Genome-wide identification of Mef2 target genes using RNA-seq. (A) Scatter plot of differential gene expression (>1.5-fold; false discovery rate corrected P ≤ .05) in pro-B cells of Mef2c/d^Δ/Δ vs control mice (Mef2c/d^+/+) in 3 independent experiments. Lin⁻CD19⁺CD43^highB220^low pro-B cells were sorted by flow cytometry for preparation of RNA followed by deep sequencing. Gene expression is presented as normalized expression data (reads per kilobase of transcript per million reads mapped). Each dot represents 1 gene. Colors indicate overexpressed (blue) and underexpressed (red) genes in Mef2c/d^Δ/Δ mice in relation to control mice. (B) Functional classification of genes significantly deregulated in pro-B cells of Mef2c/d^Δ/Δ mice. Gene ontology (GO) analysis using the STRING database was performed to identify biological processes that are enriched in deregulated genes. The number of genes per class and the statistical significance is indicated. (C-D) Relative transcript levels determined by RNA-seq of genes encoding B-cell TFs (C) and a selection of differentially regulated genes (D) in pro-B cells of Mef2c/d^Δ/Δ mice vs Mef2c/d^+/+ control mice are depicted. Shown is the mean value of 3 independent experiments relative to controls. Error bars denote ± standard deviation (SD). (E) Quantitative RT-PCR (qRT-PCR) validation of transcript levels of a number of deregulated genes in Mef2c/d^Δ/Δ mice vs Mef2c/d^+/+ mice. qRT-PCR data were normalized to Hprt expression. Error bars represent ± SD. Data were combined from at least 2 independent experiments. (F) Immunoblot analysis of whole-cell lysates of Mef2c/d-deficient (M^Δ/Δ) primary pro-B/pre-B cells (Lin⁻CD19⁺Igκ/λ⁻) in relation to pro-B/pre-B cells of Mef2c/d^+/+ mice (M^+/+). Protein levels of Klf2, Jun, and Irf4 are shown. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as a control. Data are representative of 2 independent experiments.