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EBF1 Drives Hallmark B Cell Gene Expression by Enabling the Interaction of PAX5 with the MLL H3K4 Methyltransferase Complex Charles E

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EBF1 Drives Hallmark B Cell Gene Expression by Enabling the Interaction of PAX5 with the MLL H3K4 Methyltransferase Complex Charles E www.nature.com/scientificreports OPEN EBF1 drives hallmark B cell gene expression by enabling the interaction of PAX5 with the MLL H3K4 methyltransferase complex Charles E. Bullerwell1, Philippe Pierre Robichaud1,2, Pierre M. L. Deprez1, Andrew P. Joy1, Gabriel Wajnberg1, Darwin D’Souza1,3, Simi Chacko1, Sébastien Fournier1, Nicolas Crapoulet1, David A. Barnett1,2, Stephen M. Lewis1,2 & Rodney J. Ouellette1,2* PAX5 and EBF1 work synergistically to regulate genes that are involved in B lymphocyte diferentiation. We used the KIS-1 difuse large B cell lymphoma cell line, which is reported to have elevated levels of PAX5 expression, to investigate the mechanism of EBF1- and PAX5-regulated gene expression. We demonstrate the lack of expression of hallmark B cell genes, including CD19, CD79b, and EBF1, in the KIS-1 cell line. Upon restoration of EBF1 expression we observed activation of CD19, CD79b and other genes with critical roles in B cell diferentiation. Mass spectrometry analyses of proteins co-immunoprecipitated with PAX5 in KIS-1 identifed components of the MLL H3K4 methylation complex, which drives histone modifcations associated with transcription activation. Immunoblotting showed a stronger association of this complex with PAX5 in the presence of EBF1. Silencing of KMT2A, the catalytic component of MLL, repressed the ability of exogenous EBF1 to activate transcription of both CD19 and CD79b in KIS-1 cells. We also fnd association of PAX5 with the MLL complex and decreased CD19 expression following silencing of KMT2A in other human B cell lines. These data support an important role for the MLL complex in PAX5-mediated transcription regulation. PAX5 is an essential transcription factor in B lymphocyte diferentiation that plays a role in the activation of B cell hallmark genes such as CD19, which encodes a transmembrane protein involved in B cell signaling and antigen response1. PAX5 is frst expressed at the pro-B cell stage and its expression is maintained through subsequent B cell stages until it is downregulated during the transition into plasma cells 2. PAX5 controls the switch from activated B cells into plasmablasts in part by repressing the expression of the transcription factors PRDM1 and XBP13. PAX5 also serves to repress diferentiation to other hematopoietic cell types4; for example, it represses NOTCH1 expression and thereby impairs T cell development 5. PAX5 contributes to the transcriptional activation of B-cell-hallmark genes such as CD19 and CD79b by interacting with other proteins. Tese PAX5 interacting proteins include components of the basal transcriptional apparatus such as RNA polymerase II, the TATA bind- ing protein (TBP) and TBP-associated factors (TAFs)6, as well as proteins involved in chromatin remodeling and histone modifcation7. Te KIS-1 cell line originated from a patient with Ki-1-positive (Indicating the presence of TNFRSF8, also known as CD30) difuse large B cell lymphoma (DLBCL)8. It was described as a DLBCL based on positive stain- ing for HLA-DR and CD45 and negative staining for CD20 and antigens specifc to other cell types. Class switch recombination of the JH locus (encoding a segment of the immunoglobulin heavy chain, IgH) and expression of lambda light chain suggest that the KIS-1 cell line originated from an activated B lymphocyte undergoing plasma cell diferentiation. Te KIS-1 cell line has a t(9;14)(p13;q32) translocation that brings the PAX5 coding 1Atlantic Cancer Research Institute, Pavillon Hôtel-Dieu, 35 Providence Street, Moncton, NB E1C 8X3, Canada. 2Department of Chemistry and Biochemistry, Université de Moncton, Moncton, New Brunswick, Canada. 3Present address: The Centre for Applied Genomics, Toronto, ON, Canada. *email: RodneyO@ canceratl.ca Scientifc Reports | (2021) 11:1537 | https://doi.org/10.1038/s41598-021-81000-5 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Expression of PAX5 and other B cell hallmarks in KIS-1. a Western blotting to show the expression of PAX5, CD19 and CD79b in KIS-1 whole cell extracts in comparison to three B cell lines (GM12878, RAJI and NALM-6) and a non-B lymphocyte line (K562). GAPDH is included to demonstrate equal loading. b Expression of CD19, CD20 and CD45 in KIS-1 versus GM12878 cells shown by fow cytometry. Antibody-labelled cells are indicated in purple, unlabeled cells are indicated in green. CD45 is a commonly expressed leukocyte antigen and is included as a positive control. region and its promoter into the vicinity of the strong Eµ enhancer of the IgH gene9–11, which is highly active in immunoglobulin-secreting plasma cells. Consistent with this, KIS-1 DLBCL cells have very strong expression of PAX511–13 at a time in B cell diferentiation when PAX5 is usually switched of. Nevertheless, despite high PAX5 expression, Hamada et al.12 demonstrated an absence of CD19 mRNA in KIS-1 cells, suggesting that PAX5 