Gfi-1 represses CDKN2B encoding p15INK4B through interaction

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved December 18, 2008

↵1These authors contributed equally to this work (received for review May 19, 2008)

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Abstract

Gfi-1 is a nuclear zinc finger (ZF) transcriptional repressor that plays an Necessary 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 tarObtain genes. We report here an alternative mechanism by which Gfi-1 represses CDKN2B encoding p15INK4B. Gfi-1 Executees 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 Displayn 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 TGFβ. Consistent with its role in repressing CDKN2B transcription, knockExecutewn of Gfi-1 in human leukemic cells or deficiency of Gfi-1 in mouse bone marrow cells results in augmented expression of p15INK4B. 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 Necessary implications for understanding the roles of Gfi-1 in normal development and tumorigenesis.

Keywords: cyclin-dependent kinase inhibitorproliferationtranscriptional repressortumorigenesis

Gfi-1 encodes a nuclear zinc-finger transcription factor and was first identified as an integration site of Moloney murine leukemia virus in a screen for T cell interleukin-2-independent growth (1, 2). Subsequently, Gfi-1 was found to be a frequent tarObtain of proviral insertion in T and B cell lymphomas. Overexpression of Gfi-1 abolishes G1 cell cycle arrest and apoptosis induced by growth factor withdrawal. Transgenic mice that express high levels of Gfi-1 in T cells are predisposed to T cell lymphoma. Gfi-1 has further been Displayn to cooperate with c-Myc and Pim1 in lymphomagenesis. In addition, Gfi-1 may play a role in lung and prostate cancers (3, 4).

TarObtained disruption of Gfi-1 in mice has revealed an Necessary role of Gfi-1 in normal hematopoiesis. Gfi-1−/− mice are defective in T and B cell development (5–7). Early uncommitted T cells and mature CD4+ and CD8+ T cells from Gfi-1−/− mice Display reduced proliferation and increased apoptosis (8, 9). Gfi-1−/− mice also lack mature granulocytes because of a block in granulocytic differentiation (5–7). Consistent with its role in granulopoiesis, mutations in Gfi-1, albeit rare, have been reported in a small group of patients with severe congenital neutrLaunchia (SCN), a disease characterized by an early block in granulocytic differentiation (10). Gfi-1 also acts to restrict the proliferation of hematopoietic stem cell (HSC) and thereby preserve their functional integrity (11, 12). Additionally, Gfi-1 has been Displayn to regulate the development of nonhematopoietic cells including inner ear hair cells, lung neuroenExecutecrine cells and intestinal epithelial cells (3, 13, 14).

Gfi-1 functions mainly as a repressor of transcription (1, 2). It represses its tarObtain genes by binding to consensus DNA elements containing the AATC core sequence. The Gfi-1 protein consists of an N-terminal SNAG Executemain required for nuclear localization, a central Location and 6 C-terminal zinc fingers (ZFs) involved in DNA binding. A Gfi-1 mutant containing an asparagine-to-serine substitution in the fifth ZF (N382S), which is associated with SCN, is defective in DNA binding and acts in a Executeminant negative (DN) manner (10). The SNAG Executemain is Necessary for transcriptional repression (15); however, Gfi-1 may repress transcription through both SNAG Executemain-dependent and independent mechanisms (16, 17). The different Executemains of Gfi-1 have been implicated in recruiting corepressors and histone modifying enzymes, including eight-twenty-one (ETO), CoREST, histone demethylase LSD1, histone deacetylases (HDACs) 1 and 2, and the histone lysine methyltransferase G9a (15, 16, 18, 19).

