to affect the C-tail and its post-translational modifications22,23. However, the extent and function of post-translational modifications and their connections to the C-terminal mutations remain largely unexplored.

In this study we examine the function and consequence of the C-terminal mutations and their contribution to lymphomagenesis.

Results
Lymphoma-associated mutations targeting the C-terminus of MEF2B
The MEF2B mutational landscape in DLBCL and FL displays a bimodal distribution pattern with highly recurrent missense mutations targeting the N-terminal domain of the MEF2B protein (aa 1-88), and scattered mutations of different types, including missense, frameshift, and nonsense, in the C-terminus (Fig. 1a)8–12,16. Previously, we have functionally characterized N-terminus mutants and reported that mutations targeting the C-terminus appeared to interfere with the expression of MEF2B isoform A (isoA) in favor of isoform B (isoB) or to eliminate the C-tail of the protein22,23. Based on the analysis of the currently available expanded collection of sequencing data (1,412 DLBCL and 142 FL primary cases)8–12,16, we confirm here that MEF2B C-terminal mutations represent ~30% of the total and are heterogeneous for position and type. A sizable fraction of them (n = 11/45; 24.4% of MEF2B C-terminal mutants, hereafter referred to as isoform-switch) affect the isoform balance by encoding mutant proteins that resemble the wild-type (WT) isoB, which, although less abundant (20% of the MEF2B transcripts), displays higher transcriptional activity and stability compared to isoA (Fig. 1a and Supplementary Fig. 1)22,23. A second group of mutations (n = 18/45, 40%) lead to C-tail truncated forms of the MEF2B protein by introducing frameshifts that result in early termination (G121fs, R129fs, P132fs, Y144fs, P148fs, F170fs, and A288fs), or by generating stop codons (R127X, L147X, Q164X, R171X, Y201X, R219X, and R297X) (Fig. 1a and Supplementary Fig. 1). Lastly, the C-terminal region is also targeted by a fraction of missense mutations (n = 16/45, 35.5%), the function of which remains to be explored (Fig. 1a).

Although more frequent in GCB-DLBCL, MEF2B mutations are detected in both DLBCL subtypes8–12,16 (Fig. 1b). Nonetheless, the C-terminal mutations appear to segregate by type in the DLBCL subgroups with truncating mutations (both frameshift and stop codon) significantly enriched in the ABC-DLBCL, and missense and isoform-switch mutations mostly associated with GCB-DLBCL cases (Fig. 1b). Overall, these alterations appear to replace (isoform-switch mutations) or eliminate (truncating mutations) the C-tail of MEF2B isoA protein. These observations, together with our previously reported data showing that phosphorylation of MEF2B C-tail negatively modulates MEF2B transcriptional activity22, suggest a critical functional role of the MEF2B C-terminal region.

MEF2B is phosphorylated in GCB cells
Our previous observation that MEF2B can be phosphorylated22, was confirmed and expanded to normal and malignant GCB cells using a phos-tag gel approach (Fig. 2a). In order to characterize the full pattern of MEF2B phosphorylation and map the phosphorylation sites, we performed Flag and HA sequential immunoprecipitation (IP) followed by mass spectrometry (MS) analysis in a DLBCL cell line (SUDHL10) that was engineered to express Flag-HA double-tagged MEF2B isoA. We identified recurrent phosphorylation at T196 (present in both isoA and isoB), and at the isoA-specific aa S310, T313, and S324 (Supplementary Fig. 2a), which were all confirmed in normal GC B cells (Fig. 2b). Considering the fraction of phosphorylated/unphosphorylated peptides, we estimated that phosphorylation at S310 and S324 accounted for the large majority of phosphorylated peptides (>95%) (Fig. 2b). We then engineered SUDHL10 cells to express Flag-HA double-tagged phosphorylation-deficient isoA mutants (serine or threonine residues were mutated to alanine) and analyzed changes in MEF2B phosphorylation pattern by phos-tag gel. Consistent with the MS results, the S310A and S324A mutants displayed dramatic changes in the phosphorylation pattern compared to MEF2B WT-isoA, whereas expression of T196A and T313A mutants seemed not to alter the overall phosphorylation features (Fig. 2c). These results were confirmed by phos-tag analysis in multiple DLBCL cell lines that were engineered to express the S310A and S324A mutants (Supplementary Fig. 2b).

Consistently, antibodies generated to specifically recognize MEF2B phosphorylation at S310 and S324 detected the phosphorylated WT protein upon IP (Supplementary Fig. 2c), but not the S310A or S324A phosphorylation-deficient MEF2B mutants (Fig. 2d). As expected, treatment of the protein lysate with lambda-phosphatase completely abrogated the detection (Fig. 2d). Using the phospho-specific antibodies, we confirmed that endogenous MEF2B protein is phosphorylated at S310 and S324 in both malignant and normal GCB cells (Fig. 2e). Phosphorylation at S310 and S324 occurs independently, as shown by IP followed by MS analysis or immunoblotting with phospho-specific antibodies in cell lines expressing the S310A or S324A mutants (Fig. 2d and Supplementary Fig. 2d).

In conclusion, MEF2B is abundantly phosphorylated at S310 and S324 in GCB cells.

MEF2B C-terminal mutations impair S324 phosphorylation
To further investigate the relationship between C-terminal frameshift or missense mutations and phosphorylation, we compared the predicted MEF2B mutant proteins to MEF2B isoA C-tail, which includes S310 and S324. All frameshift truncated proteins were predicted to lack the region of interest with the minimum common deletion starting at position 297 of isoA (Fig. 3a). In addition, frameshift mutations that lead to an isoform-switch, replaced the isoA tail with the isoB sequence and therefore lacked the S310 and S324 phosphorylation sites (Fig. 3a). Thus, frameshift mutations resulted in truncated mutants or in isoB-like proteins, both of which lacked the isoA-specific C-terminal phosphorylation region. The majority (12/14) of the truncated mutants displayed low or no protein expression, when exogenously expressed in DLBCL cell lines (Supplementary Fig. 3 and “Discussion”).

Several missense mutations appeared to target the phosphorylation region, in particular the aa surrounding the S324 site (Fig. 3b)8–12,16,27. In order to test the effects of these missense mutations on S324 phosphorylation, we generated SUDHL10 and OCI-Ly7 cell lines expressing double-tagged MEF2B missense mutant proteins and analyzed their phosphorylation profile by phos-tag assay in comparison to the MEF2B WT-isoA and the phospho-deficient mutants (S310A and S324A). The results showed that R322C and P325L mutants displayed a phosphorylation pattern similar to S324A mutant, suggesting impaired phosphorylation at S324 (Fig. 3c). No mutants displayed a phosphorylation pattern resembling the S310A mutant, suggesting that missense mutations are targeting phosphorylation at S324, but not at S310 (Fig. 3c). The cell lines expressing the MEF2B mutants were analyzed by immunoblotting using the pMEF2B S324 antibody, upon Flag IP, confirming abrogation (R322C and P325L) or reduction (P330L) of S324 phosphorylation by MEF2B mutants (Fig. 3d). In order to exclude that the impaired detection was due to reduced affinity of the pMEF2B S324 antibody because of the mutations affecting residues in the phosphorylation motif, we performed MS analysis in SUDHL10-Flag-HA-MEF2B-R322C cells and confirmed the lack of phosphorylation at S324 (Supplementary Fig. 2d).

Taken together, our data show that MEF2B C-terminal mutations eliminate, replace, or target the C-tail region that is phosphorylated at S324, suggesting a relevant role for S324 phosphorylation in the modulation of MEF2B function.

Enhanced binding to SWI/SNF complex and transcriptional activity of mutants
In order to investigate whether MEF2B phosphorylation at S324 affects MEF2B protein complex formation, we performed MS analysis upon MEF2B immunoprecipitation in SUDHL10 cells expressing MEF2B WT-isoA, WT-isoB, or P325L. The list of interactors was filtered for common contaminants and for proteins with low abundance (intensity values in all the analyzed samples less than 1E+04). The results confirmed the interaction of MEF2B WT-isoA with previously reported interactors, including the HUCA complex, HDAC4, HDAC5, and HDAC723,28. A subset of interactors (653/1,996; 33%) displayed affinity changes that were consistent in both MEF2B WT-isoB and P325L compared to MEF2B WT-isoA (Supplementary Data 1).

Among the interactors that displayed higher binding affinity to MEF2B WT-isoB and P325L, we identified the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex29,30, as a notable candidate among the protein complexes for which multiple subunits were detected (Supplementary Data 1). The SWI/SNF complex is involved in chromatin unwinding and therefore it may contribute to promote MEF2B transcriptional activity. Additional IP assays followed by MS analysis in normal GCB cells and in engineered DLBCL cell lines expressing MEF2B WT-isoA or P325L confirmed the enhanced interaction between MEF2B phospho-deficient mutant and multiple subunits of the SWI/SNF chromatin remodeling complex, including the SMARCA4 ATPase subunit (Supplementary Data 2). To further validate the interaction between MEF2B and SMARCA4, we performed co-IP for the endogenous proteins and identified their interaction in the nucleus of both malignant and normal GCB cells (Fig. 4a). Nuclear/cytoplasm fractionation in normal GCB cells and in DLBCL cell lines confirmed that MEF2B and SMARCA4 are detected exclusively in the nucleus (Supplementary Fig. 4a). In addition, protein proximity labeling in SUDHL10 cells engineered to express MEF2B fused with a biotinylating enzyme (MEF2B-TurBoID)31 confirmed the interaction of MEF2B/SMARCA4 in GCB cells (Supplementary Fig. 4b).

In order to investigate the transcriptional program modulated by MEF2B in cooperation with the SWI/SNF complex, we analyzed the chromatin binding profiles of MEF2B and SMARCA4 in normal human GCB cells by Cut&Run followed by sequencing. MEF2B binding profile identified by Cut&Run was largely overlapping (~55% peaks) with our previous characterization by ChIP-seq (~66% of ChIP-seq peaks were identified by Cut&Run). Of note, co-occupancy of MEF2B and SMARCA4 was detected in ~74% of SMARCA4 and ~50% of MEF2B-bound regions (Fig. 4b), which were annotated as promoters (50%; identified as -2Kb/+1Kb from the TSS), intragenic (20%) and intergenic (30%) regions. Overall, we identified 5,661 genes that were associated with the MEF2B/SMARCA4 co-bound regions and were expressed in mature B cells (Supplementary Data 3). Pathway enrichment analysis for these genes using the Hallmark, KEGG and Biocarta databases32, largely recapitulated the overall MEF2B program, including genes involved in cell cycle, DNA repair, apoptosis, and GC confinement (Supplementary Data 4)23. The same analysis on the genes associated with MEF2B in the absence of SMARCA4 did not reveal enrichment for additional pathways (Supplementary Data 4). SMARCA4 co-localizes with MEF2B in previously identified MEF2B-bound regions of the BCL6 locus both in the promoter and in the upstream super-enhancer (Fig. 4c)22,24. In addition, the SMARCA4/MEF2B co-bound targets included several DZ (i.e., CXCR4, CCND3) and LZ (i.e., CD83, CD86) markers, as well as genes involved in signaling pathways that are relevant for the affinity-based selection occurring in the LZ, including B cell receptor, NF-κB, and MAPK pathways (Fig. 4c and Supplementary Data 4).

Consistent with the MS analysis, co-IP in SUDHL10 cells expressing the phospho-deficient mutants (S324A and the lymphoma-associated R322C, P325L, and P330L) and in OCI-Ly7 isogenic clones edited to endogenously express S324A or P325L showed an increase in the amount of SMARCA4 that was immunoprecipitated by MEF2B mutants compared to the MEF2B WT-isoA, suggesting that the lack of phosphorylation promotes the interaction between MEF2B proteins and SMARCA4 (Fig. 5a, b and Supplementary Fig. 4c, d). As expected, the phospho-mimic S324E mutant did not show enhanced binding to SMARCA4 in both the overexpression and endogenous cell systems (Fig. 5a, b and Supplementary Fig. 4c, d). In addition, the double mutant S324E/P325L (EL), in which the phospho-mimic S324E was combined with the phospho-deficient P325L mutant, behaved like the single S324E mutant (Fig. 5a and Supplementary Fig. 4c). The enhanced interaction with the SWI/SNF chromatin remodeling complex may contribute to the increased transcriptional activity that is associated with the phospho-deficient mutants, as shown by luciferase reporter assay using an artificial reporter including canonical MEF2B binding sites (3xMEF2) (Fig. 5c).

Consistently, both the WT-isoB protein and the isoform-switch mutants expressing iso-B-like C-tails, which lack the S324 phosphorylation site, displayed enhanced interaction with SMARCA4 both in the over-expression and endogenous cell systems (Fig. 5d, e and Supplementary Fig. 4e, f). In addition, similar to the missense mutants, all these proteins showed an increased transcriptional activity compared to MEF2B WT-isoA (Fig. 5f). Collectively, these data show that MEF2B phosphorylation at S324 contributes to the dissociation between MEF2B and the SWI/SNF complex. C-terminal mutants, which cannot be phosphorylated at S324, display an enhanced interaction with SMARCA4 and increased transcriptional activity.

Stabilization of mutants by dissociation from the CUL3/KLHL12 complex
Among the interactors displaying lower affinity for MEF2B WT-isoB and P325L mutant, our analysis identified several subunits of the CUL3 ubiquitin E3 ligase complex, including Broad-Complex, Tramtrack and Bric a brac domain (BTB) adaptor proteins (Supplementary Data 1). In order to refine this analysis and identify proteins interacting with both MEF2B and CUL3, we performed MS analysis upon sequential immunoprecipitations of Flag (MEF2B) and CUL3 in the SUDHL10-Flag-HA-MEF2B WT-isoA cells. The results confirmed and expanded the list of MEF2B interactors that belong to the CUL3 ubiquitin E3 ligase complex (Supplementary Data 5). Of note, we identified several adaptor proteins the most abundant of which was the KLHL12 BTB-containing protein (Supplementary Data 5)33,34. These results suggest that KLHL12 may act as the adaptor protein, which recruits MEF2B in the CUL3 E3 ligase ubiquitin complex for ubiquitin-mediated degradation.

The native interaction between MEF2B, KLHL12 and CUL3 was confirmed in nuclear extracts by co-IP in both malignant and normal GCB cells (Fig. 6a). Nuclear/cytoplasm fractionation in normal GCB cells and in DLBCL cell lines confirmed expression of CUL3 and KLHL12 in both the cytoplasm and nucleus (Supplementary Fig. 5a). Protein proximity labeling in SUDHL10 cells expressing MEF2B-TurBoID protein confirmed the interaction of MEF2B/KLHL12/CUL3 (Supplementary Fig. 5b).

