Investigating The Role Of MLL2 (Mll4) In B Cell Development

The myeloid/lymphoid or mixed-lineage leukaemia 2 (MLL2) histone methyltransferase (referred to as Mll4 in mice) forms part of a large multiprotein complex, which catalyses the methylation of lysine 4 on histone H3 (H3K4). High levels of this histone modification are detected at promoter regions of actively transcribed genes. Loss-of-function mutations in MLL2 have been identified in 80-90% of follicular lymphoma (FL) cases. These mutations are distributed throughout the coding region and lead to the loss of the C-terminal catalytic SET domain and reduction in H3K4 methylation. We generated a Mll4 knockout (Mll4^-/-) mouse model to elucidate the effects of Mll4 loss on B cell development and understand how mutations in this gene contribute to FL pathogenesis.

Cre:ER^T2-mediated recombination was used to induce the deletion of exons 2–4 of Mll4 in adult mice. CD19⁺ B lymphocytes were purified from the spleens of wild-type (Mll4^WT) and Mll4^-/- mice and confirmed the loss of Mll4 mRNA and protein by quantitative RT-PCR (qRT-PCR) and immunoblotting, respectively. As expected, B cells lacking Mll4 (Mll4^ΔB/ΔB) exhibited a global reduction in H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3), compared with normal B cells. Gene expression profiling (GEP) using the GeneChip^® Mouse Genome 430 2.0 Array (n=5 Mll4^WT + 5 Mll4^-/- mice) was carried out to determine the transcriptional changes upon loss of Mll4. We identified >200 genes differentially expressed (>2-fold) between Mll4^WT and Mll4^ΔB/ΔB CD19⁺ B cells. The top 40 candidate genes (p<0.05) were verified in an independent series of experiments using qRT-PCR. We noted a significant decrease in the expression of the transcription factor lymphoid enhancer-binding factor 1 (Lef1) and the histone acetyltransferase nuclear receptor coactivator 3 (Ncoa3). The downregulation of these genes is consistent with published data, as the promoters of both are marked by H3K4me3 in normal mouse CD19⁺ B cells. Lef1 is a key regulator of lymphoid differentiation, while Ncoa3 depletion has been shown to induce B-cell lymphoma in mice as a result of constitutive NF-κB activation. A significant number of genes (p<0.001) involved in cell cycle and immune response were upregulated in Mll4^ΔB/ΔB CD19⁺ B cells. Furthermore, loss of Mll4 led to an elevated expression (2.4-fold) of activation-induced cytidine deaminase (Aicda), an enzyme required for germinal centre-derived lymphomagenesis. Immunophenotyping was used to examine the role of Mll4 at different stages of B cell development. We detected a significant reduction in the number of pre-B cells (B220⁺CD43^–IgM^–) in the bone marrow of Mll4^-/- mice (n=12), compared with their littermate controls (n=10; p<0.01), although this did not affect the number of peripheral mature splenic B cells (B220⁺IgM⁺). Mll4 loss had an adverse effect on immune response, with CD19⁺ B cells failing to induce expression of the MHC-II, CD86, CD40 and CD69 activation markers upon in vitro stimulation with lipopolysaccharide and interleukin 4.

Our findings provide the first insight into the potential mechanistic link between MLL2 loss and the onset of FL using a mouse model. Mll4^-/- mice do not develop any lymphoproliferative disorders, but show defects in B cell development and an impaired immune response. Furthermore, loss of Mll4 leads to the global depletion of H3K4 methylation in mouse B lymphocytes, thus affecting the expression of Lef1, Ncoa3 and Aicda. Although these MLL2/Mll4 target genes have defined roles in B cell biology, their contribution to the pathogenesis of FL will depend on when MLL2 mutations arise during FL development.

No relevant conflicts of interest to declare.