Interleukin 3-dependent activation of DREAM is involved in transcriptional silencing of the apoptotic Hrk gene in hematopoietic progenitor cells

Abstract

The apoptotic protein Hrk is expressed in hematopoietic progenitors after growth factor deprivation. Here we identify a silencer sequence in the 3' untranslated region of the hrk gene that binds to the transcriptional repressor DREAM in interleukin-3 (IL-3)-dependent hematopoietic progenitor cells, and abrogates the expression of reporter genes when located downstream of the open reading frame. In addition, the binding of DREAM to the hrk gene is reduced or eliminated when cells are cultured in the absence of IL-3 or treated with a calcium ionophore or a phosphatidylinositol 3-kinase-specific inhibitor, suggesting that both calcium mobilization and phosphorylation can regulate the transcriptional activity of DREAM. Furthermore, we have shown that DREAM is phosphorylated by a phosphatidylinositol 3-kinase-dependent, but Akt-independent pathway. In all cases, loss of the DREAM-DNA binding complex was correlated with increased levels of Hrk and apoptosis. These data suggest that IL-3 may trigger the activation of DREAM through different signaling pathways, which in turn binds to a silencer sequence in the hrk gene and blocks transcription, avoiding inappropriate cell death in hematopoietic progenitors.

Fig. 1. The DRE sequence of the hrk gene binds to recombinant DREAM. Radiolabeled DRE-hrk, or wild-type (wt) and mutated (mut) DRE-PD probes were incubated with recombinant human DREAM, and the formation of the binding complex was analyzed by EMSA. For competition experiments, DREAM was pre-incubated with 5- and 50-fold molar excess of unlabeled DRE-hrk probe.

Fig. 2. DREAM is expressed in FDCP-Mix and FL5.12 hematopoietic progenitor cells. (A) Nuclear extracts were analyzed by southwestern blotting using a radiolabeled DRE-hrk probe. Arrowheads indicate the monomeric and tetrameric DREAM proteins. (B) The expression of DREAM protein was analyzed by western blotting with a specific goat antibody.

Fig. 3. The expression of EGFP is blocked when DRE-hrk is inserted downstream of the reporter cDNA. Flow cytometry analysis of FDCP-Mix cells after 24 h of transfection with the reporter plasmid pCMV-EGFP containing the DREAM binding sequence of the hrk gene (DRE-hrk) or a random, nonsense sequence of the same size and nucleotide composition (DRE-NS). DNA sequences were inserted downstream of the EGFP open reading frame. Dot plots are from a representative experiment (n = 3).

Fig. 4. The expression of CAT is inhibited when DRE-hrk is located at the 3′ end of the reporter cDNA. The reporter plasmid pTK-CAT containing the DREAM binding sequence of the hrk gene (DRE-hrk), or a random, nonsense sequence of the same size and nucleotide composition (DRE-NS), or the DRE sequence of the prodynorphin gene (DRE-PD), was introduced into HEK 293 cells. After 24 h of transfection, CAT activity was determined relative to that of the pTK-CAT reporter plasmid. Data (mean ± SD) were collected from three independent transfections.

Fig. 5. Treatment with ionomycin blocks binding of DREAM to the DRE-hrk sequence and induces Hrk protein in FDCP-Mix myeloid progenitors. Cells were cultured in the absence of IL-3 or treated with ionomycin for 24 h, and then analyzed for the formation of the DREAM–DNA complex by EMSA using radiolabeled DRE-hrk (top), and for the expression of Hrk, Bad and DREAM proteins by western blotting (bottom).

Fig. 6. Inactivation of PI 3-kinase inhibits binding of DREAM to DRE-hrk and induces Hrk protein in FL5.12 lymphoid progenitors. FDCP-Mix and FL5.12 cells were cultured in the absence of IL-3 or treated with LY294002 for 24 h. An EMSA was performed using a radiolabeled DRE-hrk probe to analyze the formation of the DREAM–DNA complex (top). The expression of Hrk, Bad and DREAM proteins was analyzed by western blotting (bottom).

Fig. 7. Inactivation of PI 3-kinase induces apoptotic cell death. FDCP-Mix and FL5.12 cells were cultured in the absence of IL-3 or treated with LY294002 for 24 h, and then analyzed by flow cytometry with fluorescein isothiocyanate (FITC)-labeled annexin V. Numbers above the selected regions indicate the percentage of apoptotic cells.

Fig. 8. DREAM is phosphorylated by a PI 3-kinase-dependent pathway. FL5.12 cells were cultured in the presence of [³²P]ortho phosphate with or without LY294002. After labeling, cells were lysed and DREAM was immunoprecipitated using a specific polyclonal antibody and protein A/G–agarose beads. Phosphorylation of DREAM was determined by autoradiography.