In order to map the interaction of MEF2B and KLHL12, we performed co-IP assays on cells co-transfected with full-length or deletion mutants of both MEF2B and KLHL12 proteins (Supplementary Fig. 5c). The results showed that MEF2B full-length binds to both the N-terminus and the C-terminus of KLHL12, with the N-terminus of MEF2B being associated with the N-terminus of KLHL12. Although the MEF2B C-terminus by itself did not show binding to KLHL12 in this assay, the fact that (i) we detected binding between KLHL12 C-terminus and MEF2B full-length, (ii) the MEF2B P325L mutant displayed reduced binding affinity to KLHL12, and (iii) the canonical binding of KLHL12 to approximately ten amino acids of its substrates occurs through the C-terminus35–37, prompted us to test an oligopeptide surrounding the S324 phosphorylation region. Indeed, the binding of KLHL12 C-terminus in the MEF2B C-terminus was efficiently detected using this oligopeptide (M7 in Supplementary Fig. 5c). These results suggest that the interaction between KLHL12 and MEF2B requires the MEF2B N-terminus to retain the proper configuration of the C-terminus, and the ~270 aa C-terminus is a highly dynamic entity to be stabilized by interaction of merely ten residues with the KLHL12 Kelch domain. Thus, we mapped the interaction of MEF2B and KLHL12 to both the MEF2B N-terminal domain and the C-terminal phosphorylation region containing S324 (aa 304-333), which displayed binding to KLHL12 N-terminus (BTB-BACK domain) and C-terminus (Kelch domain), respectively (Supplementary Fig. 5c).

In order to confirm that MEF2B is recruited by KLHL12 into the CUL3 E3 ligase complex for ubiquitin-mediated degradation, we co-expressed MEF2B with the KLHL12-AMF mutant (A60E, M61E and F62A) that cannot interact with CUL338. The results showed that KLHL12-AMF mutant was unable to bind CUL3 and, when co-expressed with MEF2B, failed to mediate MEF2B degradation (Supplementary Fig. 5d, e).

Co-IP in SUDHL10 cells expressing the phospho-deficient mutants (S324A or the lymphoma-associated R322C, P325L and P330L) and in OCI-Ly7 isogenic clones edited to endogenously express S324A or P325L confirmed the impaired interaction with KLHL12 and CUL3, while the phospho-mimic S324E mutant behaved similarly to the MEF2B WT-isoA protein (Fig. 6b, c and Supplementary Fig. 6a, b). MEF2B ubiquitination, detected both in SUDHL10 cells and in transient transfection assays, was reduced in the phospho-deficient mutants consistent with the impaired interaction with the KLHL12/CUL3 complex (Fig. 6d and Supplementary Fig. 6c, d). Accordingly, we detected poly-ubiquitin chains linked to K48, a known signal for protein degradation by the proteasome39 (Fig. 6d). Ubiquitination not associated with protein degradation (i.e., K63-linked) was not detected (Supplementary Fig. 6e). Consistently, a significant increase of MEF2B protein stability and half-life was detected in SUDHL10 cells expressing the S324A, R322C, P325L or P330L mutants and in the OCI-Ly7 isogenic clones edited to endogenously express P325L (Fig. 6e, f and Supplementary Fig. 7). We note that although the S324E/P325L double mutant displays a protein stability similar to the WT (Fig. 6e), it remains deficient in its binding to KLHL12 and CUL3 (Fig. 6b), suggesting that alternative adaptor proteins may be involved in restoring the protein degradation of this artificial mutant.

The impaired interaction and ubiquitin-mediated degradation was not limited to the missense mutants affecting MEF2B phosphorylation at S324. In line with the fact that MEF2B WT-isoB lacks the S324 phosphorylation site, both the WT-isoB protein and the isoform-switch mutants displayed impaired interaction with KLHL12 and CUL3 both in the over-expression and endogenous cell contexts (Fig. 7a, b and Supplementary Fig. 8a, b). Similar to the missense mutants, the isoform-switch mutants showed a reduced ubiquitination, and extended half-life compared to MEF2B WT-isoA (Fig. 7c–e and Supplementary Fig. 8c–e).

In conclusion, these results show that MEF2B phosphorylation at S324 favors the interaction with CUL3/KLHL12, while both lymphoma-associated missense and isoform-switch frameshift mutants escape CUL3/KLHL12 complex-mediated ubiquitination and degradation.

Mef2bP297L lymphoma-associated mutant enhances GC formation in mice
In order to understand the role of MEF2B phosphorylation at S324 and its lymphoma-associated impairment in GC development and lymphomagenesis, we generated a mouse model conditionally expressing the Mef2bP297L mutant, the orthologous of human MEF2B P325L (Fig. 8a). Toward this end, the coding region of Mef2b exon 2 and part of intron 2 were replaced by a cassette including the mouse Mef2b WT coding sequence (CDS; refer to NM_001045484.2) flanked by loxP sites and followed by the mutated Mef2b CDS (encoding Mef2bP297L mutant) linked, by a P2A self-cleavage sequence, to EGFP (Supplementary Fig. 9a). Upon crossing with GC-specific Cγ1-cre mice40, the expression of WT Mef2b in GCB cells is replaced by the Mef2bP297L mutant, which is co-transcribed with EGFP. Expression of the mutated transcripts in GC B cells was confirmed by RT-PCR followed by Sanger sequencing (Supplementary Fig. 9b). As expected, EGFP was detected exclusively in GCB cells of heterozygous (HET; Mef2bP297L/+; Cγ1-Cre tg/+) and homozygous (HOMO; Mef2bP297L/P297L; Cγ1-Cre tg/+) mutant mice, proportionally to allelic dosage, but not in WT (Mef2b+/+; Cγ1-Cre tg/+) mice (Supplementary Fig. 9c-e).

Analysis of splenic B cells 10 days after sheep red blood cells (SRBC) immunization showed a significant increase of GCB cells in the mutant (HET and HOMO) mice compared to WT mice (Fig. 8b and Supplementary Fig. 9f, g), while no changes were detected in non-GCB cell populations, including follicular B cells, marginal zone B cells and plasma cells (Supplementary Fig. 9h–j). The GCB cell increase was due to an enlargement of the GC structures, not to an increased number of GCs, as shown by quantification of GC areas and numbers upon immunohistochemical staining of peanut agglutinin (PNA) (Fig. 8c). Analysis of the DZ and LZ GC B cell subpopulations showed a significant reduction in the DZ/LZ ratio, due to LZ expansion in the mutant mice (Fig. 8d–f and Supplementary Fig. 9f, k). Class-switch recombination was normal in the mutant mice, while their ability to generate high-affinity antibodies appeared slightly reduced (Supplementary Fig. 9l, m). No significant gender effect was detected since both the male and female cohorts showed statistically significant and consistent differences in GC size, DZ/LZ ratios, and LZ cell number, although the female cohort failed to reach significance regarding the increased percentage of GCB cells.

In order to characterize the transcriptional changes driven by expression of Mef2bP297L, we performed gene expression profiling of GC B cells sorted from SRBC-immunized WT and mutant (HET and HOMO) mice (at least 3 mice/genotype). We identified 137 genes that were significantly differentially expressed between WT and HOMO mice with a fold change > 1.2 (65 up- and 72 down-regulated in HOMO compared to WT; q < 0.05; Supplementary Data 6). This gene signature was consistently enriched in the expression profiles of HET mice (Supplementary Fig. 10a). Consistent with the LZ expansion phenotype, Gene Set Enrichment Analysis (GSEA) in WT vs HET or HOMO expression profiles using previously reported DZ and LZ signatures both in mouse and human41 showed significant enrichment for the LZ signatures in the mutant mice (Fig. 8g and Supplementary Fig. 10b). In particular, CD86, a marker of the LZ compartment, was significantly upregulated in the GCB cells of mutant mice (Supplementary Data 6), which was consistent with increased protein levels, as detected by cytofluorimetric analysis (Fig. 8h). Consistent with the LZ expansion, the plasma cell master regulator Prdm142,43 was among the genes that were downregulated in mutant mice (Supplementary Data 6).

In order to evaluate the effects of the enhanced interaction between Smarca4 and the phospho-deficient Mef2b mutant in vivo, we performed GSEA in the mouse transcriptomic data using the targets that we identified as co-bound by SMARCA4 and MEF2B in human GCB cells. The results showed a significant enrichment for SMARCA4/MEF2B targets in the genes up-regulated in the mutant mice, consistent with their enhanced transcriptional expression when Mef2b cannot be phosphorylated (Fig. 8i). Consistent with the pathway enrichment analysis for SMARCA4/MEF2B targets in human GCB cells (Supplementary Data 4), shared targets are enriched for genes associated with the LZ compartment also in the murine GCB cells.

A few targets which displayed MEF2B/SMARCA4 co-binding in normal GCB cells (Supplementary Data 3) and were transcriptionally affected in the murine mutant GCB cells were tested for changes in expression in the DLBCL OCI-Ly7 isogenic clones expressing endogenous P325L. Consistent with the observations in mouse GCB cells, we detected transcriptional up-regulation of CD86, CD180, MAP3K8, PAX5 and RASGRP3 in the OCI-Ly7 isogenic clones expressing endogenous P325L when compared to neutral control clones (Supplementary Fig. 10c). These results suggest that the enhanced transcriptional effects of MEF2B phospho-deficient mutant are detectable both in normal and malignant GCB cells.

Overall, these data indicate that the expression of Mef2bP297L in GC B cells pathologically alters the GC development by enforcing LZ expansion, which, in multiple instances, has been shown to represent a phenotype preceding malignant transformation23,44,45.

Mef2bP297L mutant promotes GCB cell lymphomagenesis in mice
To investigate the role of Mef2bP297L in tumor development, mice were chronically immunized with SRBC (every 2 months, for six times in total) and monitored for signs of disease over a timeframe of 17–18 months. The expression of Mef2bP297L mutant alone did not promote lymphoma development and had no impact on the tumor-free survival (Supplementary Fig. 11a, b). Since MEF2B mutations (including the phospho-deficient mutants) significantly co-occur with BCL2 rearrangements in human DLBCL and FL8,13,16,46, the Mef2b mutant mice were also crossed with Bcl2-Ig transgenic mice47. We observed that Bcl2-Ig transgenic mice (Mef2b+/+; Cγ1-Cre tg/+; Bcl2-Ig Tg) developed FL (4/19, 21%), but also more commonly plasmablastic lymphoma (PBL, 8/19, 42%), and in a few cases, multiple concomitant tumors (Fig. 9a and Supplementary Fig. 11a), as determined based on morphological features and analysis of T cell (Cd3), B cell (B220 and Pax5), GC (Bcl6) and plasma cell (Irf4) markers in the tumor tissue (Fig. 9b). In addition, expression of exogenous (human) BCL2 was confirmed in the tumors from Bcl2-Ig transgenic and compound mice (Fig. 9b). Tumors were clonal, as shown by the presence of clonally rearranged immunoglobulin loci (Supplementary Fig. 11c).

PBLs originate from B cells committed to the plasma cell lineage, and as expected, do not express GCB cell markers, including Bcl6 and Mef2b (Fig. 9b). This also applies to the mutant Mef2bP297L, which is under the control of the endogenous promoter and regulatory regions. As expected, Mef2bP297L (EGFP) expression was detected in FL and DLBCL, but not in PBL tumors (Fig. 9b). Thus, the PBL tumors are not driven by Mef2b and do not reflect its function for lymphomagenesis. In contrast, the compound mice displayed a significantly higher incidence of DLBCL (32% in HET; Bcl2 and 60% in HOMO; Bcl2 vs 12% in WT; Bcl2; Fig. 9a). Accordingly, when considering GC tumors only, the compound HET; Bcl2 mice showed a shorter tumor-free survival that reached statistical significance for the HOMO; Bcl2 mice (Fig. 9c). No differences were observed regarding the tumor types and their histological features when comparing the Mef2bP297L with the previously published Mef2bD83V cohorts23.

Although expression of the mutant Mef2bP297L did not enhance overall lymphomagenesis, the increased incidence of Mef2b-expressing tumors and the associated reduction in tumor-free-survival suggest that expression of Mef2bP297L mutant, in combination with Bcl2-deregulation, promotes malignant transformation of GC B cells.

Methods
This research complies with all ethical regulations. All in vivo experiments and procedures conformed to ethical principles and guidelines revised and approved by the Institutional Animal Care and Use Committee at Columbia University. This study includes the use of anonymized human tonsil tissue collected as residual material after diagnosis and therefore exempt from consent, as confirmed by the Columbia University Human Research Protection Office & Institutional Review Board. No material was obtained/analyzed through interaction/intervention/patient enrollment. Additional human data used in this study were previously published. As such the study represents “secondary research using data or biospecimens not collected specifically for this study” and has been defined as exempt human subject research in accordance with 45 CFR 46.

Mouse models and strains
Mice expressing the Cre recombinase under the control of the Cγ1 promoter and Bcl2-Ig transgenic mice were previously described40,47. All mouse strains were backcrossed into C57BL/6 background. Immunological responses were evaluated in immune-competent mice at ~3 months of age. Animals assigned to the tumor cohorts were monitored twice a week for tumor incidence and survival over a period of 17–18 months and were killed for analysis when visibly ill or at the end of the study. Although our approved protocol allows tumor sizes up to 20 mm, we sacrificed animals with masses of 10 mm or smaller. Both females and males were included in the experiments. Mice were housed in a dedicated pathogen-free environment with controlled temperature, humidity, and dark/light cycle.

Generation of Mef2bP297L conditional mouse model
The conditional Mef2bP297L C57BL/6 mouse was generated by Cyagen (Santa Clara, CA, USA). Briefly, the coding region of exon 2 plus partial intron 2 of Mef2b was replaced with the WT Mef2b coding DNA sequence (CDS; NCBI reference sequence: NM_001045484.2) flanked with two loxP sites and followed by a sequence encoding rabbit beta globin polyA signal. Downstream of the polyA cassette was inserted the Mef2b coding sequence carrying a C>T (nucleotide 890 of CDS) mutation (P297L amino-acid change) in exon 9. The mutated Mef2b coding region was linked to a sequence encoding enhanced green fluorescent protein (EGFP) by a 2A sequence of porcine teschovirus-1 (P2A). In the targeting vector, the neomycin cassette was flanked by self-deletion anchor (SDA) sites. C57BL/6 ES cells were used for gene targeting, and correct homologous recombination was confirmed by Southern blotting and PCR followed by sequencing. Then, the targeted mouse ES cells were injected into blastocysts derived from C57BL/6 mice to generate chimeras. The following primers were used for genotyping and amplifying the fragment with the Mef2bP297L mutation (Supplementary Fig. 9a):
F1: 5’-CTTACCATCAGGCTCCACAGATT; R1: 5’-CTTCAACATCTCC TGCTTGCTTTA; F2: 5’-CCACATTGCCTTGCATTTATTGAG; R2: 5’- AAGACCATTTGACTTTGGCCTG; R3: 5’-CCCATGGTGGCTATAACTTCG TATAAT.