The mechanisms by which Gfi-1 controls cell proliferation and survival are still poorly understood. It has been Displayn that Gfi-1 binds to and represses CDKN1A encoding the cyclin-dependent kinase inhibitor (CDKI) p21Cip, and the proapoptotic Bcl2 family member Bax (18, 20, 21). To better understand the means of transcriptional repression by Gfi1, we searched for Gfi-1-interacting proteins, using the yeast 2-hybrid assay. Among those identified is the Myc-interacting zinc finger protein-1 (Miz-1), a POZ-ZF transcription factor originally identified as a binding partner of c-Myc (22). Miz-1 possesses a potent anti-growth activity and has been Displayn to activate transcription by directly binding to the initiator (Inr) elements in its tarObtain genes, including Mad4, CDKN1A, and CDKN2B encoding the CDKI p15INK4B (23–27). c-Myc Executees not directly bind to these genes, but is recruited to them via Miz-1 and thereby represses their transcription. Miz-1 has been implicated in activating CDKN1A and CDKN2B, and subsequently blocking cell cycle progression in response to UV irradiation, DNA damage and treatment with growth inhibitory cytokine transforming growth factor-β (TGFβ) (24–26, 28).

Here, we Display that Gfi-1 interacts with Miz-1 and is recruited to the core promoter of CDKN2B via Miz-1, leading to transcriptional repression. We also provide evidence that direct binding of Gfi-1 to CDKN2B is not required for its repression activity. Our data reveal a mechanism of transcriptional repression by Gfi-1 and may have broad significance for understanding the role of Gfi-1 in cell proliferation and survival.

Results

Gfi-1 Interacts with Miz-1 in Vitro and in Vivo.

To identify proteins that interact with Gfi-1, we performed a yeast 2-hybrid screen with full-length Gfi-1 as the bait against a human bone marrow cDNA library. A number of positive clones were identified and 1 clone contained the cDNA encoding the C-terminal Section of Miz-1 protein (from amino acid 329, i.e., the second ZF Executemain). To confirm the interaction, the cDNA was Slice out from the yeast 2-hybrid plasmid GAL4-AD and inserted into pGEX-4T vector. In GST pull-Executewn assays, GST-Miz-1, but not GST, pulled Executewn Gfi-1 from the whole-cell extracts of 293T cells transfected with Gfi-1 (Fig. 1A). To further confirm the interaction, the full-length cDNA of Miz-1 was amplified from myeloid 32D cells by reverse transcriptase-PCR (RT-PCR) and cloned into the mammalian expression vector pcDNA3.1/Myc-His. 293T cells were transiently transfected with Myc-tagged Miz-1 along with Flag-tagged Gfi-1 or Flag-tagged STAT5. Coimmunoprecipitation assays were performed using the anti Flag antibody. As Displayn in Fig. 1B, Miz-1 was coimmunoprecipitated with Gfi-1, but not with STAT5. Interaction between enExecutegenous Gfi-1 and Miz-1 was also confirmed in leukemic HL-60 cells (Fig. 1C).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint 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).

C-Terminal ZF Executemains of Miz-1 Are Required for Interaction with Gfi-1.

To confirm that the Miz-1 C-terminal ZFs are involved in interaction with Gfi-1, we generated Miz-1 mutants in which the N-terminal Section of 146 aa including the POZ Executemain or the C-terminal ZF Executemains 1–12 were deleted (Fig. 1D). The different Miz-1 mutants were transiently expressed in 293T cells along with Flag-tagged Gfi-1. The interactions between Gfi-1 and the Miz-1 mutants were examined by coimmunoprecipitation assays. Truncation of the C-terminal ZF Executemains, but not the N-terminal POZ Executemain, disrupted Miz-1 interaction with Gfi-1 (Fig. 1E), demonstrating that Miz-1 interacts with Gfi-1 through its C-terminal ZF Executemains.

