The MEF2B Regulatory Network - H3K27ac and H3K4me3 ChIP-seq data. Homo sapiens

Myocyte enhancer factor 2B (MEF2B) is a transcription factor with somatic mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The recurrence of these mutations indicates that they may drive lymphoma development. However, inferring the mechanisms by which they may drive lymphoma development was complicated by our limited understanding of MEF2B’s normal functions. To expand our understanding of the cellular activities of wildtype (WT) and mutant MEF2B, I developed and addressed two hypotheses: (1) identifying genes regulated by WT MEF2B will allow identification of cellular phenotypes affected by MEF2B activity and (2) contrasting the DNA binding sites, effects on gene expression and effects on cellular phenotypes of mutant and WT MEF2B will help refine hypotheses about how MEF2B mutations may contribute to lymphoma development. To address these hypotheses, I first identified genome-wide WT MEF2B binding sites and transcriptome-wide gene expression changes mediated by WT MEF2B. Using these data I identified and validated novel MEF2B target genes. I found that target genes of MEF2B included the cancer genes MYC, TGFB1, CARD11, NDRG1, RHOB, BCL2 and JUN. Identification of target genes led to findings that WT MEF2B promotes expression of mesenchymal markers, promotes HEK293A cell migration, and inhibits DLBCL cell chemotaxis. I then investigated how K4E, Y69H and D83V mutations change MEF2B’s activity. I found that K4E, Y69H and D83V mutations decreased MEF2B DNA binding and decreased MEF2B’s capacity to promote gene expression in both HEK293A and DLBCL cells. These mutations also reduced MEF2B’s capacity to alter HEK293A and DLBCL cell movement. From these data, I hypothesize that MEF2B mutations may promote DLBCL and FL development by reducing expression of MEF2B target genes that would otherwise function to help confine germinal centre B-cells to germinal centres. Overall, my research demonstrates how observations from genome-scale data can be used to identify cellular effects of candidate driver mutations. Moreover, my work provides a unique resource for exploring the role of MEF2B in cell biology: I map for the first time the MEF2B ‘regulome’, demonstrating connections between a relatively understudied transcription factor and genes significant to oncogenesis. Overall design: ChIP-seq was performed using H3K27ac and H3K4me3 antibodies on cells expressing V5 tagged WT MEF2B and on empty vector control cells. Two biological replicates were performed.

肌细胞增强因子2B（Myocyte enhancer factor 2B, MEF2B）是一类转录因子，在弥漫性大B细胞淋巴瘤（diffuse large B-cell lymphoma, DLBCL）与滤泡性淋巴瘤（follicular lymphoma, FL）中存在K4、Y69及D83位点的体细胞突变热点。此类突变的反复出现提示其可能驱动淋巴瘤发生发展。然而，由于当前对MEF2B的正常生理功能认知有限，解析其突变驱动淋巴瘤发生的分子机制颇具挑战。

为深化对野生型（wildtype, WT）与突变型MEF2B细胞生物学活性的认知，本研究提出并检验了两项假说：（1）鉴定野生型MEF2B调控的靶基因，可明确受MEF2B活性影响的细胞表型；（2）对比突变型与野生型MEF2B的DNA结合位点、基因表达调控效应及细胞表型差异，有助于细化MEF2B突变促淋巴瘤发生的相关假说。

为验证上述假说，本研究首先鉴定了全基因组范围内野生型MEF2B的结合位点，以及野生型MEF2B介导的全转录组基因表达变化。依托上述数据，本研究鉴定并验证了全新的MEF2B靶基因。研究发现，MEF2B的靶基因涵盖MYC、TGFB1、CARD11、NDRG1、RHOB、BCL2及JUN等癌基因。通过对靶基因的分析，本研究发现野生型MEF2B可促进间充质标志物的表达，增强HEK293A细胞的迁移能力，并抑制弥漫性大B细胞淋巴瘤细胞的趋化运动。

随后，本研究探究了K4E、Y69H及D83V突变对MEF2B活性的影响。结果显示，上述三类突变均会降低MEF2B的DNA结合能力，同时削弱其在HEK293A与弥漫性大B细胞淋巴瘤细胞中促进基因表达的能力。此类突变还会降低MEF2B调控HEK293A及淋巴瘤细胞运动的能力。基于上述实验数据，本研究提出假说：MEF2B突变可能通过下调原本可限制生发中心B细胞滞留于生发中心的MEF2B靶基因表达，进而促进弥漫性大B细胞淋巴瘤与滤泡性淋巴瘤的发生发展。

总体而言，本研究阐明了如何通过基因组规模数据分析，鉴定候选驱动突变的细胞生物学效应。此外，本研究为探索MEF2B在细胞生物学中的功能提供了独特的研究资源：本研究首次绘制了MEF2B的调控组（regulome），揭示了这一研究相对较少的转录因子与肿瘤发生相关基因之间的潜在关联。

整体实验设计：针对表达V5标签野生型MEF2B的细胞及空载体对照细胞，分别使用H3K27ac与H3K4me3抗体开展染色质免疫共沉淀测序（ChIP-seq）实验。实验设置2次生物学重复。