Mouse immunization and analysis
In order to induce GC responses, both female and male 3-month-old mice were immunized by intraperitoneal injection of 1×10^9 sheep red blood cells (SRBCs, Cocalico Biologicals) or 100 μg of 4-hydroxy-3-nitrophenyl-acetyl (NP) conjugated to keyhole limpet hemocyanin (KLH, BioSearch) incomplete Freund’s Adjuvant (Sigma-Aldrich) and analyzed on day 10–12 post-immunization. To generate larger amounts of GCB cells (i.e., for gene expression profile studies), we performed two sequential injections of SRBCs (day 0, 1×10^8 SRBCs; day 5, 1×10^9 SRBC) and collected cells at day 12. This protocol yielded about three to four folds more GCB cells (~12–15% of the B cell fraction). For sorting GCB cells, B cells were first isolated from spleen by B cell isolation kit (130-090-862, Miltenyi Biotec), following manufacturer’s instructions. Then, the B cells were subjected to staining for the B cell and GC markers B220 (561086, BD Biosciences), GL7 (144613, BioLegend), and PNA (L32460, Thermo Fisher Scientific) and sorting on an Influx instrument (BD Biosciences). Only EGFP+ GCB cells were sorted from Mef2bP297L KI mice.

Spleens and other lymphoid tissues were isolated and divided into fragments, which were processed for histological staining and flow cytometry analysis. The mononuclear cells were isolated by crushing the tissue through 40 μm cell strainers in ice-cold 1X phosphate-buffered saline (PBS) + 0.5% bovine serum albumin (BSA) followed by red blood cell lysis.

For flow cytometry analysis, mononuclear cell suspensions were stained for 20 min on ice with antigen-specific fluorochrome-conjugated antibodies. Samples were acquired on an Attune (Thermo Fisher Scientific) or Canto (BD Biosciences) instruments. FlowJo Software (V10.10.0, TreeStar) was used for data analyses and plot rendering. To calculate the absolute numbers of cells within splenic B cell subsets, spleen fragments were weighted, and erythrocyte-depleted mononuclear cell suspension were counted by Trypan blue exclusion using the Countess Automated Cell Counter (Thermo Fisher Scientific). The total number of counted splenic B cells was normalized according to the weight of the specimen and then multiplied by the fraction of each subpopulation, as identified by the cytofluorimetric analyses.

Mouse analyses were performed on sub-cohorts including 2–4 mice/genotype, which were obtained from 2–4 litters born at the same time.

Immunohistochemistry
3-μm-thick formalin-fixed and paraffin-embedded (FFPE) sections were used for histological staining, performed based on established protocols23. The GC number, GC size, and spleen areas were measured by Aperio ImageScope software (Leica).

The tumor diagnosis in mice was performed by histopathological analysis of tissue sections according to the following criteria: i) follicular lymphoma (FL) was diagnosed based on the presence of nodular proliferation of B220+ (553086, BD Biosciences)/BCL6+ (5650, Cell Signaling Technology) B cells with centrocytic morphology (characterized by angulated/cleaved nuclei and inconspicuous nucleoli) and admixed centroblastic cells (round-ovoid nuclei and prominent nucleoli) detectable to varying degrees; ii) diffuse large B cell lymphoma (DLBCL) was diagnosed based on diffuse proliferation of medium to large B220+/PAX5+ (sc-1974, Santa Cruz) B cells with round-ovoid nuclei, vesicular chromatin and distinct nucleoli; iii) plasmablastic lymphoma (PBL) was diagnosed in cases showing sheets of large cells with prominent nucleoli and exhibiting expression of IRF4 (sc-6059, Santa Cruz) and absent B (B220, PAX5), GC (BCL6) or T cell (CD3) markers.

Southern blotting
High molecular weight genomic DNA was extracted from frozen tissues by DNeasy Blood & Tissue Kit (Qiagen) and 5 μg were digested with the Fast Digestion EcoRI (Thermo fisher scientific) for 3 h at 37°C. The digested DNA was resolved on 0.8% agarose gel overnight followed by transfer to nitrocellulose membrane according to standard procedures. Hybridization was performed at 37°C overnight using a 32P-labeled JH4 specific probe, as previously reported57.

Expression vectors
The plasmids pCMV-HA-MEF2B WT-isoA, pCDNA3-Flag-HA-MEF2B WT-isoA, pCDNA3-Flag-HA-MEF2B WT-isoB, pCCL-Flag-HA-MEF2B WT-isoA, pCCL-Flag-HA-MEF2B WT-isoB were previously reported22,23. pCCL-V5-TurboID was generated by subcloning the previously reported sequence encoding the V5-tagged TurboID31 into the pCCL vector. The pCCL-V5-TurboID-MEF2B WT-isoA was generated by subcloning MEF2B into the pCCL-V5-TurboID construct. pCDNA3-Flag-HA-MEF2B and pCMV-HA-MEF2B mutants were generated by site-direct mutagenesis. pCCL-Flag-HA-MEF2B mutants were constructed by subcloning from the pCDNA3-Flag-HA-MEF2B mutants. pCDNA3-EGFP were generated by subcloning EGFP cDNA into pCDNA3 vector (Invitrogen). pCDNA3-EGFP-MEF2B WT-isoA, pCDNA3-EGFP-MEF2B isoA fragments, pCDNA3-EGFP-MEF2B WT-isoB and pCDNA3-EGFP-MEF2B isoB were generated by subcloning into the pCDNA3-EGFP plasmid fragments obtained by PCR amplification from pCDNA3-Flag-HA-MEF2B WT-isoA and pCDNA3-Flag-HA-MEF2B WT-isoB. CUL3 cDNA (NM_003590.5) was amplified and inserted into pCDNA3 vector. Ubiquitin cDNA (NM_021009.7) together with V5 tag were amplified and inserted into pCDNA3 vector. KLHL12 cDNA (NM_001303051.2) was amplified and inserted into the pCMV-HA vector (Clontech). pCMV-Myc-KLHL12 and pCDNA3-Myc-CUL3 were generated by subcloning into the pCMV-Myc vector (Clontech) PCR amplicons obtained from pCMV-HA-KLHL12 and pCDNA3-Flag-CUL3. pCMV-HA-KLHL12-AMF and pCMV-Myc-KLHL12-AMF were generated by site-direct mutagenesis. pCMV-HA-SMARCA4 was generated by subcloning into pCMV-HA vector the PCR-amplified insert from pQCXIH BRGI-Flag (Addgene: #19148). All the plasmids were confirmed by Sanger sequencing.

Human normal GCB cell isolation
Human normal GC B cells were obtained from tonsils at Columbia University Medical Center. Specimens were exempt from informed consent being residual material after diagnosis and fully anonymized. The GC B cell isolation was performed as previously described58. Briefly, the tonsillar mononuclear cells (MNCs) were isolated by Ficoll-Isopaque centrifugation upon smashing the tonsil tissues. Then, GCB cells were purified by magnetic cell separation with anti-CD77-FITC antibody (BioLegend) and magnetic anti-FITC beads (Miltenyi Biotec), according to the manufacturer’s instructions (Miltenyi Biotec). The purity of GCB cells was confirmed by flow cytometry analysis with staining of anti-CD38-PE antibodies (BD Biosciences). The samples with at least 90% of GC B cell purity were used for downstream experiments.

Cell lines
HEK293T cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, 100 mg/ml penicillin, and streptomycin. The DLBCL cell lines BJAB, DB, FARAGE, HBL1, HLY1, Karpas422, OCI-LY1, OCI-LY3, OCI-LY7, OCI-LY8, RCK8, RIVA, SUDHL2, SUDHL4, SUDHL6, SUDHL7, SUDHL10 (ATCC), SUDHL1059, TMD8, U2932, VAL, WSU-DLCL2 and WSU-NHL were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FCS, 100 mg/ml penicillin and streptomycin. Cell lines were maintained at 37°C in humidified incubators in the presence of 5% CO2. All the cell lines tested negative for mycoplasma contamination. DB and SUDHL4 cell lines carry MEF2B mutations leading to the expression of the MEF2B D83V protein13. All remaining cell lines are wild-type for MEF2B.

Cell transfection and lentiviral transduction
For transient expression in HEK293T cells, polyethylenimine (PEI) (Polysciences) was used as previously described22. Briefly, DNA and PEI with amount ratio of 1:6 were incubated in 150 mM NaCl for 15 min at room temperature. DNA-PEI particles were gently added to pre-seeded HEK293T cells. After 6-h incubation at 37°C, the transfection medium was removed and replaced with fresh DMEM medium. At 48 h post-transfection, cells were harvested for downstream analysis. Lentiviral transduction was done to establish stable cell lines as performed previously23. Viral supernatants were obtained by co-transfection of HEK293T cells with lentiviral vectors with EGFP expression, a lentivirus packaging vector (Δ8.9), and a plasmid encoding vesicular stomatitis virus envelope glycoprotein (VSVg). The indicated DLBCL cell lines were infected with the viral supernatants for two rounds, and then the infected cells were subjected to EGFP+ cell sorting on a SH800 Cell Sorter (Sony).

Generation of cell lines carrying endogenous mutations
The Alt-R CRISPR-Cas9 System (Integrated DNA Technologies) was used to edit OCI-Ly7 cells by introducing missense (S324A, S324E, and P325L) and isoform-switch (P256fs and P273fs) mutations in the MEF2B locus, as well as irrelevant mutations in intron 1 of PPP1R12C gene (neutral control), as reported previously15. Briefly, a ribonuclear protein (RNP) complex including specific crRNA and homology-directed repair (HDR) donor template, a tracer RNA labeled with ATTO550 and Alt-R SpCas9 Nuclease V3 was formed according to the manufacturer’s protocol. The Neon NxT Electroporation System (Thermo Fisher Scientific) was used to deliver the RNP complex into OCI-Ly7 cells (electroporation condition: 1700 V, 20 ms and 1 pulse; buffer R). After 24 h, the top 70% of ATTO550-positive cells were sorted on an Influx Cell Sorter (BD Biosciences) and single-cell plated. Recovered single-cell clones were selected and analyzed by Sanger sequencing upon genomic DNA isolation and PCR amplification for the specific mutated region. In brief, clone pellets were resuspended in 100 μL of lysis buffer (10 mM Tris-HCL pH 7.5; 10 mM NaCl; 10 mM EDTA; 0.5% Sarcosyl) containing 0.5 mg/mL Proteinase K and incubated at 55°C overnight. Genomic DNA was precipitated by adding 100 μL of cold EtOH/75 mM NaCl mixture followed by centrifugation at 956 g for 20 min at 4°C. Finally, precipitated DNA was washed twice with cold 70% EtOH and centrifuged at 956 g at 4°C for 10 min, air dried for 15 min and resuspended in molecular grade water for downstream analyses. PCR amplification of the targeted regions was performed using Taq polymerase and touch-down PCR conditions. Correct editing of recovered clones was assessed by using Mutation Surveyor (v. 4.0.8).

The sequences of crRNAs and HDR donor templates are as the following:
crRNA for MEF2B P325L: CAAGTCTGAGCGCCTCTCTC
HDR donor template for MEF2B P325L: AGGGGAAGGTCTTAGGAAAGTCGCCGGGGCCCCCGGGGGCAA GAGAGAGGCGCTCAGACTTGATGCTGACTGGGGGGGTCGGCGG
crRNA for MEF2B S324A and S324E: CAAGTCTGAGCGCCTCTCTC
HDR donor template for MEF2B S324A: GGAAGGTCTTAGGAAAGTCGCCGGGGCCCCCGGGGGCTGGAGC GAGGCGCTCAGACTTGATGCTGACTGGGGGGGTCGGCGGG
HDR donor template for MEF2B S324E: GGAAGGTCTTAGGAAAGTCGCCGGGGCCCCCGGGGGCTGGTTC GAGGCGCTCAGACTTGATGCTGACTGGGGGGGTCGGCGGG
crRNA for MEF2B P256fs: GGTTAGGGGATGTACCTGGG
HDR donor template for MEF2B P256fs: CGCTTCCCAGGTGGGGCAGGGCAGGTTAGGGGATG TACCTGGGGGCCTCCGGGGAGGAAGGGGAAGCTCCCCAGTGGGGGT
crRNA for MEF2B P273fs: CTTGTTGCAGCCCCCCACCC
HDR donor template for MEF2B P273fs: CCTGGGAGACCCTCCACCGCCCCCTGGCTTGTTGCAGCCCCCA CCCTGGCCCCCTGGCAGCCCTCGAGGGGTGATGGGCCCCC
crRNA for PPP1R12C intron mutation: CCAGCGAGTGAAGAC GGCAT
HDR donor template for PPP1R12C intron mutation: GAAGGCCATCCTAAGAAACGAGAGATGGCACAGGCCCCAGAA GGAGAAGGAAAAGGGAACCCAGCGAGTGAAGACGCCATGCGGTT GGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGA GGATCCGCTCAGAGGACATC

The sequences of primers used for PCR amplification of the edited regions are as following:
S324A, S324E, P325L Forward: CTGGCTTCCTTCCCTTCCTC
S324A, S324E, P325L Reverse: CGTCACTGTTGGGTCTTCTC
P256fs Forward: CCTCTCTTCTCACCAAGCCA
P256fs Reverse: CACTTACCTGGGCTGGGAG
P273fs Forward: GTACATCCCCTAACCTGCCC
P273fs Reverse: CAAAATAGCTGCCCATCCGT
PPP1R12C Forward: GGGTGTCCAGGCAAAGAAAG
PPP1R12C Reverse: CCTTCAGGTTCCGTCTTCCT

Luciferase reporter assay
The luciferase reporter constructs (3xMEF2 WT-Luc and 3xMEF2 Mutant-Luc) were generated by cloning into the pGL4.26 vector (Promega) in our lab as previously described23. HEK293T cells were transiently transfected by PEI (Polysciences) with pCMV-HA-MEF2B WT or mutant plasmids along with the pGL4.26–3xMEF2 reporter construct and a renilla luciferase control reporter (pGL4.54-renilla). At 48 h post transfection, cells were harvested and lysed. Luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (E1960, Promega), according to the manufacturer’s instructions in a Glomax plate reader (Promega).