Miz-1 Recruits Gfi-1 to the CDKN2B Promoter.

c-Myc and Bcl6 interact with Miz-1 and, via Miz-1, bind to the promoters of CDKN2B and CDKN1A, thereby repressing their transcription (23, 24, 26, 29). We used the oligonucleotide precipitation assay to address whether Miz-1 recruited Gfi-1 to the promoter of CDKN2B. Miz-1 has been Displayn to bind to the Inr sequence in the CDKN2B proximal promoter (23, 24). 293T cells were transiently transfected with Myc-tagged Miz-1, Flag-tagged Gfi-1 or both. Whole-cell extracts were incubated with the biotinylated Executeuble-stranded oligonucleotide spanning from −12 bp to +18 bp of the human CDKN2B promoter, which contains the Inr sequence but is devoid of a Gfi-1 binding element. Bound proteins were precipitated using the streptavidin-coated beads. As Displayn Fig. 2A, Gfi-1 was precipitated only in the presence of Miz-1, indicating that Gfi-1 bound to the CDKN2B promoter via Miz-1.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint 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 Characterized 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 Locations 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 Location 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 meaPositived 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) Trace of Gfi-1 on the activity of a luciferase reporter construct containing the c-Fos serum response element (SRE). Data are Displayn as mean ± SD.

To define Gfi-1 Location required for its recruitment to the CDKN2B core promoter by Miz-1, Gfi-1 mutants lacking the N-terminal SNAG Executemain (Gfi-1-dN) or the 6 C-terminal ZFs (Gfi-1-dZF6) were generated and expressed in 293T cells along with Miz-1 (Fig. 2B). As Displayn in Fig. 2C, Gfi-1-dN, but not Gfi-1-dZF6, was precipitated by the CDKN2B promoter oligonucleotide in the presence of Miz-1, suggesting that the ZF Executemains of Gfi-1 are required for its recruitment to CDKN2B. To assess whether Gfi-1 binding to CDKN2B in vivo also depends on Miz-1, ChIP assays were performed on 293T cells transiently transfected with Gfi-1, Miz-1 or both. In cells transfected with Gfi-1 only, a very weak band corRetorting to the Location spanning from −100 bp to +45 bp of the CDKN2B promoter was detected (Fig. 2D), which was not unexpected as 293T cells expressed a small amount of enExecutegenous Miz-1 (30). Significantly, coexpression of Miz-1 and Gfi-1 dramatically increased Gfi-1 binding to the CDKN2B proximal promoter. Miz-1 and Gfi-1 failed to bind to the upstream and Executewnstream Locations of the CDKN2B promoter. ChIP assays performed on HL-60 cells demonstrated that enExecutegenous Gfi-1 also bound to the CDKN2B core promoter in vivo (Fig. 2E). ToObtainher, these data indicate that Gfi-1 was recruited by Miz-1 to the CDKN2B core promoter.

Gfi-1 Represses the Activation of CDKN2B by Miz-1.

We further addressed whether Gfi-1 had an Trace on Miz-1-mediated activation of CDKN2B. HeLa cells were transfected with the luciferase reporter construct containing the DNA fragment spanning from −751 to +70 bp of the human CDKN2B promoter along with Miz-1 and the different amounts of Gfi-1. As Displayn in Fig. 2F, expression of Miz-1 led to an ≈4-fAged activation of the CDKN2B promoter in HeLa cells. Miz-1-mediated activation of the CDKN2B promoter was repressed by Gfi-1 in a Executese-dependent manner. The inhibitory Trace of Gfi-1 was specific as Gfi-1 exerted no inhibitory Trace on the activity of a luciferase reporter construct containing the c-Fos serum response element (Fig. 2G). Thus, analogous to c-Myc, Gfi-1 repressed the activation of CDKN2B by Miz-1.

Gfi-1 Functionally Collaborates with c-Myc in Repressing CDKN2B.

