Mef2C Maintains B Cell Homeostasis Through the Regulation of DNA Repair Machinery

Abstract 278

B lymphocytes are central mediators of humoral immunity as they are able to bind specifically to an antigen through B cell receptor (BCR) and generate antibodies against the antigen. During aging, specific reduction of bone marrow pre-B cells and defects in BCR rearrangement result in declined B lymphopoiesis and contribute to immunosenescence.

One novel regulator of B cell homeostasis is Mef2C, a MADS-box transcription factor that activates target genes by binding to the MEF2 consensus binding site. Using VavCre⁺ Mef2C^fl/fl mice that ablate Mef2C in hematopoietic cells, we observed a specific reduction in the bone marrow pre-B cell already in young adults – a phenotype that resembles B cell aging. Loss of Mef2C resulted in higher percentage of late apoptotic and necrotic (AnnexinV+, 7AAD+) pre-B cells, suggesting a requirement for Mef2C in pre-B cell survival.

To investigate the mechanism that Mef2C uses to regulate B lymphoid homeostasis, we performed gene expression study. Gene Ontology analysis of Mef2C dependent genes in pre-B cells showed a significant down-regulation of DNA repair and cell cycle genes. Furthermore, by searching for the MEF2 consensus binding site, we identified critical regulators of cell cycle (Ccnb1&2,E2F2, E2F6) and DNA damage response (Ercc8, H2afx, Mre11a, Rad51ap1, Rad54l, Rpa1) as putative Mef2C direct targets in pre-B cells. To functionally assess the requirement for Mef2C in DNA damage response, we performed radio-sensitivity analysis. Upon 6Gy irradiation, Mef2C deficient mice showed a dramatic ablation of peripheral blood B cells as compared to controls while their T and myeloid cells remained largely unaffected. These data suggest that Mef2C regulates DNA repair machinery to protect B lymphocytes against irradiation induced stress.

Pre-B cell stage is a vulnerable step of B lymphopoiesis as successful BCR rearrangement is required for the pre-B cell receptor (pre-BCR) to support pre-B cell survival and expansion. DNA repair is a critical step during V(D)J recombination. Interestingly, in absence of Mef2C, V(D)J initiators (Rag1&2) and critical DNA repair effectors (Ku70, Xrcc4 & Lig4) failed to be upregulated during pro-B to pre-B transition as they did in controls. Furthermore, loss of Mef2C in both pro- and pre-B cells resulted in decreased expression of surrogate light chain genes (VpreB1&2) and recombined IgH μ chain.

These findings define Mef2C as a lineage specific DNA repair regulator that links the classical DNA repair machinery to B cell factors and guards B lymphoid homeostasis at an inherently precarious developmental stage.