Immunoprecipitation and immunoblotting
Whole-cell protein extracts were isolated using the following immunoprecipitation (IP) buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA pH 8.0, 0.05% NP40, 0.2% Triton X-100, 30 mM beta-glycerophosphate, 0.5 mM PMSF, 50 mM sodium fluoride, 1 mM sodium orthovanadate and protease inhibitor cocktail (Sigma-Aldrich). For nuclear extraction, cells were lysed with cytoplasmic extraction buffer (10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 0.5% NP40, and protease inhibitor cocktail) for 10 min on ice. Upon centrifugation for 10 min at 500×g, the supernatant was transferred to a new tube as the cytoplasmic fraction and subjected to further centrifugation at 20000×g for 10 min. The nuclei pellet was washed with cytoplasmic extraction buffer three times (500×g for 10 min), and then nuclear extraction buffer (10 mM HEPES, 1.5 mM MgCl2, 420 mM NaCl, 1 mM DTT, 20% Glycerol and protease inhibitor cocktail) was added to lyse for 30 min on ice. The nuclear fraction was obtained with centrifugation at 20000×g for 15 min.

Lysates were incubated overnight at 4°C with anti-Flag (A2220, Sigma-Aldrich) or anti-HA affinity beads (F2426, Sigma-Aldrich) or indicated antibodies. In the latter case lysates were supplemented with Protein G beads (17-0618-01, Cytiva) for 2 h at 4°C. Beads were washed 5 times in a modified IP buffer containing 400 mM NaCl. Immuno-complexes were eluted with 2.5 mg/ml MEF2B, Flag, or HA peptides (Synthesized by Genemed Synthesis, Inc.; San Antonio, TX, USA) or in 100 mM Glycine (pH 2.5) buffer. If proteins were eluted by 100 mM Glycine, neutralization was further performed by adding 20% (volume of Glycine solution) 500 mM Tris-HCl (pH 8). Eluted samples were concentrated by Vivaspin Protein Concentrators (Cytiva) or Amicon® Ultra Centrifugal Filters (Millipore), if elution volume was greater than 30 ul. The protein lysates or IP eluents were resolved on Tris-Glycine mini gels (Thermo Fisher Scientific), and subsequently transferred on a 0.45 μm nitrocellulose membrane. Membranes were blocked for 1 h in TBS (20 mM Tris, pH 7.4 and 150 mM Nacl) with 0.05% Tween-20 and 5% non-fat dry milk. The membranes were incubated overnight at 4°C with the primary antibodies against MEF2B (custom23 and NBP3-03920, Novus Biologicals; 1:1000 dilution), pMEF2B S310 (custom; 1:1000 dilution), pMEF2B S324 (custom; 1:1000 dilution), Actin (A5441, Sigma-Aldrich; 1:3000 dilution), Vinculin (SAB4200080, Sigma-Aldrich; 1:2000 dilution), Lamin B1 (12586, Cell Signaling Technology; 1:1000 dilution), HA (3724, Cell Signaling Technology; 1:1000 dilution), Myc (sc-40, Santa Cruz; 1:1000 dilution), Flag (P2983, Sigma-Aldrich; 1:1000 dilution), EGFP (2956, Cell Signaling Technology; 1:1000 dilution), Ubiquitinylated proteins (04-263, Sigma-Aldrich; 1:1000 dilution), K48-linkage Specific Polyubiquitin (8081, Cell Signaling Technology; 1:1000 dilution), K63-linkage Specific Polyubiquitin (5621, Cell Signaling Technology; 1:1000 dilution), V5 (13202, Cell Signaling Technology; 1:1000 dilution), SMARCA4 (49360, Cell Signaling Technology; 1:1000 dilution), CUL3 (A301-109A, Bethyl Laboratories; 1:1000 dilution) and KLHL12 (9406, Cell Signaling Technology; 1:1000 dilution). After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (NA931 and NA934, Cytiva; 1:5000 dilution) or Trueblot secondary antibodies (18-8816-33 and 18-8817-33, Rockland; 1:5000 dilution) for 1 h at room temperature. Detection was performed by ECL or Dura Reagent (Thermo Fisher Scientific) followed by film exposure. ImageJ software was used to quantitate band density.

TurboID-mediated biotinylated protein pull-down
SUDHL10 cells stably expressing V5-TurBoID or V5-TurBoID-MEF2B WT-isoA were treated with 50 μM biotin (B4639, Sigma-Aldrich) for 24 h. Then, the cells were subjected to nuclear fraction isolation, as described in the Immunoblotting section. The nuclear extracts were incubated with Streptavidin Magnetic Beads (88817, Thermo Fisher Scientific) at 4°C overnight. Subsequently, the beads were subjected to three rounds of washing (1st round: 2% SDS; 2nd round: 50 mM pH 7.5 HEPES, 0.5 M NaCl, 1 mM EDTA, 0.1% Deoxycholic acid and 1% Triton x100; 3rd round: 10 mM pH 7.5 Tris-HCl, 0.25 M LiCl, 1 mM EDTA, 0.1% Deoxycholic acid and 1% NP40). Finally, the beads were boiled before proceeding to electrophoresis on Tris-Glycine gels followed by immunoblotting.

Phos-tag gel analysis
Whole cell lysates were extracted by EDTA- and phosphate-free lysis buffer (50 mM Tris-Hcl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, EDTA-free cOmplete protease inhibitor and phosSTOP phosphatase inhibitor). The samples were loaded into the 7.5% SuperSep Phos-tag™ gels (192-18001, Wako Chemicals) and electrophoresis was performed at 100 V on ice using freshly prepared Tris-MOPS buffer (50 mM Tris, 50 mM MOPS, 0.5% SDS and 5 mM sodium bisulfite). After electrophoresis, the gel was soaked three times in NuPAGE transfer buffer (Thermo Fisher Scientific) for 10 min/each. Protein transfer to 0.45 μm PVDF membrane (Millipore) was performed using the wet-tank method (Bio-Rad) at 150 mA for 16 h in the cold room. Antibody incubation and signal detection were performed using standard immunoblotting procedures.

Protein half-life measurement
SUDHL10 cells stably expressing N-terminal double-tagged Flag-HA-MEF2B WT or mutants were treated with 100 μg/ml cycloheximide (Sigma-Aldrich) at different time points and collected at the same time. To confirm proteasome-mediated degradation, 5 μM MG132 (Cayman Chemicals) was added in combination with cycloheximide treatments for 12 or 24 h. Cells lysates were resolved by Tris-Glycine gels and analyzed by immunoblotting with anti-HA and anti-vinculin antibodies. MEF2B half-life was quantified using the exponential regression function of densitometry values measured using the ImageJ processing software.

Ubiquitination assay
HEK293T cells were transfected with indicated plasmids by PEI, and then cultured for 48 h. 4 h before cell harvest, 10 μM MG132 was added into the medium for ubiquitin accumulation. For ubiquitin detection in DLBCL cells, SUDHL10 cells stably expressing N-terminal double-tagged Flag-HA EV or Flag-HA-MEF2B isoA-WT were treated with 5 μM MG132 for ~16 h. Both the HEK293T and SUDHL10 cells were subjected to lysis and then denatured at 95°C for 15 min using a modified IP buffer containing 2 μM NEM (N-Ethylmaleimide; Sigma-Aldrich) and 1% SDS. Subsequently, sonication was performed to shear genomic DNA (Bioruptor, Diagenode). The SDS in the cell lysate was quenched by adding 10% Triton-100 and diluted to 0.1% SDS with IP buffer containing 2 μM NEM. Flag IP was performed overnight, and the beads were boiled before electrophoresis on Tris-Glycine gels followed by immunoblotting.

RNA extraction, cDNA synthesis, and RT-PCR
Total RNA was extracted using the Trizol Reagent (Thermo Fisher Scientific), RNeasy Kit (Qiagen), or the Nucleospin RNA XS RNA Isolation Kit (Macherey-Nagel), according to the manufacturer’s instructions. cDNA synthesis was performed with 1–3 μg of total RNA using the SuperScript II First-Strand Synthesis System (Thermo Fisher Scientific). PCR products were analyzed by electrophoresis on agarose gel. Quantitative retrotranscription q(RT)-PCR was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific) on a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). The primer sequences are as following:
Exogenous Flag-HA-MEF2B:
Forward: GATCCAAGCCACCATGTACC; Reverse: CAGAGCACGCT CAGCTCATA
CD86:
Forward: TTGATAATGGGATGAATGGAAGGA; Reverse: CGTAGG ACATCTGTAGGCTAAG
CD180:
Forward: CACCTCCTGGGATCAGATGT; Reverse: TGGTAGAGTGT CAGGGATTTCA
MAP3K8:
Forward: GGCCGCAGATGCAATCTTCTTA; Reverse: TGGCTTTGC AGATACTGCGTT
PAX5:
Forward: GTCCCAGCTTCCAGTCACAG; Reverse: CGGAGACTCC TGAATACCTTCG
RASGRP3:
Forward: GGGAAAAGCCTGTCTGCTGTT; Reverse: GCTCCAGAA AGTGAGGTGCT
GAPDH:
Forward: CATTTCCTGGTATGACAACGAA; Reverse: AGGGGTCTA CATGGCAACTG

RNA-seq
Total RNA extracted from sorted murine GCB cells were quantified and subject to quality controls using a Bioanalyzer (Agilent). ~200 ng RNA was used for library preparation and sequencing by Azenta Life Sciences (South Plainfield, NJ, USA). RNA-seq reads were mapped to the Mus musculus (mm10/GRCm38) genome reference using the hisat2 prebuilt genome index60. Genome-mapped reads were aligned to exons on the mm10 (or GRCh38) transcriptome reference61 based on the information in the genomic BAM files, using featureCounts62 to produce abundance tables. We sanitized the transcriptome references (exon only) by removing read-through genes, anti-sense elements, miRNA, and rRNA. The count tables were subsequently normalized to produce transcript per million (TPM) tables. Differentially expressed genes were determined using DESeq263 with the following filters: FDR < 0.05 (after Benjamini-Hochberg correction) and absolute fold change (FC) > 1.2.

Cut and run
Nuclei were isolated from GCB cells freshly purified from human tonsil using a nuclear extraction buffer (20 mM pH 7.9 HEPES, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, and 1 mM MnCl2). Cut&Run sample preparation was performed using the CUTANA ChIC/CUT&RUN Kit version 3.1 (14-1048, EpiCypher), following the manufacturer’s instructions. Briefly, activated ConA Beads (EpiCypher) were added to the nuclei samples followed by incubation with 0.5 μg of IgG (13-0042, EpiCypher), SMARCA4 (49360, Cell Signaling Technology), MEF2B (custom), H3K27Ac (C15410196, Diagenode) and H3K4me3 (13-0041, EpiCypher) antibodies to capture protein/DNA complexes. Subsequently, pAG-MNase (EpiCypher), added to all samples, was activated by CaCl2 to cleave target chromatin. DNA purification was performed using CUTANA™ DNA Purification Kit to capture DNA fragments >50 bp. Library preparation was performed with 5 ng of CUT&RUN enriched DNA (or the total sample volume if DNA output was less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit version 1.0 (14-1001, EpiCypher) following the manufacturer’s instructions.

After quantification, using Qubit™ fluorometer with 1X dsDNA HS Assay Kit (Invitrogen), as per manufacturer’s instructions, and analysis of the library fragment size, using the High Sensitivity DNA Kit (Bioanalyzer), libraries were pooled and sequenced using HiSeq 3000/4000 Illumina paired-end sequencing. Paired-end samples were aligned to the GRCh38 genome using hisat264 with the following options: hisat2, no mixed, no discordant, phred33, minins 10, and maxins 700. After alignment, the BAM files were converted to BED files using bedtools bamtobed with the bedpe option. The BED files were filtered to keep properly paired fragments <1000 bp in length. MACS3 was used to find peaks in the filtered BED files using the matching IgG samples as control with the following options: macs3 callpeak, format BEDPE, and keep dup all ghs q 0.05.

Sample preparation for mass spectrometry (MS) analysis
The protein samples for MS analysis were purified from normal GCB cells and SUDHL10 cells stably expressing the N-terminal double-tagged Flag-HA-MEF2B WT or mutants according to the IP procedure. The IP eluate for interactor identification was separated on 4–12% Tris-Glycine gel (Thermo Fisher Scientific) for Coomassie blue (Thermo Fisher Scientific) staining, and all the bands were excised. For protein modification analysis, the IP eluates were subject to electrophoresis using 10% Tris-Glycine gel (Thermo Fisher Scientific) and ~42 KDa bands were excised. The excised protein gel slices were used for in-gel digestion as previously described65, with minor modifications. Gel slices were washed with 1:1 Acetonitrile and 100 mM ammonium bicarbonate for 30 min then dehydrated with 100% acetonitrile for 10 min until shrunk. The excess acetonitrile was then removed, and the slices dried in a speed vacuum at room temperature for 10 min. Gel slices were reduced with 5 mM DTT for 30 min at 56°C in an air thermostat, cooled down to room temperature, and alkylated with 11 mM IAA for 30 min with no light. Gel slices were then washed with 100 mM of ammonium bicarbonate and 100% acetonitrile for 10 min each. Excess acetonitrile was removed and dried in a speed-vacuum for 10 min at room temperature and the gel slices were re-hydrated in a solution of 25 ng/μl trypsin in 50 mM ammonium bicarbonate for 30 min on ice and digested overnight at 37°C in an air thermostat. Digested peptides were collected and further extracted from gel slices in an extraction buffer (1:2 ratio by volume of 5% formic acid:acetonitrile) at high speed, shaking in an air thermostat. The supernatants from both extractions were combined and dried in a speed vacuum. Peptides were dissolved in 3% acetonitrile/0.1% formic acid.

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) for interactor identification
Peptides were separated within 80 min at a flow rate of 400 nl/min on a reversed-phase C18 column with an integrated Captive Spray Emitter (25 cm × 75 μm, 1.6 μm, Ion Opticks). Mobile phases A and B were using 0.1% formic acid in water and 0.1% formic acid in ACN. The fraction of B was linearly increased from 2 to 23% within 70 min, followed by an increase to 35% within 10 min and a further increase to 80% before re-equilibration. The timsTOF Pro was operated in PASEF mode66 with the following settings: Mass Range 100 to 1700 m/z, 1/K0 range 0.6 to 1.6, Ramp time 100 ms, lock duty cycle to 100%, Capillary Voltage 1600 V, Dry Gas 3 l/min, Dry Temp 200°C, PASEF settings: 10 MS/MS Frames (1.16 s duty cycle), charge range 0–5, the active exclusion for 0.4 min, Target intensity 20,000, Intensity threshold 2500, CID collision energy 59 eV. A polygon filter was applied to the m/z and ion mobility plane to select features most likely representing peptide precursors rather than singly charged background ions.