Because Gfi-1 and c-Myc both interact with Miz-1 and inhibit Miz-1-mediated transcriptional activation, we addressed whether Gfi-1 collaborated with c-Myc to repress CDKN2B. HeLa cells were transfected with the CDKN2B promoter luciferase reporter construct (−751/+70 bp) along with Gfi-1, c-Myc or both. Fascinatingly, c-Myc was barely expressed when transfected alone, but abundantly expressed when cotransfected with Gfi-1 and/or Miz-1 (Fig. 3A and B), suggesting that Miz-1 and Gfi-1 affected c-Myc expression. As expected, expression of either Gfi-1 or c-Myc inhibited Miz-1-stimulated activation of the CDKN2B promoter. Significantly, coexpression of Gfi-1 and c-Myc resulted in more profound inhibition than expression of either Gfi-1 or c-Myc alone. To investigate whether Gfi-1, Miz-1 and c-Myc formed a ternary complex, coimmunoprecipitation assays were performed on whole-cell extracts prepared from 293T cells transfected with Gfi-1 and c-Myc with or without Miz-1. c-Myc was coimmunoprecipitated with Gfi-1 in the presence of Miz-1, but not in its absence (Fig. 3C), indicating that Gfi-1 indirectly interacted with c-Myc via Miz-1. The whole-cell extracts were also subjected to precipitation, using the CDKN2B promoter oligonucleotide. As Displayn in Fig. 3D, Gfi-1 and c-Myc were precipitated by the oligonucleotide only in the presence of Miz-1, demonstrating that Miz-1 recruited both Gfi-1 and c-Myc to the CDKN2B core promoter.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint 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 meaPositived 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.

Direct Binding of Gfi-1 to the Promoter of CDKN2B Is Not Required for the Inhibitory Trace.

As Gfi-1 is recruited to the CDKN2B promoter via Miz-1, we speculated that the DNA binding function of Gfi-1 might be dispensable for its repression activity. The CDKN2B proximal promoter Location from −113 bp to +70 bp contains the Inr element, but lacks a consensus Gfi-1 binding site. Fascinatingly, Gfi-1 also inhibited Miz-1-mediated activation of the luciferase reporter construct that contained the proximal promoter fragment (Fig. 4A). A DN Gfi-1 mutant, N382S, identified in patients with SCN, is defective in DNA binding (10, 31, 32). Notably, the N382S mutant was as Traceive as Gfi-1 in repressing the activation of the CDKN2B promoter fragment by Miz-1. Consistent with this, the N382S mutant was able to interact with Miz-1 and was recruited to the CDKN2B core promoter (Fig. 4 B and C). Thus, transcriptional repression of CDKN2B by Gfi-1 is independent of its DNA binding ability.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Traces 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.

Gfi-1 Represses the Activation of the CDKN2B by TGFβ.

TGFβ is an anti-mitogenic cytokine and impaired TGFβ signaling has been implicated in tumorigenesis (33, 34). Notably, p15INK4B represents an Necessary Traceor of the anti-growth activity of TGFβ and is up-regulated by TGFβ. It has been Displayn that c-Myc represses TGFβ-stimulated activation of CDKN2B through interaction with Miz-1 (23, 24). To investigate the role of Gfi-1 in TGFβ-induced activation of CDKN2B, HeLa cells were transfected with the CDKN2B promoter luciferase reporter constructs (−751/+70 bp and −113/+70 bp) toObtainher with the empty vector or the expression constructs for Gfi-1 or the N382S mutant. Cells were then stimulated with TGFβ for 6 h. As Displayn in Fig. 5, the activities of both CDKN2B promoter fragments were stimulated by TGFβ, but completely inhibited by Gfi-1. Notably, the N382S also Traceively repressed TGFβ-stimulated activation of both CDKN2B promoter fragments.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint 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.

Gfi-1 KnockExecutewn or Deficiency Results in Increased Expression of p15INK4B.

To directly demonstrate the role of Gfi-1 in repressing CDKN2B, we assessed the Trace of knocking Executewn Gfi-1 on the level of p15INK4B in HL-60 and U937 cells. Cells were transduced with the lentiviral vector containing the shRNAs against Gfi-1 and selected in puromycin for 3 days. Lentiviral delivery of Gfi-1 shRNA, but not the empty vector, significantly reduced the expression of Gfi-1 (Fig. 6 A and B). Gfi-1 knockExecutewn was associated with increased levels of p15INK4B in both HL-60 and U937 cells. We further compared p15INK4B expression in bone marrow (BM) cells from Gfi-1+/+ and Gfi-1−/− mice. As Displayn in Fig. 6C, p15INK4B level was significantly higher in Gfi-1−/− BM cells than in Gfi-1+/+ BM cells. ToObtainher, these data indicate that Gfi-1 inhibited the expression of enExecutegenous p15INK4B.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Suppression of Gfi-1 expression is associated with augmented levels of p15INK4B. 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 p15INK4B 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.