is not sufcient to drive expression of this hallmark B cell gene. We reasoned that restoration of expression of a missing protein partner of PAX5 might restore CD19 expression in KIS-1 DLBCL cells. We here report an expanded characterization of gene expression in KIS-1 cells, confrm the lack of CD19 expression and demonstrate reduced expression of other B cell hallmark genes including CD79b and EBF1. Exog- enous expression of the transcription factor EBF1, a transcription factor required for the expression of certain PAX5-regulated genes14, is sufcient to restore expression of CD19, CD79b and other B cell-specifc genes to KIS-1 DLBCL cells. We further demonstrate that this transcriptional activation is mediated in part by increased association of PAX5 with the MLL (mixed-lineage leukemia) H3K4 methyltransferase complex, including the catalytic component KMT2A, in the presence of EBF1. Our results also support a role for the MLL complex, in association with PAX5 and EBF1, for B cell-specifc transcription regulation in other human B cell lines. Results KIS-1 cells lack hallmark B cell gene expression. Te KIS-1 DLBCL cell line was previously reported to have high expression of PAX5 mRNA and PAX5 protein11–13 and undetectable expression of CD19 mRNA12. We used Western blot analyses to compare the protein expression level of PAX5, CD19 and CD79b (also PAX5- regulated), in KIS-1 cells in addition to several other B cell lines (Fig. 1a). PAX5, CD19 and CD79b are all absent in K562 (a non-B Chronic Myelogenous Leukemia cell line). PAX5 is expressed more strongly in KIS-1 cells than in the other B cell lines investigated. By contrast, CD19 and CD79b are absent in KIS-1 cells but are expressed in GM12878 (an Epstein-Barr Virus-transformed B lymphocyte), RAJI (Burkitt lymphoma) and Nalm-6 (Acute Lymphoblastic Leukemia) cells. We further demonstrate the lack of CD19 and CD20 (another B-lymphocyte- specifc cell surface protein) expression in KIS-1 cells by fow cytometry (Fig. 1b). Tese results confrm and extend previous fndings and characterize KIS-1 as having lost B cell specifc gene expression despite strong expression of PAX5. To further characterize gene expression in KIS-1 DLBCL cells we used next-generation sequencing, specif- cally RNA-seq, to compare this cell line to the RAJI cell line, which has much lower PAX5 expression yet strong expression of both CD19 and CD79b (Fig. 1a). 2913 genes are diferentially expressed (with a log 2 fold change of ≥ 2.5 (5.7x) and False Discovery Rate (FDR) value of < 0.05) between the two lines: 1557 genes are downregu- lated and 1356 genes are upregulated in KIS-1 cells relative to RAJI cells (Supplementary Table 1). Results of genes most diferentially expressed in each cell line is shown in Table 1. PAX5 is expressed 2.4 × more strongly in KIS-1, consistent with the Western blot results (Fig. 1a). Te top ten genes down-regulated in KIS-1 cells according to this analysis include heavy- and light-chain immunoglobulin genes and CD79b. CD19 and CD20 are also strongly and signifcantly down-regulated (fold changes of 2815 × and 22x, respectively) in KIS-1 cells, as are SPi1 and EBF1 (1015 × and 1932x, respectively). SPi1 and EBF1 are transcription factors essential for B cell diferentiation whose expression is normally terminated during the plasmablast transition. Figure 2 summarizes the diferentially-expressed genes between these two cell lines. 1317 of the 1557 down-regulated genes in KIS-1 were mapped using DAVID 2.015 to fve KEGG pathways with a fold enrichment of at least 2.5: Primary immu- nodefciency (hsa05340), Osteoclast diferentiation (hsa04380), B cell receptor signaling pathway (hsa04662), Arginine and proline metabolism (hsa00330), and NF-κB signaling pathway (hsa04064). Te B cell receptor signalling pathway is represented by 10 genes: CD19, CD79b, SYK, CD72, BTK, NFATC1, VAV1, FCGR2B, FOS/ AP-1 and BCAP. Tese data clearly demonstrate that KIS-1 DLBCL cells lack gene expression characteristic of PAX5-expressing B cells like RAJI, despite having an elevated level of PAX5 expression. Scientifc Reports | (2021) 11:1537 | https://doi.org/10.1038/s41598-021-81000-5 2 Vol:.(1234567890) www.nature.com/scientificreports/ 2 KIS1 mean RAJI mean log Fold Change p value padj IGHM 0 166,655.1998 − 19.7346 3.78E−37 1.94E−34 IGHV3-21 0 19,103.9253 –16.6097 1.04E−26 1.51E−24 ST14 0 10,358.3341 –15.7266 2.96E−24 3.43E−22 CD79B 0.5649 23,122.6324 –15.4408 1.20E−24 1.44E−22 TCL1A 0 6160.0247 −14.9768 3.72E−22 3.60E−20 IGHV1-69 0 3511.1722 −14.1658 4.98E−20 3.80E−18 IDH2 0 3159.8325 −14.0138 1.48E−19 1.06E−17 SERPINA9 0.5649 8432.8389 − 13.9856 1.68E−20 1.35E−18 IGKV3-20 0 3035.7311 − 13.9559 1.76E−19 1.25E−17 IGHG1 0 2997.9458 − 13.9379 1.40E−19 1.02E−17 SEMA4C 1468.6114 0 13.0477 2.95E−17 1.61E−15 ALDH1A1 4257.3685 0.5 13.1376 2.70E−17 1.48E−15 ATP9A 30,003.313 3.2228 13.1626 1.62E−67 1.25E−63 BLVRA 1651.8437 0 13.2173 1.02E−17 6.08E−16 ARNTL2 1911.8631 0 13.428 1.31E−17 7.69E−16 MSC 2551.8359 0 13.8449 1.37E−18 8.79E−17 TMEM173 2734.9963 0 13.9447 1.49E−19 1.07E−17 EOMES 3040.8551 0 14.0977 7.49E−20 5.60E−18 LHX2 3201.0322 0 14.1717 3.33E−20 2.58E−18 ANKRD30A 5323.245 0 14.9054 1.01E−20 8.40E−19 Table 1.
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