LC-MS/MS data analysis for interactor identification
Acquired PASEF raw files were analyzed using the MaxQuant environment v. 2.0.3.0 and Andromeda for database searches at default settings67. The default is used for the first search tolerance and main search tolerance (20 ppm and 4.5 ppm, respectively). MaxQuant was set up to search with the reference human proteome database (UP000005640) downloaded from UniProt. MaxQuant performed the search trypsin digestion with up to 2 missed cleavages. Peptide, site, and protein false discovery rates (FDR) were all set to 1% with a minimum of 1 peptide needed for identification. The following modifications were used for protein identification and quantification: Carbamidomethylation of cysteine residues (+57.021 Da) was set as static modifications, while the oxidation of methionine residues (+15.995 Da), deamidation (+0.984) on asparagine and glutamine were set as a variable modification. Results obtained from MaxQuant were used to identify MEF2B complex formation. Moreover, the common protein contaminants including keratins, trypsin, serum albumin, and actin were excluded as previously reported68.

LC-MS/MS analysis for MEF2B phosphorylation
The dried peptides were dissolved in 10 μl of 3% acetonitrile/0.1% formic acid and 500 ng analyzed on an Orbitrap Fusion mass spectrometer coupled to a Dionex Ultimate 3000 (Thermo Fisher Scientific) using the PRM method69. Peptides were separated on an EASY-Spray C18 25 cm column (Thermo Fisher Scientific). Peptides elution and separation were achieved at a non-linear flow rate of 300 nl/min using a gradient of 5%-30% of buffer B (0.1% v/v formic acid, 100% acetonitrile) for 100 min with a temperature of the column maintained at 40°C during the entire experiment. The Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer was used for peptide tandem mass spectroscopy (MS/MS). MS data were acquired with combined two scan events corresponding to a full scan and a MS2 method targeting the specific MEF2B-unmodified and phospho-modified peptides. The target value for the full scan MS spectra was 1×10^6 ions in the 450–900 m/z range with a maximum injection time of 60 ms and resolution of 120,000 at 200 m/z. The tMS2 method were acquired with an isolation width of 1.6 m/z, a resolution of 30,000 on an Orbitrap, a target AGC value of 1.0×10^6, fragmented by higher-energy C-trap dissociation with a normalized collision energy of 27 eV, and a maximum injection time of 110 ms.

Data analysis was performed using Thermo Xcalibur v4.5.445.18 and the area under the curve of selected precursor ions summed to determine the abundance of the respective MEF2B-unmodified and modified phosphopeptides.

Enzyme-linked immunosorbent assay (ELISA)
ELISA for NP-specific antibodies using plates coated with NP5 or NP25 in BSA (Biosearch Technologies; n5050) was done as previously described70. Peripheral blood was obtained before immunization and 14 d after immunization. The levels of serum IgG were measured by ELISA using unlabeled anti-mouse Ig antibodies (Southern Biotech) as capture reagent, Alkaline phosphatase (AP)-labeled anti-mouse lg subclass-specific antibodies (Southern Biotech) as developing reagents, and 4-methylumbelliferyl phosphate as AP substrate.