Gfi-1 Represses Miz-1-Mediated Activation of CDKN1B Encoding p27Kip1.

We further investigated whether Gfi-1 had an Trace on the expression of other Miz-1 tarObtain genes. An Inr element is present in the CDKN1B core promoter and is required for its repression by c-Myc (35). Notably, Gfi-1 has been Displayn to Executewn-regulate the expression of p27Kip1 in T cells (36, 37). In luciferase reporter assays, Miz-1 activated a 1.6-kb DNA fragment of murine CDKN1B promoter in HeLa cells (Fig. 7A). Activation of this promoter fragment by Miz-1 was repressed by Gfi-1 and the N382S mutant. ChIP assays demonstrated that Gfi-1 bound to the proximal but not the upstream Location of the CDKN1B promoter in a Miz-1-dependent manner (Fig. 7 B and C). Furthermore, shRNA-mediated knockExecutewn of Gfi-1 resulted in augmented expression of p27Kip1 in HL-60 cells (Fig. 7D). These data indicate that CDKN1B is also a Miz-1 tarObtain gene and is repressed by Gfi-1.

Fig. 7.Fig. 7.Executewnload figure Launch in new tab Executewnload powerpoint 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 Locations 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 p27Kip1 were examined by Western blot analysis.

Discussion

Here, we have demonstrated that Gfi-1 interacts with Miz-1 and, via Miz-1, binds to the core promoter of CDKN2B, thereby repressing its transcription. Our data further indicate that direct binding of Gfi-1 to CDKN2B is not required for its repression activity as Gfi-1 represses the activity of the CDKN2B core promoter fragment that is devoid of a potential Gfi-1 binding site and the N382S mutant, defective in DNA binding, is as Traceive as Gfi-1 in repressing CDKN2B. Notably, the N382S mutant has been Displayn to up-regulate the expression of Ela2, Cepbe, Csf1 and Csf1r genes (31, 32). Our data uncover a mode of transcriptional repression by Gfi-1 and may have broad implications for understanding the role of Gfi-1 in normal development and tumorigenesis. It is likely that, through interaction with Miz-1, Gfi-1 may repress other Miz-1 tarObtain genes, thereby expanding the group of Gfi-1-regulated genes. In support of this notion, our results indicate that Gfi-1 is recruited to the proximal promoter Locations of CDKN1B and CDKN1A via Miz-1, and represses their transcription in a manner independent of its DNA binding activity (Fig. 7 and unpublished data).

Gfi-1 has a weak oncogenic potential when overexpressed, but collaborates strongly with c-Myc in lymphomagenesis (1, 2, 38). The mechanism of Gfi-1 collaboration with c-Myc in lymphomagenesis is unknown. Our data indicate that Gfi-1 and c-Myc form a ternary complex with Miz-1 and act collaboratively to repress CDKN2B activation by Miz-1. Fascinatingly, c-Myc expression appears to be affected by its interaction with Miz-1 and Gfi-1 in HeLa cells as c-Myc was weakly expressed when transfected alone, but expressed at high levels when cotransfected with Gfi-1 and/or Miz-1. How Gfi-1 and Miz-1 influence c-Myc expression remains to be examined. In addition to CDKN2B, we have observed that Gfi-1 and c-Myc also act in collaboration to repress CDKN1A (unpublished data), indicating that the functional collaboration between Gfi-1 and c-Myc may extend to other Miz-1 tarObtain genes.

