Validation of GABP target genes and predicted role of GABP in maintaining HSC quiescence. (A) Heatmaps of select GABP-activated direct target genes from the transcriptomic analysis. Color-coded scale bars denote Z-scores. Validation of GABP binding and transcript changes of all these genes is summarized in Table 1. (B) Validation of expression changes of select genes on protein levels. BM cells from pIpC-treated mice were sequentially surface-stained and intracellularly stained with fluorochrome-conjugated antibodies against GABPα, Bcl-2, Pten, Brm, or Brg1. The expression of each protein in LSKs is shown in red lines, with shaded histograms denoting isotype controls. For GABPα and Pten staining, self-conjugated normal rabbit IgG was used as an isotype control, and for Brm and Brg1 staining, self-conjugated normal goat IgG was as an isotype control. The values in histograms indicate the percentages of LSKs expressing indicated proteins. Data are representative from 3 independent experiments analyzing 3-6 pairs of Mx1Cre-GABPα^FL/− and control mice. (C) Proliferation status in myeloid progenitors and LSK cells. Four to 6 days after pIpC treatment, Mx1Cre-GABPα^FL/+ and Mx1Cre-GABPα^FL/− mice were pulsed with BrdU via intraperitoneal injection for 18 hours. BM cells were surface-stained followed by intracellular detection of BrdU uptake. The percentage of BrdU⁺ cells in each subset is shown. (D) Increased proliferation of GABPα-deleted LSKs and myeloid progenitors. Data were pooled results from 3 independent experiments with 5 mice of each genotype analyzed. (E) Heatmaps of select genes that do not have GABPα binding within 2 kb of TISs but are affected in expression by GABPα deficiency. Color-coded scale bars showing Z-scores are the same as in panel A. The gene-expression changes were validated by quantitative RT-PCR in panel F. (G) Proposed model for the roles of GABP in regulating HSC activity. A GABP-controlled gene regulatory module in HSCs is illustrated, showing GABP auto-regulation, potential interregulation with other key transcription factors and epigenetic modification molecules, and coregulation of downstream effector genes involved in HSC survival, self-renewal, quiescence, and differentiation. Solid red and blue lines denote direct and indirect regulatory connection confirmed in this study, respectively. Dashed lines denote possible interaction in the regulatory module. Solid black lines are regulatory roles based on literature. Arrows indicate positive regulation, and lines ending in bars indicate negative regulation.