Genome-wide identification of Klf2 target genes. (A) Experimental setup for transcriptome analysis of Klf2 gain-of-function vs loss-of-function. For gain-of-function analysis, pro-B cells were transduced with retroviral vectors expressing blue fluorescent protein (BFP) with (+Klf2) or without Klf2 and sorted. For loss-of-function analysis, pro-B cells (Lin⁻CD19⁺B220^medCD43^hiIgL⁻) of Klf2^Δ/ΔCd79a^hCre/+ mice (Klf2^Δ/Δ) or control mice (Cd79a^hCre/+) were sorted by flow cytometry, RNA was isolated and sequenced. (B) Reciprocal deregulation of genes in loss-of-function (Klf2^Δ/Δ vs Klf2^+/+) and gain-of-function (+Klf2 vs +BFP) experiments in pro-B cells shown as fold change. Colors indicate 20 activated Klf2 target genes (blue) and 162 repressed Klf2 target genes (red). (C) Gene expression changes shown as fold change of genes encoding for AP1 family members (Jun/b/d, Fos/b) and early response genes (Egr1-3, Ier2) in Klf2 loss-of-function and gain-of-function experiments. (D) Schematic representation of the role of Mef2c/d proteins upon pre-BCR stimulation. Phosphorylation of Mef2c/d by Erk5 via pre-BCR signaling activates several transcriptional circuits. Mef2c/d activates the expression of Klf2 and IEGs (Egr1/2, Ier2, Jun/Fos). Klf2 suppresses IEG expression and thereby blocks pre-BCR–induced cell proliferation (negative feed-forward loop). In a positive feed-forward loop, Mef2c activates Egr and Irf4, the gene products of which collaborate with Mef2c and several key pre-B-cell regulatory factors to drive pre-B-cell differentiation.