Statistics
GraphPad Prism v. 9.0 software was used for statistical analyses. One-way analysis of variance (ANOVA) paired with Dunnett’s Multiple Comparison Test, Mann-Whitney test, unpaired two-tailed T-test, or one-sample T-test were used to assess statistically significant differences between groups. Fisher’s Exact test was used to assess significance of MEF2B mutation distribution and tumor incidence. Detailed information of the statistical test, number of replicates, and number of animals (defined as n) used in each experiment, as well as the definition of center and dispersion are reported in the figures and in the legends. Significance was associated to a p ≤ 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study by Yu et al explores the molecular and physiological impact of mutations targeting\nMEF2B in its C-terminus, in the context of B cell lymphomas. MEF2B is mutated in ~10% of\nFL and GCB-DLBL (two most common forms of lymphoma), with a third of mutations\naffecting its C-terminus. Authors deploy an impressive collection of approaches and models\n(from classical biochemistry, to proteomics, epigenetics, and animal studies), to achieve an\nin-depth and comprehensive characterization of these mutations. The inclusion of multiple\nand well-thought-out controls greatly contributes to the robustness of the observations. The\nstudy starts by characterizing post-translational modifications in WT MEF2B, and then\nexplores how different classes of mutations alter these modifications, impacting the stability,\nbinding partners, and function of MEF2B in lymphoma cells.\nRegarding the study’s significance, FL and GCB-DLBCL typically exhibit relatively good\nprognosis, compared to other lymphoma types. From the data presented, it is unclear whether\nC-terminus MEF2B mutations associate with more aggressive forms of these diseases\n(relapse/resistance) in the clinic. Furthermore, the idea that MEF2B mutants could be\nspecifically targeted to treat lymphomas is attractive in concept, but does not appear readily\napplicable, given the structural diversity and complex behavior induced by different\nmutations, as described here. Hence, the detailed mechanistic characterization of the\nmutations is the biggest perceived contribution of this study, which still holds major value.\nFrom a conceptual standpoint, it clearly illustrates: a) how lymphoma mutations may hijack\ndifferent functions in normal B cells, and b) how different types of mutations on the same\ngene may converge at the molecular and/or physiological level.\n*There are some points that, if addressed, could add further value and/or improve clarity:\n- If available, reporting on MEF2B mutations status in all cell models (or at the very least in\nSUDHL10) would help the interpretation of results.\n- Regarding MEF2B/SMARCA4 interactions at the chromatin level:\no The chromatin profiling of MEF2B/SMARCA4 in normal B cells (Figure 4) is interesting,\nbut may not reflect the behavior of these factors that in the context of tumor cells (where both\nthe chromatin landscape and other factors are dysregulated). While additional Cut&Run\nassays may be out of scope, authors should at least conduct qChIPs, to validate co-occupancy\n(or lack of) at selected loci.\no Related to the point above, it would be valuable to show that, in the presence of MEF2B\nmutants in tumor cells: a) SMARCA4 recruitment to selected chromatin locus would change,\nand b) expression of selected genes of interest would change. This would allow authors to\nfurther support a role for the MEF2B/SMARCA4 association in driving a disrupted\ntranscriptional program.\no In addition to the reported pathway enrichment analysis for MEF2B/SMARCA4 occupied\ngenes, the same analysis could be run for MEF2B-only locus/genes, to better understand\nwhich programs driven by MEF2 may require the co-recruitment of SMARCA4.\n- The authors claim that S324 phosphorylation contributes to the dissociation of\nMEF2B/SMARCA4 (line 216). However, the S324E mutant does not show significant\ndifferences to the WT protein (ex. 5a, 6b), whereas, based on the statement, a gain in the\ninteraction would have been expected. Authors should reconcile these\nstatement/observations. Along these lines, it would be helpful to investigate if the half-life for\nS324E is reduced as compared to WT.\n- In various sections, the authors try to convey a direct equivalency between the missense\nmutation in P352L, and the loss of phosphorylation in S324. However, effects of this and\nother mutations appear consistently more dramatic than those of the S324A variant (ex.\nSMARCA4 interactions, transcriptional effects, etc.). This suggests that the effects of the\nmissense mutations cannot be solely explained by loss of S324 phosphorylation. If a double\nmutant P352L/S324E was studied, would this reverse the effects of the P352L mutation\nalone?\n- The HOMO and HET mice show comparable expansion in GCB (ex. Fig 7a, e, f). Would\nauthors have expected to see a more exacerbated phenotype, specially considering differences\nat ulterior stages (Fig 7)?\n- The text mentions that HET GCB were profiled by RNASeq, however data is not presented\nor discussed. Given that the magnitude of GC expansion and DZ/LZ bias is comparable, were\nthere also differences at the transcriptional levels between HET vs WT (and HOMO)?.\n- Based on the Cut&Run experiments, BCL6 appear to be co-occupied by\nMEF2B/SMARCA4. Intuitively, P352L would then favor BCL6 expression, which could\nsupport the observed GC expansion. Was BCL6 expression altered in the mutant GCB? From\nthe RNASeq data, it doesn’t appear to be the case.\n- In Figure 7i, were all MEF2B-bound genes used as background list to determine\nenrichment, or were MEF2B/SMARCA4 sites compared to the whole genome? If the latter,\ncomparison is inadequate. Moreover, given that there is a bias in GC populations (LZ over\nDZ), is the enrichment for MEF2B/SMARCA4 occupied genes a feature specific to the\nmutant, or would WT LZ show a similar enrichment over WT DZ?\n- The statement that a LZ expansion, in itself, represents a pre-malignant state (line 313) is\nnot well supported. Studies referenced describe additional features in mutant cells that\nconvey malignancy: blockage in differentiation, independence from T cells, aberrant\nproliferation/cell death, increased rate of mutation, etc. Authors should modify the statement,\nor show evidence that P352L GCBs harbor bona fide pre-malignant features.\n- The statement that MEF2B mutations promote lymphomagenesis (line 339) is not accurate.\nInstead, mutations appear to change the trajectory of the cells, in favor of other lymphoma\ntypes. Also, PBL tumors in human are considered to have an early GC origin (based, for\nexample, on the presence of SHM). Could the authors comment on this?\n*Other comments\n- MW markers should be added to all protein blots.\n- In blots where authors use asterisks to flag “non-specific bands”, how was this determined?\n- When conducting densitometry analysis, consider normalizing expression of phosphorylated\nprotein to total MEF2B levels. For example, in Figure 3d, both S324 and total levels for\nMEF2B appear lower for the P330L variant.\n- Was there a particular criterion to select SUDHL10 as the main tumor model in the study?\n- Figure 2b could be improved by reporting results separately for transformed and normal\nGCBs.\n- Inclusion of full-size blots for the custom antibodies presented in the study would help\nassess their specificity.\n- In the Sanger sequencing characterization of the HET mice, wouldn’t a mixed peak (C/T) be\nexpected?\n- In the representative flow cytometry plots, what does FASmidGL7neg population\nrepresent?\n- Authors should report if any differences between female and male mice were observed,\nconsidering known differences in the magnitude of the immune response across sexes.\n- In Figure 6e, T274fs maintains >50% interaction with CUL3, and 80% interaction\nKHLHL12, but its half-life is still significantly higher than WT, and comparable to other\nmutants where interactions are completely lost. How do the authors interpret this?\n- Also, in Figure 6e, for P273fs interaction with KLHLSL12 is largely preserved (~70%), but\nthat with CUL3 is lost (~40%). Are these then independently regulated?\n- Authors claim that the ability to generate high-affinity antibodies in HET/HOMO mice was\nunchanged, but supplementary Figure 7k shows a statistical significance.\n- RNA-Seq claims to have been derived from littermates. Probability of getting 4x WT/WT\nand 3x MUT/MUT mice from a single regular-sized litter appear unlikely (unless offspring\ndoes not follow Mendelian ratios).\n- Methods mention an immunization with NP-KLH, and intracellular staining of markers for\nflow cytometry, but these data are not presented.\n- It would be useful to include, as a supplementary item, survival curves for all animals\nregardless of lymphoma type (in addition to those with GC tumors shown in Fig 8c).\n- Number of animals in Figs 8a and 8c don’t match. Where these different cohorts?", "reviewer_2": "Yu et al., studied mutations in the C-terminal MEF2B region that result in a phosphorylation\ndeficit of MEF2B isoformA, increasing transcriptional activity as well as protein stability.\nThey show the localization of MEF2B phosphorylation at the C-tail and how this position is\nmutated in patients and cell lines. Using MS, they discovered that unphosphorylated mutated\nMEF2B as well as isoformB associate with a chromatin remodeling complex (SWI/SNF\nSMARCA4) and increases its transcriptional activity. Independently of that, their MS\nexperiment revealed KLHL12/Cul3 as another interactor of MEF2B and show that mutated\nMERF2B leads to hindered interaction with KLHL12 and less ubiquitination, stabilizing the\nMEF2B transcription factor.\nFinally, they present a mouse model in which the lymphoma-associated MEF2B mutant,\nwhich lacks the ability to be phosphorylated at the C-terminus, leads to enlargement of the\ngerminal center and enhanced lymphomagenesis in these mice when bred to Bcl2 transgenic\nbackground.\nOverall, they show biologically and clinically relevant findings on the MEF2B function and\nan interesting connection with the chromatin remodeling complex and KLHL12/Cul3, that I\nfeel are potentially worth publishing in Nature Communications, after however addressing the\nfollowing issues.\nMajor issue:\nKey findings, which include interactions and protein stability experiments are derived from\nimmunoblot data. As the authors do not indicate how often these results were obtained\n(independent replicates), it is hard to get a feeling for the reproducibility of the results. Please\nindicate then number of replicates performed and quantify, where appropriate. Additionally,\nthe interactions of MEF2B with SMARCA4 and KLHL12 are shown by immunoprecipitation\nand immunoblot analysis - immunofluorescence imaging or a PLA assay would give great\ninsight into the localization as well as the physiological interaction of the proteins and would\nstrengthen the manuscript.\nThe authors claim that KLHL12/Cul3 ubiquitinates MEF2B, triggering its proteasomal\ndegradation. There are strong leads that this degradation process occurs via the ubiquitin\nproteasome system but it is not shown beyond doubt. It would strengthen the claim\nsubstantially, if the authors investigated the ubiquitin chain type (e.g. K48-linkage type)\nattached to MEF2B. Additionally, a proteasome inhibitor like MG-132 or bortezomib should\nbe included to their protein stability assays (cycloheximide treatments), to investigate\nwhether the simultaneous inhibition of the proteasome recues the degradation of MEF2B (or\nits variants). Speaking of the protein stability assays, it seems essential to include/show the\ndegradation of a well-known, short lived-protein (e.g. MCL1 or PLK1) as control. This way\nthe reader would be assured that the inhibition of protein synthesis was successful and\noccurred to a similar extend for every single MEF2B variant tested. There is a discrepancy in\nthe protein stability data presented by the authors. In Figure 6g and Extended Data Fig. 5e-f\nthere is rather little degradation of MEF2B after 24hrs of CHX-treatment, while in Figure\nEx5d, the half live of IsoA is around 6hrs. Please address this experimentally by including the\nsuggested controls or discuss accordingly.\nThe work presented explains the mechanism of action of C-terminally mutated MEF2B in\nlymphomagenesis. A question that is neither addressed experimentally nor discussed, is the\nphysiological role of the phosphorylation of Serine 324 and why it is not a mutated in any\nprimary tumor samples presented. This should be added to the discussion.\nIn the discussion Yu et al. state that the presented mechanism provides multiple opportunities\nfor the development of targeted therapeutic approaches. Could the authors specify and\nelaborate on this, to make it a stronger point? Since most patients seem to have truncating\nmutations of the C-terminal region of MEF2B, two different therapeutic approaches might be\nnecessary. Please discuss how exactly you would target the protein in these patients.\nMinor issues:\nIt is stated that MEF2B is phosphorylated at the C-tail of isoform A and that patients have\ndifferent truncating and isoform-switch mutations, resulting in different phosphorylation\npatterns of MEF2B (Fig. 2a +c, 3c). The authors also state that depending on the MEF2B\nisoform and C-tail mutation, ubiquitination by KLHL12 changes (Fig. 6c+f). Please indicate\nthe kDa in those blots to better comprehend the size changes in these experiments.\nIn paragraph 160-162 the authors describe a mass spectrometry experiment in SUDHL10\ncells that confirmed the lack of phosphorylation at S324. Please include the data/figure or a\nreference to this MS experiment, as it is unclear which data the authors are referring to.\nIn figure 4a, a co-IP of endogenous MEF2B and SMARC4 proteins is depicted to show their\ninteraction in the nucleus. Simply blotting for the nuclear protein lamin, without the use of\nother marker proteins and/or showing cellular fractions, does not qualify to state, that a\nnuclear interaction takes place. Please include IPs in other cellular fractions or show this\nusing additional methods (e.g PLA or IF).\nAfter finding SMARCA4 as a potential interactor of MEF2B by mass spectrometry, Yu et al.\nstate that phosphor-deficient MEF2B mutants (S324A) show an increased interaction with\nSMARCA4 via Co-IP (Fig. 5a+c). The quantification seems based on one experiment alone.\nAs this seems like an important finding, please quantify from multiple independent\nexperiments.\nIn figure 5a a phospho-mimic (S324E) is included as another control to show that there is no\nincreased interaction with SMARCA4. To strengthen their point and see if see the phospho-\nmimic could decrease transcriptional activity (figure 5b), please include the phosphor,-mimic\nin this experiment. Please discuss why the non-phos mutant S324A has the smallest effect on\nthe MEF2 reporter system, if the functional relevance is mediated by lower phosphorylation\non exactly this site.\nThe authors further state, that MEF2B interacts with KLHL12 via the N- as well as the C-\nterminus of MEF2B. C-terminus phosphodeficient MEF2B has a decrease binding affinity\ntowards KLHL12 (Figure. 6b+e). Again, the quantification is based on one blot, please\nquantify this in triplicates.\nIn figure 6c, impaired MEF2B-KLHL12/Cul3 binding due to phospho-deficiency of the C-\nterminus results in reduced ubiquitination of the MEF2B phospho-mutants in HEK293T cells.\nCan the authors also show the ubiquitination in a DLBCL cell line like SUDHL10? In\naddition, the inclusion of the phospho-mimic mutant (S324E) from previous experiments in\nthis ubiquitination experiment as control would be helpful.", "reviewer_3": "The manuscript by Yu et al entitled “MEF2B C-terminal mutations enhance transcriptional\nactivity and stability to drive B cell lymphomagenesis” revealed MEF2B phosphorylation at\nits c-tail S324 site is impaired by lymphoma-associated mutations. Mechanistically, the\nauthors showed that lack of phosphorylation by the surrounding mutations enhances the\ninteraction of MEF2B with the SWI/SNF chromatin remodeling complex and increases the\nstability of the mutant proteins during to a reduction in their interaction with the\nCUL3/KLHL12 ubiquitin complex. Consistently, mice expressing a phosphorylation-\nimpairing MEF2B mutant display GC enlargement and when crossed with Bcl2 transgenic\nmice, develop GC-derived lymphomas. Overall, the data support the conclusion of the\nmanuscription and most of experiments are well controlled with high quality results. Thus,\nthe manuscript is appropriate for Nature Communication. However, the following concerns\nshould be addressed.\n1. A major weakness of the manuscript will have to do with the fact that most of mechanistic\nstudies were performed using ectopic expression. While certain experiments using this\napproach is well justified, those with naturally occurring mutants should be repeated using\nrelevant cell lines at the endogenous levels for protein interaction, genome-wide distribution\nand stability/ubiquitination (ideally using isogenic cell lines).\n2. Along this line, correlative studies should be performed to correlate the mechanistic studies\nwith the phenotypes observed using the genetic mouse models. A comparison between\ntumors developed from those generated using N vs. C -tails mutants would have provided\nsubstantial insights into biology of MEF2B in the context of lymphomagenesis.\n3. Although examples in Figure 4c are convincing, Figure 4b is not very informative. An\noverlap profile might be more informative to support the authors’ conclusions.\n4. The data as presented in Extended Data Fig. 4 is counterintuitive because the interaction in\nnormal GC B cells appears to be more robust compared with the P325L mutant. This does not\nsupport an oncogenic role of MEF2B mutant based on the model proposed.\n5. SWI/SNF mutations are prevalent in both DLBCL and FL. Based on the model proposed,\none would predict a mutual exclusivity between SWI/SNF mutations and those that affect\nS324 phosphorylation. This should be examined.\n6. To better justify the combination used to generate the mouse model, the authors may\nconsider examine the co-occurrence between MEF2B mutants that affect S324\nphosphorylation and BCl2 re-arrangement."}, "author_response": "RESPONSE: As detailed in the revised Methods (page 24, line 557-559), all cell lines used as\nmodels in this work do not carry endogenous mutations in the MEF2B gene. No cell lines with\nmutations targeting the C-terminus have been reported.\n- Regarding MEF2B/SMARCA4 interactions at the chromatin level:\no The chromatin profiling of MEF2B/SMARCA4 in normal B cells (Figure 4) is interesting, but may not\nreflect the behavior of these factors that in the context of tumor cells (where both the chromatin\nlandscape and other factors are dysregulated). While additional Cut&Run assays may be out of\nscope, authors should at least conduct qChIPs, to validate co-occupancy (or lack of) at selected loci.\nRESPONSE: We were unable to establish robust qChIP assays in normal or malignant GC B cells.\nTherefore, we performed Cut&Run assays for MEF2B and SMARCA4 in the lymphoma cell line\nSUDHL10 (which we extensively used in multiple experiments). The results showed that about 60%\nof the regions co-bound by MEF2B and SMARCA4 in SUDHL10 cells are also detected in the normal\nGC B cells. This significant overlap supports that the large majority of the MEF2B/SMARCA4 program\nis retained in tumor cells.\no Related to the point above, it would be valuable to show that, in the presence of MEF2B mutants in\ntumor cells: a) SMARCA4 recruitment to selected chromatin locus would change, and b) expression\nof selected genes of interest would change. This would allow authors to further support a role for the\nMEF2B/SMARCA4 association in driving a disrupted transcriptional program.