Gfi-1 represses CDKN2B encoding p15INK4B through interaction with Miz-1

Abstract

Gfi-1 is a nuclear zinc finger (ZF) transcriptional repressor that plays an important role in hematopoiesis and inner ear development, and has been implicated in lymphomagenesis. Gfi-1 represses transcription by directly binding to the consensus DNA sequence in the promoters of its target genes. We report here an alternative mechanism by which Gfi-1 represses CDKN2B encoding p15(INK4B). Gfi-1 does not directly bind to CDKN2B, but interacts with Miz-1 and, via Miz-1, is recruited to the core promoter of CDKN2B. Miz-1 is a POZ-ZF transcription factor that has been shown to mediate transcriptional repression by c-Myc. Like c-Myc, upon recruitment to the CDKN2B promoter, Gfi-1 represses transcriptional activation of CDKN2B by Miz-1 and in response to TGFbeta. Consistent with its role in repressing CDKN2B transcription, knockdown of Gfi-1 in human leukemic cells or deficiency of Gfi-1 in mouse bone marrow cells results in augmented expression of p15(INK4B). Notably, Gfi-1 and c-Myc are both recruited to the CDKN2B core promoter and act in collaboration to repress CDKN2B. Our data reveal a mechanism of transcriptional repression by Gfi-1 and may have important implications for understanding the roles of Gfi-1 in normal development and tumorigenesis.

Fig. 1.

Gfi-1 interacts with Miz-1 in vitro and in vivo. (A) Whole-cell extracts of 293T cells transfected with the Flag-tagged Gfi-1 were incubated with GST or GST-Miz-1 fusion protein, and examined for Gfi-1 by Western blot analysis (Upper). GST proteins were stained with GelCode Blue Stain reagent (Lower). (B) 293T cells were transfected with Flag-tagged Gfi-1 or Flag-tagged STAT5 along with Myc-tagged Miz-1. Whole-cell extracts were subjected to immunoprecipitation, using the anti-Flag antibody followed by immunoblotting with the anti-Myc antibody (Top). The expression of the transfected proteins was examined using the indicated antibodies (Middle and Bottom). (C) Nuclear extracts from HL-60 cells were immunoprecipitated using the anti-Miz-1 antibody or an irrelevant species-matched antibody before immunoblotting for Gfi-1 and Miz-1. (D) Schematic diagrams of Miz-1, its mutants and GST-Miz-1 fusion protein. (E) 293T cells were transfected with Flag-tagged Gfi-1 along with the different forms of Myc-tagged Miz-1. Whole-cell extracts were examined using the indicated antibodies (Right), or immunoprecipitated using the anti-Flag antibody before immunoblotting (Left).

Fig. 2.

Gfi-1 represses Miz-1-mediated activation of the CDKN2B promoter through interaction with Miz-1. (A) 293T cells were transfected as indicated. Whole-cell extracts were subjected to precipitation, using the CDKN2B promoter oligonucleotide, and examined for the indicated proteins. (B) Schematic diagrams of Gfi-1 and the mutants. (C) The 293T cells were transfected as indicated and oligonucloetide precipitation assays were carried out as described in A. (D) 293T cells were transfected with the indicated expression constructs. ChIP experiments were carried out using the antibodies against Gfi-1, Miz-1, or an irrelevant species-matched antibody. The indicated regions of CDKN2B were examined by PCR. (E) ChIP assays were carried out on HL-60 cells, using the anti Gfi-1 antibody or an irrelevant species-matched antibody. The core promoter region of CDKN2B (−100/+45 bp) was amplified by PCR. (F) HeLa cells were transfected with the CDKN2B promoter (−751/+70 bp) luciferase reporter construct along with Miz-1 and increasing amounts of Gfi-1. Luciferase activities were measured and normalized for the cotransfected SV40 β-gal activities. In separate experiments, HeLa cells were transfected as above and examined for expression of Gfi-1 and Miz-1 by Western blot analysis. (G) Effect of Gfi-1 on the activity of a luciferase reporter construct containing the c-Fos serum response element (SRE). Data are shown as mean ± SD.

Fig. 3.

Gfi-1 collaborates with c-Myc in repressing the CDKN2B promoter activity. (A and B) HeLa cells were transfected with the CDKN2B promoter luciferase reporter construct (−751/+70 bp) along with Gfi-1 and c-Myc without or with Miz-1. Luciferase activities were measured 36 h later and normalized for β-gal activities. Expression of Gfi-1, Miz-1, and c-Myc was examined in separate transfection assays. (C and D) 293T cells were transfected as indicated. Whole-cell extracts were subjected to immunoprecipitation, using the anti-Flag antibody (C), or precipitation, using the CDKN2B promoter oligonucleotide (D), and examined for the indicated proteins.

Fig. 4.

Effects of Gfi-1 and the N382S mutant on Miz-1-mediated activation of the CDKN2B core promoter. (A) HeLa cells were transfected with the CDKN2B promoter (−113/+70 bp) luciferase reporter construct along with Miz-1 and Gfi-1 or the N382S mutant before assay for luciferase activities. Expression of Miz-1 and Gfi-1 proteins was examined in separate transfection assays. (B) 293T cells were transfected with Miz-1 with or without the N382S mutant. Immunoprecipitation was performed using the anti Gfi-1 antibody before immunoblotting for Miz-1 (Top). Expression of the proteins was confirmed (Middle and Bottom). (C) Oligonucleotide precipitation was performed on whole-cell extracts of 293T cells transfected with the indicated expression constructs, using the CDKN2B promoter oligonucleotide. Bound proteins were examined using the anti Gfi-1 and anti Miz-1 antibodies.

Fig. 5.

Gfi-1 represses TGFβ-induced activation of the CDKN2B promoter. HeLa cells were transfected with the CDKN2B promoter luciferase reporter constructs containing the CDKN2B promoter fragments covering −751 bp to +70 bp (A) and −113 bp to +70 bp (B), along with Gfi-1 or the N382S mutant. Cells were subsequently incubated in the absence or presence of TGFβ for 6 h before assay for luciferase activity.

Fig. 6.

Suppression of Gfi-1 expression is associated with augmented levels of p15^INK4B. HL-60 (A) and U937 (B) cells were infected with the empty lentivirus (Ctr) or the lentiviruses containing the shRNAs against Gfi-1. The expression of Gfi-1 and p15^INK4B were examined by Western blot analysis after selection of the cells in puromycin for 3 days. (C) Whole-cell extracts were prepared from Gfi-1^+/+ and Gfi-1^−/− BM cells and examined for p15INK4B expression.

Fig. 7.

Gfi-1 represses activation of CDKN1B by Miz-1. (A) HeLa cells were transfected with the CDKN1B promoter luciferase reporter construct along with Miz-1, Gfi-1 or the N382S mutant. Luciferase activities were normalized for the co-transfected SV40 β-gal activities. (B) 293T cells were transfected with the indicated expression constructs. ChIP experiments were carried out using the antibodies against Gfi-1, Miz-1 or an irrelevant species-matched antibody (Ctr). The indicated regions of CDKN1B were examined by PCR. (C) ChIP assays were carried out on HL-60 cells using the anti Gfi-1 antibody or an irrelevant species-matched antibody. (D) HL-60 cells were infected with the empty lentivirus (Ctr) or the lentiviruses containing the shRNA against Gfi-1 and selected in puromycin. Individual clones were obtained by limiting dilution. The expression of Gfi-1 and p27^Kip1 were examined by Western blot analysis.