MEF2C protects bone marrow B-lymphoid progenitors during stress haematopoiesis

Introduction

Sustained B lymphopoiesis through various stress conditions is essential for maintaining a functional immune system. B lymphopoiesis occurs in bone marrow where B-lymphoid progenitors undergo V(D)J recombination to generate B-cell receptors (BCRs)1,2,3,4. The success of V(D)J recombination is critical for humoral immunity as diverse BCRs are required to recognize antigens and generate antibodies. V(D)J recombination is initiated by creating DNA double strand breaks (DSBs) by RAG recombinases at the border of recombining gene segments5,6. After rearrangement, the DSBs are repaired by non-homologous end joining (NHEJ) machinery7,8. Defective DNA repair during this process results in cell death or genetic lesions9, making B lymphopoiesis inherently vulnerable. To ensure genomic integrity, B-lymphoid progenitors tightly regulate cell survival and exclude cells with abnormal rearrangement10. This homeostatic balance is altered during physiological ageing11,12,13 due to reduced V(D)J recombination efficiency14,15 and increased B-lymphoid progenitor death16, which contributes to the impaired immune function during ageing.

The haematopoietic system encounters various stress factors that necessitate rapid proliferation of stem/progenitor cells to replenish the blood/immune system17. The regeneration of the haematopoietic system under such situations is called stress haematopoiesis and can be induced by bone marrow transplantation18, radiation and chemotherapy19, bleeding20 and infection21. In addition to investigating the effects of stress on haematopoietic stem cell maintenance, several studies have focused on stress erythropoiesis and identified multiple unique signals that regulate this process22. However, little is known how other haematopoietic lineages secure proficient progenitor proliferation and differentiation during stress.

Studies have identified myocyte enhancer factor 2C (MEF2C) as a regulator of the B-lymphoid system. MEF2C is a MADS box transcription factor originally discovered as a regulator of cardiogenesis and myogenesis23. In bone marrow, Mef2c is highly expressed by common lymphoid progenitors (CLPs) and B-lymphoid cells, whereas Mef2c expression is minimal in T cells, granulocytes and erythrocytes24. Deletion of Mef2c by B-cell-specific Cd19-Cre showed that MEF2C is required for BCR-induced proliferation of splenic B cells25,26,27; however, as the deletion of Mef2c was not complete in bone marrow B-cell progenitors, this model cannot be used to evaluate the presence of B-cell progenitor defects. Deletion of Mef2c using Mx1-Cre and PIPC treatment followed by transplantation or culture led to a severe reduction in the number of B cells, whereas myeloid cell numbers were increased, indicating a role for MEF2C in myeloid/lymphoid fate choice24. We previously showed that haematopoietic deletion of Mef2c using Vav-Cre results in a reduction of bone marrow B-cell progenitors, especially pre-B cells, without overtly affecting the peripheral B-cell pool during homeostasis28. A requirement for MEF2C within bone marrow B-lymphoid cells was also documented using B-cell-specific Mb-1-Cre. This led to a reduction of B cells in both bone marrow and spleen of neonates. Although peripheral cellularity of B cells was corrected in adult mice, bone marrow B lymphopoiesis remained compromised29. Another study showed that MEF2C acts redundantly with MEF2D, and that MEF2C/D are activated by pre-BCR signalling. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis showed that MEF2C directly binds to several pre-B-cell genes, and possibly regulates them together with other B-cell regulators such as E2A and IKAROS30. Although these studies show a requirement for MEF2C in B-lymphoid progenitors, the cellular and molecular mechanisms through which MEF2C protects bone marrow B lymphopoiesis are mostly unknown. Moreover, little is known about MEF2C function during stress. This question is particularly intriguing asMef2c-deficient mice share features of B-lymphoid ageing28, which is characterized by reduction of bone marrow B-cell progenitors, whereas peripheral B-cell cellularity can initially be maintained through compensatory mechanisms11,12,13.

Here we report that MEF2C protects bone marrow B-lymphoid progenitors by augmenting the transcription of factors crucial for DNA repair and V(D)J recombination, thereby ensuring efficient bone marrow B lymphopoiesis. Loss of Mef2c severely compromises B-lymphoid recovery after sub-lethal irradiation or 5-fluorouracil (5-FU) injection, documenting a critical function for MEF2C during regenerative stress. We find that MEF2C binds directly to regulatory regions of genes encoding DNA repair and V(D)J factors, as well as B-cell transcription factors in mouse B-cell progenitors and human B-lymphoblasts, co-localizing with epigenetic marks representing active enhancers and promoters. ATAC-sequencing (ATAC-seq) shows that MEF2C is required for proper chromatin accessibility of regulatory regions of its target genes in mouse pre-B cells. These findings uncover a central role for MEF2C as a transcriptional activator of DNA repair and B-cell regulatory machinery in B-lymphoid progenitors, and establish that MEF2C-dependent transcriptional mechanisms are required to secure efficient bone marrow B-cell production during stress haematopoiesis.

Results

MEF2C maintains the cellularity of B-lymphoid progenitors

To define the processes that MEF2C regulates in B-lymphoid cells,Mef2cfl/fl was conditionally deleted in mice using Vav-Cre28,31. Unless otherwise stated, middle-aged mice (7–11-month-old, males and females) were used for the analyses, as our prior study showed that the reduction in Mef2c-deficient B-cell progenitors was most notable at this age28. Analysis of larger groups of mice confirmed a reduction of bone marrow B cells in Mef2c-deficient mice, while B-lymphocytes in blood and spleen were not significantly affected (Supplementary Fig. 1). In addition to the reduction of pre-pro-B and pre-B cells in Mef2c-deficient mice observed previously28, analysis of a larger cohort of mice suggested that all bone marrow B-lymphoid progenitor stages (pre-pro-B, pro-B and pre-B) were significantly reduced (Fig. 1a). The reduction of all bone marrow B-cell progenitor subsets was also confirmed by immunophenotyping B-cell development as defined by Hardy et al.32(Supplementary Fig. 2a). In contrast, the cellularity of mature recirculating B cells (sIgM+ or Fr.F) in bone marrow was not affected (Fig. 1a). Moreover, Vav-Cre Mef2cfl/fl mice showed no significant difference in the percentage of CLPs, defined as Lin−c-KitloAA4.1+IL-7Rα+Flt3+ (Fig. 1b) or Lin−IL-7R+Sca-1loc-Kitlo (Supplementary Fig. 2b). These data suggested that MEF2C has a critical function in maintaining the integrity of bone marrow B-lymphoid progenitor compartment.

Figure 1: MEF2C maintains the integrity of bone marrow B-lymphoid compartment.

(a) Flow cytometric analysis of bone marrow B-lymphoid progenitor fractions in Vav-CreMef2cfl/fl mice as compared with control mice revealed reduction of all B-lymphoid progenitors in Mef2c-deficient mice while the mature B cells in the bone marrow (sIgM+) were not affected (n≥11). (b) Flow cytometric analysis suggested that loss of Mef2c does not affect the frequency of bone marrow common lymphoid progenitors (CLP) (n≥7). (c) Flow cytometric analysis of caspase 3/7 activation in bone marrow B-lymphoid progenitors documented increased cell death in Mef2c-deficient B-lymphoid progenitors while total bone marrow and sIgM+ cells were not affected (n≥7). (d) Caspase 3/7 activation was not increased in Mef2c-deficient CLP (n=8). All mice were analysed at 7–11 months of age and both male and female mice were included. Data shown are the mean±s.d. of two or more independent experiments. NS, not significant, *P<0.05, **P<0.01 and ***P<0.001, unpaired t-test.