MEF2C Protects Bone Marrow B Lymphopoiesis during Stress Hematopoiesis

Author(s): Wang, Wenyuan | Advisor(s): Mikkola, Hanna K.A. | Abstract: The integrity of the immune system is critical for health as it serves as the guardian to protect an organism from foreign pathogens. Because most mature immune cells have a short lifespan, the lifelong replenishment of the immune system depends on the hematopoietic stem cell (HSC). In order to adapt to various stress situations that occur throughout life, the immune system needs to have mechanisms to protect its integrity and ensure rapid regeneration during such stress situations.One key protective mechanism that is required to protect the immune system from various stress factors is the DNA repair system. Such mechanisms are of particular importance in the development of B lymphocytes, which are central mediators of humoral immunity. During development, the B cell progenitors in the bone marrow (BM) go through a series of rearrangements of immunoglobulin genes to generate diverse B cell receptors that can recognize foreign antigens. The nature of this rearrangement process is the generation and repair of DNA double strand breaks (DSBs), and failure of this process causes cell death of BM B lymphoid progenitors, which makes the BM B lymphoid compartment inherently vulnerable. My thesis research focused on the identification of novel mechanisms that protect B lymphopoiesis, especially upon stress situations. Through my research, I identified transcription factor MEF2C as a guardian of BM B lymphoid progenitors and showed that MEF2C is critical for maintaining the integrity of the immune system through regulation of DNA repair and V(D)J recombination during both homeostasis and stress hematopoiesis. Hematopoietic deletion of Mef2c in mice reduced the survival and cellularity of BM B cell progenitors downstream of common lymphoid progenitor (CLP), while peripheral B cells remained unaltered in homeostatic conditions. Intriguingly, this phenotype was reminiscent of the B lymphoid defects observed during aging. Loss of Mef2c severely compromised the recovery of BM and peripheral B cells after sub-lethal irradiation and 5-FU induced BM ablation, while the recovery of T lymphoid and myeloid cells was unaffected. These data imply that MEF2C protects B cell progenitor survival, especially upon proliferative stress. Microarray of Mef2c deficient B cell progenitors showed down-regulation of DNA repair genes, including sensors of DSB and effectors in homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways. Comet assay revealed excessive DNA damage specifically in B cell progenitors in Mef2c deficient BM, while CLPs, mature B cells, T cell progenitors or myeloid cells were unaffected. γH2AX staining showed increased DSBs in Mef2c deficient pre-B cells. These data show that MEF2C regulates DNA repair specifically in B cell progenitors. NHEJ repair is also required for V(D)J recombination in BM B cell progenitors. Loss of Mef2c impaired the proper induction of Rag initiators and key NHEJ factors during B cell progenitor transition, and reduced the recombination efficiency of both heavy and light chains, uncovering a novel function for MEF2C in V(D)J recombination. ChIP-Seq in human B lymphoblasts showed that MEF2C directly binds to genes encoding critical factors of DSB repair and V(D)J machinery. MEF2C binding strongly correlated with the binding of co-activator and enhancer epigenetic marks, suggesting that MEF2C functions by boosting gene activation through these enhancers. These data define MEF2C as a lineage specific regulator of DNA repair machinery that protects B lymphoid progenitor homeostasis.In the HSC compartment, deletion of Mef2c resulted in a reduction of HSCs. Microarray analysis revealed down-regulation of critical HR DSB repair and nucleotide excision repair (NER) factors as well as cell cycle regulators in Mef2c deficient HSCs. During stress hematopoiesis induced by irradiation, higher cell death was observed in Mef2c deficient HSCs. These preliminary data suggested a potential function of MEF2C also in regulating DNA repair and protecting the integrity of the HSC pool. Future studies will be needed to explore the specific cellular and molecular mechanism that MEF2C utilizes in protecting the HSC compartment.