\nRESPONSE: Regarding the change in SMARCA4 recruitment (point a), we were unable to establish\nrobust qChIP assays, but this aspect was indirectly addressed by measuring changes in the\nexpression of selected targets (point b) showing MEF2B/SMARCA4 co-occupancy by Cut&Run\nassays in normal GC B cells. Toward this goal, we generated isogenic lymphoma cell lines expressing\nthe phospho-deficient P325L mutant from the MEF2B endogenous locus and showed upregulation of\nMEF2B/SMARCA4 targets in the mutant clones compared to their isogenic controls (Supplementary\nFig. 8o). These results were consistent with the increased expression observed also in murine GC B\ncells engineered to express a phospho-deficient mutant Mef2b (Fig. 8i) and are now reported in the\nResults section (page 14, lines 333-339).\no In addition to the reported pathway enrichment analysis for MEF2B/SMARCA4 occupied genes, the\nsame analysis could be run for MEF2B-only locus/genes, to better understand which programs driven\nby MEF2 may require the co-recruitment of SMARCA4.\nRESPONSE: In the revised version, we performed pathway enrichment analysis on genes displaying\nbinding for MEF2B but not SMARCA4. The results show that, although several pathways (including\nBCR and PI3K/AKT signaling pathways) are uniquely enriched for genes co-bound by\nMEF2B/SMARCA4, no pathways are uniquely affected by genes displaying MEF2B-only occupancy,\nsuggesting that at the pathway level all MEF2B-driven programs involve its interaction with\nSMARCA4. The pathways uniquely affected by the MEF2B/SMARCA4 targets are now highlighted in\nthe revised Supplementary Table 4. In addition, the Results section has been revised to include this\ninformation (page 9, lines 197-198).\n- The authors claim that S324 phosphorylation contributes to the dissociation of MEF2B/SMARCA4\n(line 216). However, the S324E mutant does not show significant differences to the WT protein (ex.\n5a, 6b), whereas, based on the statement, a gain in the interaction would have been expected.\nAuthors should reconcile these statement/observations. Along these lines, it would be helpful to\ninvestigate if the half-life for S324E is reduced as compared to WT.\nRESPONSE: We have confirmed that no significant differences are detected between the MEF2B\nwild-type (WT) and the artificial phospho-mimic S324E mutant regarding the transcriptional activity\n(revised Fig. 5c) as well as the protein stability (revised Fig. 6d-e). The results were also confirmed in\nthe newly established OCI-Ly7 isogenic clones expressing the artificial S324E mutant from the\nendogenous locus (new Figs. 5b and 6c and Supplementary Figs. 4d and 6b), suggesting that the\nS324E restores a WT-like phenotype. These observations may not be completely unexpected\nconsidering MEF2B heterodimerization in the wild-type context and the possibility that phosphorylated\nheterodimers may function similarly to phosphorylated homodimers.\n- In various sections, the authors try to convey a direct equivalency between the missense mutation in\nP352L, and the loss of phosphorylation in S324. However, effects of this and other mutations appear\nconsistently more dramatic than those of the S324A variant (ex. SMARCA4 interactions,\ntranscriptional effects, etc.). This suggests that the effects of the missense mutations cannot be solely\nexplained by loss of S324 phosphorylation. If a double mutant P352L/S324E was studied, would this\nreverse the effects of the P325L mutation alone?\nRESPONSE: As suggested by the reviewer, we generated SUDHL10 cells expressing the\nP325L/S324E double mutant and analyzed them in parallel with cells expressing the MEF2B wild-\ntype (WT) protein or the single mutants (P325L or S324E). The cells expressing the double mutant\nbehaved similarly to those expressing MEF2B-WT regarding the protein stability (revised Fig. 6e and\nSupplementary Fig. 6d) and the transcriptional activity (revised Fig. 5c), suggesting that the\nphosphomimic mutant rescues these features driven by P325L. We note that although the interaction\nwith SMARCA4 is restored to levels similar to the WT (revised Fig. 5a) as expected, the\nP325L/S324E double mutant remains deficient in its binding to KLHL12 (revised Fig. 6b), suggesting\nthat alternative adaptor proteins may be involved in restoring the protein degradation of this artificial\nmutant.\n- The HOMO and HET mice show comparable expansion in GCB (ex. Fig 7a, e, f). Would\nauthors have expected to see a more exacerbated phenotype, specially considering differences\nat ulterior stages (Fig 7)?\nRESPONSE: We do not have an explanation for the lack of significant differences between HET and\nHOMO mice regarding the mild phenotype of GC B cell expansion (Fig. 8, in the revised manuscript),\nespecially considering the more exacerbated lymphoma phenotype observed in the HOMO mice (Fig.\n9, in the revised manuscript).\n- The text mentions that HET GCB were profiled by RNASeq, however data is not presented\nor discussed. Given that the magnitude of GC expansion and DZ/LZ bias is comparable, were there also\ndifferences at the transcriptional levels between HET vs WT (and HOMO)?\nRESPONSE: The transcriptional signature discriminating GC B cells of WT and HOMO mice was also\nobserved in the HET mice, as shown in the newly added GSEA plots (Supplementary Fig. 8m and\nResults, page 14, lines 316-317).\n- Based on the Cut&Run experiments, BCL6 appear to be co-occupied by\nMEF2B/SMARCA4. Intuitively, P352L would then favor BCL6 expression, which could\nsupport the observed GC expansion. Was BCL6 expression altered in the mutant GCB? From\nthe RNASeq data, it doesn’t appear to be the case.\nRESPONSE: No significant differences in BCL6 expression were observed in bulk GC B cells at\ntranscript (Supplementary Table 6), or protein level, as measured by intracellular staining and FACS\nanalysis (data not shown). However, we cannot exclude that BCL6 de-regulation may occur in a GC B\ncell subpopulation and differences are not captured in the overall bulk analysis.\n- In Figure 7i, were all MEF2B-bound genes used as background list to determine\nenrichment, or were MEF2B/SMARCA4 sites compared to the whole genome? If the latter,\ncomparison is inadequate.\nRESPONSE: We may not have presented the data clearly leading to some misunderstanding. Figure\n8i (former Fig. 7i) reports the results of gene set enrichment analysis (GSEA) performed to evaluate\nwhether the MEF2B/SMARCA4 co-bound targets are transcriptionally affected in GC B cells\nexpressing the WT versus those expressing the Mef2bP297L mutant protein. The enrichment is\nmeasured by ranking all the assessed transcripts in the RNAseq profiles and evaluating any\ndistribution bias along the ranked list. This information is now detailed in the figure legend (page 44,\nlines 1037-1039).\nMoreover, given that there is a bias in GC populations (LZ over DZ), is the enrichment for\nMEF2B/SMARCA4 occupied genes a feature specific to the mutant, or would WT LZ show a similar\nenrichment over WT DZ?\nRESPONSE: To investigate this aspect, we performed GSEA using published murine DZ and LZ\nexpression data and we observed an enrichment toward the LZ for MEF2B/SMARCA4 co-bound\ntargets. In our view, these results support that indeed the program driven by the MEF2B/SMARCA4\ncomplex is of relevance in driving the LZ expansion phenotype that is observed in the mice (revised\nResults page 14, lines 330-332).\n- The statement that a LZ expansion, in itself, represents a pre-malignant state (line 313) is\nnot well supported. Studies referenced describe additional features in mutant cells that\nconvey malignancy: blockage in differentiation, independence from T cells, aberrant\nproliferation/cell death, increased rate of mutation, etc. Authors should modify the statement,\nor show evidence that P352L GCBs harbor bona fide pre-malignant features.\nRESPONSE: We modified the sentence (page 15, lines 341-342) to clarify that the LZ expansion is a\nphenotype observed in multiple instances (including in the Mef2bD83V mouse model) to precede\nmalignant transformation.\n- The statement that MEF2B mutations promote lymphomagenesis (line 339) is not accurate.\nInstead, mutations appear to change the trajectory of the cells, in favor of other lymphoma\ntypes. Also, PBL tumors in human are considered to have an early GC origin (based, for\nexample, on the presence of SHM). Could the authors comment on this?\nRESPONSE: We revised the statement (page 16, lines 368-371) to more precisely convey the\nmessage that the reported increased lymphoma incidence is restricted to Mef2b-expressing tumors\n(DLBCL and FL), as opposed to bcl2-driven malignancies. Regarding the late GC origin of human\nPBL (from cells committed to plasma cell differentiation, as reported in the 2008 WHO classification;\nsee Campo et al. Blood 2011), we showed that deregulated Bcl2 expression in mice (Bcl2-Ig\ntransgenic mice) leads to the development of PBL malignancies with mutated and unmutated V\nregions, suggesting that in mice these tumors can have a GC-dependent or -independent origin.\n*Other comments\n- MW markers should be added to all protein blots.\nRESPONSE: Protein blots have been revised accordingly.\n- In blots where authors use asterisks to flag “non-specific bands”, how was this determined?\nRESPONSE: The “non-specific bands” were determined as following: for phospho-specific antibodies\n(Fig. 2e), the specificity (or lack of it) was defined by immunoprecipitation and treatment with lambda-\nphosphatase, now shown in the revised Supplementary Fig. 2c; for KLHL12 (Fig. 6a) the lack of\nspecificity was determined based on incorrect size and detection in the negative control (IgG).\n- When conducting densitometry analysis, consider normalizing expression of phosphorylated\nprotein to total MEF2B levels. For example, in Figure 3d, both S324 and total levels for\nMEF2B appear lower for the P330L variant.\nRESPONSE: As detailed in the revised figure legends, IP quantifications of phosphorylated MEF2B\n(Fig. 3d) or MEF2B interactors (Figs. 5, 6 and 7) are normalized by the proper loading control and\ntotal MEF2B protein.\n- Was there a particular criterion to select SUDHL10 as the main tumor model in the study?\nRESPONSE: We have extensively used this cell line model in our previous studies on MEF2B\nmutants (Ying, Nat. Immunol. 2013; Brescia, Cancer Cell 2018). Therefore, we decided to expand our\nstudy to the C-terminal mutations in the same cell system. The SUDHL10 cell line was initially\nselected because established from a DLBCL case, carries wild-type MEF2B gene and expresses\nMEF2B protein.\n- Figure 2b could be improved by reporting results separately for transformed and normal\nGCBs.\nRESPONSE: Note that Figure 2b reports the quantitation obtained only in normal GC B cells (not in\ntransformed cells). We have revised the figure legend (page 38, lines 897-898) to clarify this point.\nMass-spectrometry analysis performed in transformed SUDHL10 cells confirmed the prevalence of\nphosphorylation at S310, followed by S324 and at a lower level of T313 and T196. This data are now\nshown in the revised Supplementary Fig. 2a.\n- Inclusion of full-size blots for the custom antibodies presented in the study would help\nassess their specificity.\nRESPONSE: We report in the revised Supplementary Fig. 2c the blots used for the custom antibody\nvalidation, showing the specificity of the detection upon MEF2B immunoprecipitation, but not in\nimmunoblotting of total protein lysates.\n- In the Sanger sequencing characterization of the HET mice, wouldn’t a mixed peak (C/T) be\nexpected?\nRESPONSE: No, a mixed peak should not be detected because this RT-PCR was designed to detect\nexpression of the mutated allele (indeed it is not detected in the WT mice, Supplementary Fig.8b top\npanel), as detailed in the Methods (page 20), in the scheme reported in the Supplementary Fig. 8a,\nand in the corresponding figure legend.\n- In the representative flow cytometry plots, what does FASmidGL7neg population\nrepresent?\nRESPONSE: We have found that the FASmid/GL7neg population, observed in the representative\nexperiment that had been originally used for the figure, is a technical artifact due most likely to a high\nvoltage setting. This same population is equally observed across all genotypes and in all cells,\nincluding non-B cells. Although this artifact does not affect the identification of GC B cells, we have\nreplaced the representative dot plots with those of mice in which this technicality was corrected\n(revised Supplementary Fig. 8f).\n- Authors should report if any differences between female and male mice were observed,\nconsidering known differences in the magnitude of the immune response across sexes.\nRESPONSE: We have re-analyzed the data upon splitting the cohort by gender (males, n=26;\nfemales, n=24). Although the female cohort fails to reach significance regarding the increased\npercentage of GC B cells, both the male and female cohorts show statistically significant and\nconsistent differences in GC size, DZ/LZ ratios, and LZ cell number. We have included these\nobservations in the Results (page 13, lines 308-311) and Methods (page 21, line 470) sections.\n- In Figure 6e, T274fs maintains >50% interaction with CUL3, and 80% interaction\nKHLHL12, but its half-life is still significantly higher than WT, and comparable to other\nmutants where interactions are completely lost. How do the authors interpret this?\n- Also, in Figure 6e, for P273fs interaction with KLHLSL12 is largely preserved (~70%), but\nthat with CUL3 is lost (~40%). Are these then independently regulated?\nRESPONSE: We speculate that for these specific mutants the interaction with KLHL12 does not\ntrigger efficient degradation because KLHL12/CUL3 is unlikely to be the only complex involved in\nMEF2B degradation. It is possible that other adaptor(s) and/or cullin family members may be involved\nin the process and become of relevance in the case of selected mutations.\n- Authors claim that the ability to generate high-affinity antibodies in HET/HOMO mice was\nunchanged, but supplementary Figure 7k shows a statistical significance.\nRESPONSE: The text has been revised accordingly (page 13, lines 306-308).\n- RNA-Seq claims to have been derived from littermates. Probability of getting 4x WT/WT\nand 3x MUT/MUT mice from a single regular-sized litter appear unlikely (unless offspring\ndoes not follow Mendelian ratios).\nRESPONSE: To clarify this point we revised the Methods section (page 22, lines 493-494) as\nfollowing: “Mouse analyses were performed on sub-cohorts including 2-4 mice/genotype, which were\nobtained from 2-4 litters born at the same time”.\n- Methods mention an immunization with NP-KLH, and intracellular staining of markers for\nflow cytometry, but these data are not presented.\nRESPONSE: Data about mice immunized with NP-KLH were displayed in former Supplementary Fig.\n6k (current Supplementary Fig. 8k). The intracellular staining methods have been removed in the\nrevised version, because indeed no data on intracellular staining are included in the manuscript.\n- It would be useful to include, as a supplementary item, survival curves for all animals\nregardless of lymphoma type (in addition to those with GC tumors shown in Fig 8c).\nRESPONSE: As requested, we now show the survival curve for all mice, regardless of tumor\nphenotype (revised Supplementary Fig. 9b).\n- Number of animals in Figs 8a and 8c don’t match. Where these different cohorts?\nRESPONSE: The figure legend (page 44, lines 1046-1047) has been revised to clarify this point. The\ndifferences are due to the fact that Fig. 9a (former Fig. 8a) reports the “tumors”, while Fig. 9c (former\nFig. 8c) displays the survival for the “mice” bearing the tumors. Six mice displayed more than one\ntumor type, therefore the tumor counts are larger than the mouse counts.\n\nReviewer #2 (Remarks to the Author):\nYu et al., studied mutations in the C-terminal MEF2B region that result in a phosphorylation deficit of\nMEF2B isoformA, increasing transcriptional activity as well as protein stability.\nThey show the localization of MEF2B phosphorylation at the C-tail and how this position is mutated in\npatients and cell lines. Using MS, they discovered that unphosphorylated mutated MEF2B as well as\nisoformB associate with a chromatin remodeling complex (SWI/SNF SMARCA4) and increases its\ntranscriptional activity. Independently of that, their MS experiment revealed KLHL12/Cul3 as another\ninteractor of MEF2B and show that mutated MERF2B leads to hindered interaction with KLHL12 and\nless ubiquitination, stabilizing the MEF2B transcription factor.\nFinally, they present a mouse model in which the lymphoma-associated MEF2B mutant, which lacks\nthe ability to be phosphorylated at the C-terminus, leads to enlargement of the germinal center and\nenhanced lymphomagenesis in these mice when bred to Bcl2 transgenic background.\nOverall, they show biologically and clinically relevant findings on the MEF2B function and an\ninteresting connection with the chromatin remodeling complex and KLHL12/Cul3, that I feel are\npotentially worth publishing in Nature Communications, after however addressing the following\nissues.\nMajor issue:\nKey findings, which include interactions and protein stability experiments are derived from\nimmunoblot data. As the authors do not indicate how often these results were obtained (independent\nreplicates), it is hard to get a feeling for the reproducibility of the results. Please indicate then number\nof replicates performed and quantify, where appropriate.\nRESPONSE: All experiments were repeated at least twice independently, as detailed in the figure\nlegends. We now display the quantifications for each experiment directly in the immunoblots and/or\nas bar/dot plots reporting average and standard deviation (Supplementary Figs. 4c,e, 6a, 7a).\nAdditionally, the interactions of MEF2B with SMARCA4 and KLHL12 are shown by\nimmunoprecipitation and immunoblot analysis - immunofluorescence imaging or a PLA assay would\ngive great insight into the localization as well as the physiological interaction of the proteins and\nwould strengthen the manuscript.\nRESPONSE: To confirm co-localization and interaction, we first analyzed the expression of each\ninteractor in cytoplasmic and nuclear fractions and then performed protein-proximity labeling using\nthe TurboID system (Branon et al., Nat. Biotechnology 2018). The data confirmed nuclear localization\nof MEF2B and SMARCA4 and both cytoplasmic and nuclear localization for CUL3 and KLHL12 (new\nSupplementary Figs. 4a and 5a). The protein-proximity labeling in SUDHL10 cells using MEF2B-\nTurboID provided an independent confirmation of these interactions (new Supplementary Figs. 4b\nand 5b).\nThe authors claim that KLHL12/Cul3 ubiquitinates MEF2B, triggering its proteasomal degradation.\nThere are strong leads that this degradation process occurs via the ubiquitin proteasome system but\nit is not shown beyond doubt. It would strengthen the claim substantially, if the authors investigated\nthe ubiquitin chain type (e.g. K48-linkage type) attached to MEF2B.\nRESPONSE: Consistent with the degradation phenotype, we confirmed the involvement of the K48\nubiquitin chain type (revised Figs. 6d and 7c).\nAdditionally, a proteasome inhibitor like MG-132 or bortezomib should be included to their protein\nstability assays (cycloheximide treatments), to investigate whether the simultaneous inhibition of the\nproteasome recues the degradation of MEF2B (or its variants). Speaking of the protein stability\nassays, it seems essential to include/show the degradation of a well-known, short lived-protein (e.g.\nMCL1 or PLK1) as control. This way the reader would be assured that the inhibition of protein\nsynthesis was successful and occurred to a similar extend for every single MEF2B variant tested.