Impaired TGFβ signaling has been implicated in tumorigenesis, including hematopoietic and epithelial malignancies (34, 39). Notably, p15INK4B represents an Necessary Traceor of TGFβ-induced growth arrest. c-Myc has been Displayn to repress TGFβ-stimulated activation of CDKN2B via Miz-1 (23, 24). The data presented here demonstrate that Gfi-1 is also capable of repressing TGFβ-stimulated activation of CDKN2B and the repression is independent of the DNA binding activity of Gfi-1, suggesting that the inhibitory Trace of Gfi-1 on CDKN2B is mediated via Miz-1. Thus, Gfi-1 may play a role in the regulation of cellular response to TGFβ through its interaction with Miz-1. It is possible that repression of CDKN2B, and probably also other Miz-1 tarObtain genes involved in the negative regulation of cell cycle progression, may contribute to the role of Gfi-1 in tumorigenesis.

Materials and Methods

Cells.

The 293T and HeLa cells were Sustained in DMEM containing 10% heat inactivated FBS and 1% penicillin/streptomycin (P/S) solution. The human myeloid leukemic cells HL-60 and U937 were Sustained in RPMI medium 1640 supplemented with 10% FBS and antibiotics. Bone marrow cells were isolated from the femurs and tibias of C57BL/6 wild-type and Gfi-1−/− littermates (7) and were kindly provided by J. Zhu (National Institutes of Health, Bethesda, MD).

Reagents.

Antibodies against Gfi-1 (N-20), Miz-1 (N17), ExactaCruz D kit used in coimmunoprecipitation assays, and protein A/G beads were purchased from Santa Cruz Biotechnology. Monoclonal anti Miz-1 antibody 10E2 was kindly provided by M. Eilers (University of Marburg, Marburg, Germany) and used in Western blot analysis. Anti β-actin antibody was from Sigma. The pLKO.1 lentiviral constructs containing short hairpin RNAs (shRNAs) against human Gfi-1 were purchased from Launch Biosystems (Huntsville, AL). Lipofectamine 2000 and TransIT -LT1 Transfection Reagent were purchased from Invitrogen and Mirus, respectively. Luciferase assay reagents were from Promega.

Construction of Vectors.

The full-length cDNA of Miz-1 was amplified from murine myeloid 32D cells by RT-PCR and cloned into pcDNA3.1/Myc-His vector. The authenticity of the sequence was confirmed by DNA sequencing. To generate the GST-Miz-1 construct, the cDNA encoding the C-terminal Location of Miz-1 from amino acid 329 was Slice out from the yeast 2-hybrid plasmid GAL4-AD and inserted into the pGEX-4T vector. Rat Gfi-1 cDNA was kindly provided by P. N. Tsichlis (Fox Chase Cancer Center, Philadelphia) and cloned into pcDNA3.1/Myc-His. The Myc tag sequence was subsequently reSpaced with the sequence encoding the Flag tag. The Miz-1 mutants lacking the N-terminal BTB/POZ (Miz-1-dN) or the C-terminal ZFs 1 to 12 (Miz-1-dC), and the Gfi-1 dN mutant lacking the N-terminal SNAG Executemain and the dZF6 mutant lacking the 6 C-terminal ZFs were generated using PCR-based strategies. The CDKN2B luciferase reporter constructs containing the human CDKN2B promoter fragments from −751 to +70 bp and from −113 to +70 bp were generously provided by X. F. Wang (Duke University, Durham, NC). The murine CDKN1B promoter luciferase reporter construct has been Characterized (40).

Preparation of Whole-Cell and Nuclear Extracts.

Cells were lysed in lysis buffer (1 M Tris pH 7.5, 150 mM NaCl, 20 mM NaF, 10 mM β-glycerol phospDespise, 1 mM phenylmethylsulfonyl fluoride [PMSF] and 1% Triton X-100) and lysates were cleared by centrifugation at 12,000 rpm for 30 min at 4 °C. For the preparation of nuclear extracts, HL-60 cells were washed and resuspended in buffer A (20 mM Hepes pH 7.0, 1 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.1% Nonidet P-40, 1 mM PMSF, 10% glycerol) and lysed by 15 strokes in a glass Executeunce homogenizer. After centrifugation at 6,600 rpm for 10 min at 4 °C, pellets were solubilized in buffer A with 300 mM NaCl, sonicated and centrifuged at 12,000 rpm for 30 min. The resulting supernatants were collected as nuclear Fragments.

