Ikaros-responsive genes in pre-B cells. (A) Scheme of adult B lymphocyte development in the context of hematopoiesis. Black arrows indicate developmental progression toward and within the lymphoid lineages, gray arrows indicate alternative lineage choices that are not depicted here. (B) Regulation of the Ikaros gene family during hematopoiesis and B-cell development. Data are normalized array counts from www.immgen.org. (C) Schematic representation of the inducible Ikaros construct and the experimental system. Ikaros-ERt2 is expressed at levels comparable with endogenous Ikaros (supplemental Figure 1D) and tethered in the cytoplasm until the addition of 4-hydroxy tamoxifen (4-OHT) triggers its release into the nucleus, where Ikaros is visible at DAPI-dense pericentromeric heterochromatin.30 (D) Nuclear translocation of inducible Ikaros (2 h) precedes G1 arrest (16 h). Immunofluorescence images of DAPI and hemagglutinin-Ikaros (HA-Ik) are representative of 2 experiments. DNA content was determined by flow cytometry of propidium iodide-stained cells at the indicated time points (representative of 3 experiments). (E) Bar graphs show the percentage of G1, S, and G2/M cells at the indicated time points (n = 3, mean ± SD). (F) Repression of the Ikaros target gene Igll1 is initiated within 2 h of Ikaros induction as determined by quantitative reverse transcription-polymerase chain reaction (n = 5, mean ± SD). (G) The relationship between Ikaros-responsive genes at 2 h, 6 h, and 48 h. (H) Genes that change their expression in response to Ikaros at early time points are more likely to be down-regulated.