\nRESPONSE: We have repeated the protein stability assays including MYC, as a well-known short-\nlived protein (half-life 30’), and treatment with the MG132 proteasome inhibitor (revised\nSupplementary Figs. 6d and 7c). The results confirmed that the cycloheximide treatment was\nefficient and MYC degradation was detected in all experiments, while MG132-mediated proteasome\ninhibition rescued this effect.\nThere is a discrepancy in the protein stability data presented by the authors. In Figure 6g and\nExtended Data Fig. 5e-f there is rather little degradation of MEF2B after 24hrs of CHX-treatment,\nwhile in Figure Ex5d, the half live of IsoA is around 6hrs. Please address this experimentally by\nincluding the suggested controls or discuss accordingly.\nRESPONSE: As shown in the revised Supplementary Figs. 6d and 7c, MEF2B half-life is consistent\n(about 6.5 hrs). The apparent inconsistencies were due to a less efficient cycloheximide treatment in\none of the former experiments leading to a reduced overall degradation.\nThe work presented explains the mechanism of action of C-terminally mutated MEF2B in\nlymphomagenesis. A question that is neither addressed experimentally nor discussed, is the\nphysiological role of the phosphorylation of Serine 324 and why it is not a mutated in any primary\ntumor samples presented. This should be added to the discussion.\nRESPONSE: We have revised the Discussion to address the physiologic role of S324\nphosphorylation and to emphasize that although we identified several mutations consistently\nimpairing phosphorylation at S324, none of these alterations directly impact this amino acid in primary\ntumors (page 17, lines 379-380). We noted that not every base is mutated with the same frequency\n(page 17, lines 380-383).\nIn the discussion Yu et al. state that the presented mechanism provides multiple opportunities for the\ndevelopment of targeted therapeutic approaches. Could the authors specify and elaborate on this,\nto make it a stronger point? Since most patients seem to have truncating mutations of the C-terminal\nregion of MEF2B, two different therapeutic approaches might be necessary. Please discuss how\nexactly you would target the protein in these patients.\nRESPONSE: Our goal was to emphasize that N-terminus and phospho-deficient mutants affect\ndifferent protein complexes and therefore these differences should be considered if therapeutic\ntargeting of MEF2B is considered. We revised the Discussion to clarify this aspect (page 18, lines\n399-400).\nMinor issues:\nIt is stated that MEF2B is phosphorylated at the C-tail of isoform A and that patients have different\ntruncating and isoform-switch mutations, resulting in different phosphorylation patterns of MEF2B\n(Fig. 2a +c, 3c). The authors also state that depending on the MEF2B isoform and C-tail mutation,\nubiquitination by KLHL12 changes (Fig. 6c+f). Please indicate the kDa in those blots to better\ncomprehend the size changes in these experiments.\nRESPONSE: Protein blots have been revised accordingly.\nIn paragraph 160-162 the authors describe a mass spectrometry experiment in SUDHL10 cells that\nconfirmed the lack of phosphorylation at S324. Please include the data/figure or a reference to this\nMS experiment, as it is unclear which data the authors are referring to.\nRESPONSE: The data are now shown in the revised Supplementary Fig. 2e.\nIn figure 4a, a co-IP of endogenous MEF2B and SMARC4 proteins is depicted to show their\ninteraction in the nucleus. Simply blotting for the nuclear protein lamin, without the use of other\nmarker proteins and/or showing cellular fractions, does not qualify to state, that a nuclear interaction\ntakes place. Please include IPs in other cellular fractions or show this using additional methods (e.g\nPLA or IF).\nRESPONSE: Cytoplasmic and nuclear fractionations were performed and confirmed that MEF2B is\nexclusively detected in the nucleus, where all other interactors are also detected (new\nSupplementary Figs. 4a and 5a). In addition, the protein-proximity labeling in SUDHL10 cells using\nMEF2B-TurboID provided an independent confirmation of these interactions (new Supplementary\nFigs. 4b and 5b).\nAfter finding SMARCA4 as a potential interactor of MEF2B by mass spectrometry, Yu et al. state that\nphosphor-deficient MEF2B mutants (S324A) show an increased interaction with SMARCA4 via Co-IP\n(Fig. 5a+c). The quantification seems based on one experiment alone. As this seems like an\nimportant finding, please quantify from multiple independent experiments.\nRESPONSE: The figure displayed a representative plot, but the experiment was repeated at least two\ntimes. The revised Supplementary Fig. 4c displays now the quantification from all experiments,\nincluding averages and standard deviations.\nIn figure 5a a phospho-mimic (S324E) is included as another control to show that there is no\nincreased interaction with SMARCA4. To strengthen their point and see if see the phospho-mimic\ncould decrease transcriptional activity (figure 5b), please include the phosphor,-mimic in this\nexperiment. Please discuss why the non-phos mutant S324A has the smallest effect on the MEF2\nreporter system, if the functional relevance is mediated by lower phosphorylation on exactly this site.\nRESPONSE: We have now included the S324E mutant in the transcriptional assay, showing that it\nbehaves similar to MEF2B-WT (revised Fig. 5c). Regarding the extent of induction in the reporter\nassay, we confirmed that the artificial S324A and the lymphoma-associated R330L mutants display a\nsmaller induction compared to the P325L and R322C mutants. The smallest effect observed for the\nS324A and R330L mutants may be due to multiple factors, including the possibility that P325L and\nR322C provide additional conformational changes that further enhance the interaction with\nSMARCA4, as shown by the increased relative binding observed in these mutants (Fig. 5a,b and\nSupplementary Fig. 4c,d).\nThe authors further state, that MEF2B interacts with KLHL12 via the N- as well as the C-terminus of\nMEF2B. C-terminus phosphodeficient MEF2B has a decrease binding affinity towards KLHL12\n(Figure. 6b+e). Again, the quantification is based on one blot, please quantify this in triplicates.\nRESPONSE: The figure displayed a representative plot, but the experiments were repeated at least\ntwo times. The revised Supplementary Fig. 6a displays now the quantification from all experiments,\nincluding averages and standard deviations.\nIn figure 6c, impaired MEF2B-KLHL12/Cul3 binding due to phospho-deficiency of the C-terminus\nresults in reduced ubiquitination of the MEF2B phospho-mutants in HEK293T cells. Can the authors\nalso show the ubiquitination in a DLBCL cell line like SUDHL10? In addition, the inclusion of the\nphospho-mimic mutant (S324E) from previous experiments in this ubiquitination experiment as\ncontrol would be helpful.\nRESPONSE: We have now included the S324E mutant in the ubiquitination assays (revised Fig. 6d)\nand detected MEF2B ubiquitination in SUDHL10 cells expressing MEF2B-WT (Supplementary Fig.\n6c).\n\nReviewer #3 (Remarks to the Author):\nThe manuscript by Yu et al entitled “MEF2B C-terminal mutations enhance transcriptional activity and\nstability to drive B cell lymphomagenesis” revealed MEF2B phosphorylation at its c-tail S324 site is\nimpaired by lymphoma-associated mutations. Mechanistically, the authors showed that lack of\nphosphorylation by the surrounding mutations enhances the interaction of MEF2B with the SWI/SNF\nchromatin remodeling complex and increases the stability of the mutant proteins during to a reduction\nin their interaction with the CUL3/KLHL12 ubiquitin complex. Consistently, mice expressing a\nphosphorylation-impairing MEF2B mutant display GC enlargement and when crossed with Bcl2\ntransgenic mice, develop GC-derived lymphomas. Overall, the data support the conclusion of the\nmanuscription and most of experiments are well controlled with high quality results. Thus, the\nmanuscript is appropriate for Nature Communication. However, the following concerns should be\naddressed.\n1. A major weakness of the manuscript will have to do with the fact that most of mechanistic studies\nwere performed using ectopic expression. While certain experiments using this approach is well\njustified, those with naturally occurring mutants should be repeated using relevant cell lines at the\nendogenous levels for protein interaction, genome-wide distribution and stability/ubiquitination\n(ideally using isogenic cell lines).\nRESPONSE: To address this issue, we generated isogenic OCI-Ly7 cell lines by introducing point\nmutations in the MEF2B endogenous locus and selecting single-cell clones. Using these isogenic\ncells, we have confirmed that both missense and frame-shift mutants display enhanced interaction\nwith SMARCA4 (Fig. 5b,e and Supplementary Fig. 4d,f) and defective interaction with\nKLHL12/CUL3 complex (Figs. 6c, 7b and Supplementary Figs. 6b and 7b) leading to an increase in\nprotein stability (Figs. 6f and 7e and Supplementary Figs. 6e and 7d). Overall, the assays initially\nperformed on cell lines stably expressing the exogenous WT or mutants are fully confirmed in the\nisogenic cell lines edited in the endogenous locus.\n2. Along this line, correlative studies should be performed to correlate the mechanistic studies with\nthe phenotypes observed using the genetic mouse models. A comparison between tumors developed\nfrom those generated using N vs. C -tails mutants would have provided substantial insights into\nbiology of MEF2B in the context of lymphomagenesis.\nRESPONSE: Although a direct comparison of the tumor cohorts generated from the N- and C-\nterminus mutant was not performed, mostly for economic reasons related to the re-establishment of a\nMef2bD83V tumor cohort, we have characterized the tumors using the same approaches (clonal\nrearrangements, immunophenotypic characterization by FACS and immunohistochemistry) and no\nsignificant differences were detected in the two cohorts regarding the type of tumors triggered by the\ndistinct mutations. We have included this information in the revised Results section (page 16, line\n366-367).\n3. Although examples in Figure 4c are convincing, Figure 4b is not very informative. An overlap profile\nmight be more informative to support the authors’ conclusions.\nRESPONSE: In the revised Fig. 4b, we have added overlap/heat map plots to show the co-location\nof MEF2B and SMARCA4 peaks. However, we kept also the original distribution plots, because the\nheat maps/overlap plots are not a suitable alternative for analyzing the secondary distal peak regions\nas this would require centering the regions ~100 kb from the reference peak midpoints and a plotting\nrange of +/- 1 Mb.\n4. The data as presented in Extended Data Fig. 4 is counterintuitive because the interaction in\nnormal GC B cells appears to be more robust compared with the P325L mutant. This does not\nsupport an oncogenic role of MEF2B mutant based on the model proposed.\nRESPONSE: We do not agree with the data interpretation. The intensity signal cannot be compared\nacross different cell types and experiments. The higher values detected in the GC B cells compared\nto SUDHL10 cells do not reflect a stronger interaction in normal versus tumor cells.\n5. SWI/SNF mutations are prevalent in both DLBCL and FL. Based on the model proposed, one\nwould predict a mutual exclusivity between SWI/SNF mutations and those that affect S324\nphosphorylation. This should be examined.\nRESPONSE: We have tested the co-occurrence between mutations targeting SMARCA4 and MEF2B\nphosphorylation in a cohort including 1,483 DLBCL/FL patients of which 51 and 20 had mutations in\nSMARCA4 or MEF2B, respectively. Although the numbers are too small to reach statistical\nsignificance, no co-occurrence was observed. This information is now reported in the Discussion\n(page 19, lines 434-437).\n6. To better justify the combination used to generate the mouse model, the authors may consider\nexamine the co-occurrence between MEF2B mutants that affect S324 phosphorylation and BCl2 re-\narrangement.\nRESPONSE: We confirmed the significant co-occurrence of BCL2 translocations and MEF2B\nmutations affecting phosphorylation, in a cohort of 1,268 DLBCL/FL patients. This information is now\nreported in the Results (page 15, lines 348-349) and Discussion (page 19, lines 434-437)\nsections."}, {"round": 2, "reviewer_comments": {"reviewer_2": "1. Yu et al., now show quantifications for supplementary figure 4 (only N=2), supplementary\nfigure 6 (N=3) and supplementary figure 7 (only N=2). We asked them for at least triplicates\nto conduct statistical analysis. For the N=3 testing in supplementary figure 6: why are\nstatistical tests missing? Was the result not significant? Additional quantifications especially\nfor the in vivo ubiquitination assays are necessary to prove their point of increased\nubiquitylation in figure 6d and 7c.\n2. Using a fractionated immunoblot, they show that both SMARCA4 and MEF2B are located\nin the nucleus and not in the cytosol, and using a proximity dependent biotinylating assay,\nthey confirm the interaction between SMARCA4 and MEF2B in supplementary figure 4a and\n5a as well as supplementary figure 4b and 5b. The same is true for the localization and\ninteraction of MEF2B and KLHL12. While the requested immunofluorescence imaging\nwould have further confirmed the interaction in the nucleus, the conducted experiments\nappear sufficient.\n3. The authors showed the involvement of K48 ubiquitin chains by performing an in vivo\nubiquitylation assay and blotting for total ubiquitin as well as for K48 ubiquitin in figure 6d\nand 7c, which supports their claim that it is a degradation phenotype. It seems like they only\nconducted this experiment once which is not sufficient. Triplicates with statistical\nquantifications are missing again. Including a blot input level of ubiquitin to show that the\nobserved increase of ubiquitin in the IP is not due to different ubiquitin input levels would\nalso help confirming their point. In addition to prove that this is mostly K48 ubiquitin chain\ntypes, blotting for other chain types such as K63 would make sure that this is a specific\nobservation and not background or an K48 antibody issue. We also suggested to conduct a\nTUBE assay in SUDHL10 or OCI LY7 cells, but in the revised manuscript the authors only\nshow increase ubiquitylation in their in vivo ubiquitin assays in HEK293T cells.\n4. Treating cells for 24 hours with CHX and MG132 seems very long and most likely all cells\nwill be dead by the end of the experiment. What is the rationale behind this long timepoint.\nAs we suggested, they included MG-132 and showed the rescue of MEF2B after treatment.\nThey also included MYC as a control, but there was no rescue of MYC with the addition of\nMG-132. Using one of our suggested controls (PLK1 or MCL-1) and showing a rescue of\nthese with MG-132 treatment is necessary to validate the CHX experiment. Also, why is\nthere no rescue of MEF2B after 24 hours of MG-132 but after 12 hours?\nMy other concerns have been resolved.", "reviewer_3": "This reviewer thanks the authors for addressing the concerns raised in the initial review. I\nhave a couple remaining points for the authors to consider:\n1) For point #2: While the type of tumors triggered by the distinct mutations is the same, if at\nall possible, the differential mechanisms between N vs. C-tails mutants should be directly\ncompared. This is the key taking home message of the paper.\n2) For point #5: Please show the data that support the discussion on page 19, lines 434-437."}, "author_response": "RESPONSE: Now all quantifications include data from at least 3 independent experiments and\nstatistical analyses (Revised Supplementary Figures 4c,e, 6a, 8a).\nAdditional quantifications especially for the in vivo ubiquitination assays are necessary to prove\ntheir point of increased ubiquitylation in figure 6d and 7c.\nRESPONSE: Ubiquitin assays have been performed at least 3 times independently, with\nconsistent results. We now show the quantitation for each experiment in the new\nSupplementary Figure 8c. It should be noted that although the quantification of the ubiquitin\nsignal (a broad smear ranging all possible sizes) cannot provide accurate estimates, MEF2B-\nisoB and all tested mutants displayed a significant (p<0.05) reduction in the overall and\nproteasome-associated K48-linked ubiquitination.\nsuch as K63 would make sure that this is a specific observation and not background or an K48\nantibody issue.\nRESPONSE: As suggested, we have performed immunoblotting including antibodies detecting\nK63-linked ubiquitination. Although the input signal is clearly detected, no signal was observed\nupon MEF2B immunoprecipitation; therefore, MEF2B does not show evidence of K63-linked\nubiquitination (new Supplementary Figure 6e).\nWe also suggested to conduct a TUBE assay in SUDHL10 or OCI LY7 cells, but in the revised\nmanuscript the authors only show increase ubiquitylation in their in vivo ubiquitin assays in\nHEK293T cells.\nRESPONSE: The reviewer had originally asked for showing “the ubiquitination in a DLBCL cell\nline like SUDHL10” and indeed we had already provided in our first revision the results of\nMEF2B ubiquitination in SUDHL10 cells expressing MEF2B-WT (Supplementary Fig. 6c).\n4. Treating cells for 24 hours with CHX and MG132 seems very long and most likely all cells\nwill be dead by the end of the experiment. What is the rationale behind this long timepoint.\nRESPONSE: The length of the time course (up to 24 hours) was due to the fact that some\nlymphoma-associated MEF2B mutants are extremely stable and detectable even after 24h of\nCHX treatment (i.e. see frame-shift mutants in Supplementary Figure 8d).\nAs we suggested, they included MG-132 and showed the rescue of MEF2B after treatment.\nThey also included MYC as a control, but there was no rescue of MYC with the addition of MG-\n132. Using one of our suggested controls (PLK1 or MCL-1) and showing a rescue of these with\nMG-132 treatment is necessary to validate the CHX experiment. Also, why is there no rescue of\nMEF2B after 24 hours of MG-132 but after 12 hours?\nRESPONSE: MYC protein displays a short half-life (about 30 minutes, similar to the suggested\ncontrol MCL1), therefore already 2hr after CHX treatment, no protein can be detected. We now\nshow that, as expected, MG132 allows to rescue MYC expression up to 2hr after CHX\ntreatment, while for the more stable MEF2B protein the rescue is still visible at 12hr (new\nSupplementary Figure 7a).\nThis reviewer thanks the authors for addressing the concerns raised in the initial review. I have\na couple remaining points for the authors to consider:\n1) For point #2: While the type of tumors triggered by the distinct mutations is the same, if at all\npossible, the differential mechanisms between N vs. C-tails mutants should be directly\ncompared. This is the key taking home message of the paper.\nRESPONSE: In the revised Discussion (page 17-18, lines 398-412), we have expanded the\nsection discussing the differences between N- and C-terminus mutants.\n2) For point #5: Please show the data that support the discussion on page 19, lines 434-437.\nRESPONSE: In the new Supplementary Table 7, we now report the source data relative to\nMEF2B C-terminal mutations in relationship to SMARCA4 mutations and BCL2 translocations in\nlymphoma patients.\n3"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have now adequately all my remaining points and I recommend publication.", "reviewer_3": "The comment on the direct comparison was addressed by discussion (instead of experimental data). This reviewer supports the acceptance of the manuscript for publication."}, "author_response": "RESPONSE: No action required."}]

Decision letter