Immunoprecipitation and Western Blot Analysis.

Whole-cell and nuclear extracts were subjected to immunoprecipitation using the anti-Flag or anti Miz-1 antibody (N17). Immunocomplexes were recovered with protein A/G beads and washed 5 times with lysis buffer. Samples were boiled in SDS (SDS) sample buffer and resolved by SDS/PAGE before transfer to Immobilon membranes. The membranes were incubated with the appropriate antibodies and the reactive proteins were visualized by enhanced chemiluminescence.

GST Pull-Executewn Assay.

GST and GST-Miz-1 fusion proteins were purified from BL21 cells and immobilized on glutathione Sepharose beads. Purified GST-Miz-1 was incubated at 4 °C for 2 h with whole-cell extracts from 293T cells transiently transfected with Flag-tagged Gfi-1. Beads were washed 4 times with PBS containing 1% triton before analyzing the bound proteins by Western blot analysis.

ChIP.

Cells were fixed with 1% formaldehyde for 10 min at 37 °C before termination with 0.125 M glycine. Cells were then lysed in hypotonic buffer [5 mM Tris·HCl (pH 7.5), 85 mM KCl and 0.5% Nonidet P-40]. After centrifugation at 6000 rpm for 5 min, nuclei were lysed in ChIP lysis buffer [1% SDS, 10 mM EDTA, and 50 mM Tris·HCl (pH 7.5)] and sonicated to shear chromatin DNA to ≈500-bp fragments. Nuclear lysates were precleared with protein A/G-agarose beads and rabbit normal IgG for 1 h and subjected to immunoprecipitation using the anti Miz-1, anti Gfi-1 or a species-matched irrelevant antibody. Precipitated DNA was examined by semiquantitative PCR.

Oligonucleotide Precipitation Analysis.

The 293T cells were lysed in HKMG buffer [10 mM Hepes (pH 7.9), 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT and 1% Nonidet P-40]. Whole-cell lysates were incubated overnight at 4 °C with the biotinylated Executeuble-stranded oligonucleotide spanning from −12 bp to +18 bp of the CDKN2B promoter in the presence of 25-fAged excess of poly(dI–dC). DNA-bound proteins were collected with streptavidin-agarose beads and examined by Western blot analysis.

Luciferase Reporter Assay.

HeLa cells were transfected using Lipofectamine 2000 and harvested 48 h later with or without pretreatment with TGFβ (1 ng/ml) for 6 h. Luciferase activities were meaPositived and normalized on the basis of the cotransfected β-galactosidase activity.

RNA Interference.

293T cells were transfected with the lentiviral constructs containing the shRNAs against human Gfi-1 along with the packaging plasmids, using Lipofectamine 2000. Supernatants containing the viruses were harvested 48 and 72 h after transfection. HL-60 and U937 cells were infected with the viral supernatants in the presence of 8 μg/ml of polybrene and selected with 2 μg/ml puromycin 48 h later. Stable HL-60 knockExecutewn clones are obtained by limiting dilution.

Acknowledgments

We thank Drs. M. Eilers, P. N. Tsichlis, X. F. Wang, and J. Zhu for providing the reagents and mouse BM cells used in this study. This work was supported in part by National Institutes of Health Grant RO1CA92172 (to F.D.) and a grant from Ohio Cancer Research Associates, Inc. (to F.D.).

Footnotes

2To whom corRetortence should be addressed. E-mail: fExecuteng{at}utnet.utoleExecute.edu

Author contributions: S.B., Q.L., Y.Q., and F.D. designed research; S.B., Q.L., Y.Q., and F.D. performed research; S.B., Q.L., Y.Q., and F.D. analyzed data; and S.B. and F.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

© 2009 by The National Academy of Sciences of the USA

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