Mechanisms of MEF2C-Dependent Transcription in Lymphoid Differentiation

Hematopoiesis is a multi-step developmental process that requires an intricate coordination of signal relays and transcriptional regulation to give rise to all blood lineages in the organism. Hematopoietic stem/progenitor cells (HSPCs) can be driven to differentiate along three main lineages: myeloid, erythroid, and lymphoid. One of the earliest lineage decisions for HSPCs is whether to adopt the lymphoid or myeloid fate. Despite the discovery of several transcription factors required for different lineages of hematopoietic differentiation, the understanding of how gene expression allows HSPCs to adopt the lymphoid fate still remains incomplete. A study using an inducible hematopoietic-specific (Mx1-Cre) KO mouse line found that Myocyte Enhancer Factor 2C (MEF2C) is required for multi-potent HSPCs to differentiate into the lymphoid lineage (Stehling-Sun et al, 2009). However, the mechanisms of how MEF2C is activated and in turn, drives lymphoid fate specification are not known.

Through a candidates approach with co-expression and co-immunoprecipitation, we have identified Early B Cell Factor 1 (EBF1) to be a specific interacting partner of MEF2C, and not other B cell specific transcription factors such as E12, E47, or PAX5. Genome-wide survey of MEF2C and EBF1 binding sites via chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in a proB cell line revealed that these two sequence-specific transcription factors co-occupy the promoters and intragenic regions of many B cell specific genes such as Il7ra, Myb, Foxo1, Ets1, Ebf1 itself, and Pou2af1. Regulatory regions of Il7ra and Foxo1 derived from the ChIP-seq data, as well as an artificial enhancer containing trimerized MEF2C and EBF1 binding sites, were examined in luciferase reporter assays and found to be sufficient to drive transcription from a minimal reporter in 293T cells. Further, this activation was co-dependent on MEF2C and EBF1 expression. The functional relevance of MEF2C binding was further supported by gene expression analyses of MEF2C-regulated B lineage genes in Mx1-Cre Mef2c KO mice compared to WT mice. Consistent with ChIP-seq and luciferase reporter assays, Myb, Ebf1, Il7ra, and Foxo1 all had significantly decreased expression levels in MEF2C-null HSPCs as well as B lineage progenitor cells, compared to sex-matched littermate control mice.

Interestingly, myeloid gene expression in Mef2c-KO mice was increased compared to WT control. MEF2C ChIP-seq in a murine HSPC line revealed that it binds myeloid lineage gene targets that are not regulated by MEF2C in proB cells. These results suggest that MEF2C can repress myeloid gene expression in HSPCs. To elucidate the mechanism of this functional switch, we tested the requirement for MAPK pathways to phosphorylate and activate MEF2C at three previously identified residues in order to drive B cell differentiation. Inhibition of p38 MAPK (p38i), but not ERK1/2/5, decreased the potential of HSPCs to differentiate into B220+CD19+ B cells cultured with cytokines that drive this particular lineage fate. Instead, p38i-treated progenitor cells gave rise to more myeloid cells. 65% of this phenotype was rescued by over-expressing a phosphomimetic mutant of MEF2C that can bypass p38 inhibition. Furthermore, MEF2C is known to bind class II HDAC proteins to repress gene expression, providing a possible mechanism for its repression of myeloid transcription program. Supporting this mechanism, phosphomimetic and HDAC-binding double mutant of MEF2C can rescue p38 inhibition phenotype almost 100%.

Taken together, this study elucidated the molecular mechanisms of a key lymphoid-specific lineage fate determinant, MEF2C. We discovered that p38 MAPK converts MEF2C from a transcriptional repressor to an activator by phosphorylation in B cell specification, which can be applied to understanding other cell differentiation processes regulated by this important stress-induced signaling pathway. Furthermore, we identified MEF2C’s binding and co-activating partner EBF1, several novel B cell specific targets that it activates in proB cells, and a novel myeloid transcription program that it represses in hematopoietic progenitors. Therefore, these results expanded our understanding of the intricate transcription network that regulates B cell differentiation.