Fig 1.
MEF2C co-immunoprecipitates with EBF1, but not other bHLH factors.
FLAG-tagged WT, phosphomimetic (EED), or phosphorylation-deficient (AAA) MEF2C were co-transfected into 293T cells with Myc-tagged EBF1 (A), E12 or E47 (B), or PU.1 (C). After FLAG-IP, the immunocomplexes were blotted with anti-FLAG or anti-Myc antibodies, as indicated. (D) FLAG-tagged WT MEF2C was co-transfected into 293T cells with Myc-tagged EBF1; FLAG-IP with or without benzonase treatment and blotted with anti-Myc antibodies; the image was cropped for clarity from the same blot. Right panel: ethidium bromide gel showing all nucleic acids were degraded following benzonase treatment.
Fig 2.
MEF2C and EBF1 co-occupy many B lineage genes.
(A) Venn diagram of MEF2C and EBF1 overlapping ChIP-seq peaks in pre-B cells. MEF2C peaks are those commonly shared in two separate ChIP experiments, as shown in S2A Fig. (B) ChIP-qPCR validation of MEF2C binding near Ebf1 and Foxo1 genes. (C, D) Representative MEF2C and EBF1 ChIP-seq profiles at Mef2c and Myb loci, with the corresponding antibody used in the ChIP indicated (g for goat, and rb for rabbit); blue arrow on the input track indicates the position of the gene; red lines denote the highest called peak using MACS1.4. (E) Venn diagram of overlapping MEF2C ChIP-seq peaks in hematopoietic progenitor cells (HPCs) and in pre-B cells; two separate ChIP experiments were included for each cell type.
Fig 3.
MEF2C and EBF1 can functionally co-regulate their common targets.
(A) Relative luciferase activities of either WT pGL4.23-Il7ra (black bars) or pGL4.23-Il7ra containing mutated MEF2C binding site (white bars) in 293T cell lysates expressing FLAG-tagged WT, EED, or AAA MEF2C, and/or Myc-tagged EBF1, as indicated on the x-axis. Renilla luciferase was used as internal control, and the experiments were performed in biological triplicates, each with technical triplicates; asterisk denotes p-value<0.5 and double-asterisk denotes p-value<0.01 (student t-test, one-tail, paired). (B) Relative luciferase activities of WT pGL4.23-Il7ra in 293T cell lysates expressing the same MEF2C constructs as (A), along with Myc-tagged E12 or E47; experiments were performed in technical triplicates. (C) Luciferase reporter activities of 293T cell lysates transfected with pGL4.23-trimerized EBF1 and MEF2C binding sites with either MEF2C or EBF1 alone or in combination. Cells were transfected with either WT (WT_3xE/M, black bars) construct or a construct with mutated EBF1 and MEF2C binding sites (Mutant_3xE/M, white bars); the mutations are indicated in black in the scheme; experiment was performed in technical triplicates. (D) Relative expression levels of Il7ra and Ebf1 in mouse lineage-depleted progenitor (lin-) cells that over-express either empty vector (EV), WT, or EED MEF2C, two days after transduction; summary of two biological duplicates is shown. (E) Relative expression levels of Il7ra and Pou2af1 in lin- cells that over-express either EV, WT MEF2C, EBF1 individually, or MEF2C and EBF1 incombination. Summary of two biological duplicates is shown.
Fig 4.
Relative expression levels of various hematopoietic genes in different lineages.
(A) Relative expression levels of Mef2c in LKS and B220+ (all B lineage) cells sorted from Mef2c-KO mice (gray bars) or WT littermates (black bars). (B) Relative expression levels of Ebf1 in LKS, CLPs, and B220+ cells sorted from same mice as in (A). (C)-(F) Relative expression levels of various B cell genes (Myb, Pou2af1, Il7r, and Rag1) and myeloid genes (Klf7, Csf1r, and Jun) in LKS, CLPs, Pre-B, or B220+ cells sorted from Mef2c-KO mice (white bars) or WT littermates (black bars) as determined by qRT-PCR. Experiments were performed in either technical triplicates or biological triplicates.
Fig 5.
In vitro B cell differentiation of lineage negative progenitor cells (lin-).
(A) Schematic for differentiation experiments. (B) Representative FACS plots of undifferentiated lin- cells and of those differentiated for 14 days; top panel—B220 and CD19 cell surface markers; bottom panel—forward scatter and Gr1 cell surface marker; undifferentiated cells were cultured in stimulation media; B cell differentiation cells were either untreated (DMSO), or treated with p38 MAPK inhibitor (SB-p38i), or ERK1/2/5 inhibitor (U0126-ERKi); the percentages of CD19, B220, or Gr1-positive cells are indicated. (C) Representative result of day 14 B cell differentiation of cells over-expressing empty vector (EV), WT, or EED MEF2C, treated with DMSO or SB-p38i; differentiation was measured by the expression of cell surface markers B220 and CD19 (left) or Gr1 (right). Two separate experiments were performed with similar results.