Targeted transcriptional repression of Gfi1 by GFI1

Published online May 6, 2004 2508±2519 Nucleic Acids Research, 2004, Vol. 32, No. 8 DOI: 10.1093/nar/gkh570

Targeted transcriptional repression of G®1 by GFI1 and GFI1B in lymphoid cells Loretta L. Doan1,2, Susan D. Porter3, Zhijun Duan4, Marcella M. Flubacher5, Diego Montoya1,2, Philip N. Tsichlis6, Marshall Horwitz4, C. Blake Gilks3,7 and H. Leighton Grimes1,2,8,* 1

Institute for Cellular Therapeutics, 2Department of Biochemistry and Molecular Biology and 8Department of Surgery, University of Louisville, KY, USA, 3Genetic Pathology Evaluation Centre, University of British Columbia, Vancouver, BC, Canada, 4Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA, 5Molecular Oncology, Fox Chase Cancer Center, Philadelphia, PA, USA, 6Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA, USA and 7Department of Pathology, Vancouver General Hospital, BC, Canada Received February 10, 2004; Revised and Accepted April 6, 2004

ABSTRACT Growth factor independence-1 (GFI1) and GFI1B are closely related, yet differentially expressed transcriptional repressors with nearly identical DNA binding domains. GFI1 is upregulated in the earliest thymocyte precursors, while GFI1B expression is restricted to T lymphopoiesis stages coincident with activation. Transgenic expression of GFI1 potentiates T-cell activation, while forced GFI1B expression decreases activation. Both mice and humans with mutant G®1 display lymphoid abnormalities. Here we describe autoregulation of G®1 in primary mouse thymocytes and a human T-cell line. GFI1 binding to cis-element sequences conserved between rat, mouse and human G®1 mediates direct and potent transcriptional repression. In addition, dramatic regulation of G®1 can also be mediated by GFI1B. These data provide the ®rst example of a gene directly targeted by GFI1 and GFI1B. Moreover, they support a role for auto- and trans-regulation of G®1 by GFI1 and GFI1B in maintaining the normal expression patterns of G®1, and suggest that GFI1B may indirectly affect T-cell activation through repression of G®1. INTRODUCTION Growth factor independence-1 (G®1) and G®1B are two closely related oncogenes that play different but pivotal roles in hematopoiesis. G®1-de®cient mice display both thymic and peripheral T lymphopenia, with severe abnormalities in pre-T-cell development (1±3). Furthermore, mutation of G®1 in humans induces defects in T lymphocyte function and development (4). G®1B is necessary for the development of megakaryocytes and for de®nitive erythropoiesis (5). G®1B de®ciency is embryonic lethal by day E15 (5). Subsequently,

no thymic or T-cell phenotype has been reported for G®1B de®ciency; however, forced expression of GFI1B induces perturbed T-cell development and function (6). In accordance with the distinct hematopoietic phenotypes of G®1 and G®1B de®ciency, one or the other factor is predominantly expressed in hematopoietic tissues of normal adult animals (7). G®1 is highly expressed in thymus, the site of T-cell development, while G®1B is the predominant factor expressed in spleen. Both factors are expressed in bone marrow (7). The distinct physiological functions of G®1 and G®1B may seem somewhat surprising given the similarities of their de®ned biochemical functions (7,8). GFI1 and GFI1B are two members of a family of zinc-®nger transcriptional repressors that are characterized by the presence of the SNAG (found in the Snail and GFI1 family of proteins) repression domain (8). Transcriptional repression by both GFI1 and GFI1B requires an intact SNAG domain (8). Speci®cally, transient transcription assays demonstrate that mutation of proline to alanine at amino acid 2 (P2A) ablates transcriptional repression of GFI1 and GFI1B (8). Furthermore, having nearly identical zinc®nger DNA binding domains, GFI1 and GFI1B bind the same consensus DNA sequence (7,9). Despite the extensive similarities of these proteins, each contains a region unique in amino acid sequence, the activities of which are yet to be de®ned. We therefore expect that these two factors have both redundant and unique biological roles in blood cell development. GFI1 is expressed throughout T-cell development, and transgenic expression of GFI1 in T cells causes an increase in the response to activation mediated through the T-cell receptor (TCR) (6,10,11) or interleukin 2 (IL-2) (6). GFI1B is induced at stages of T lymphopoiesis that are coincident with TCR activation, and forced transgenic expression of GFI1B in T cells leads to decreased T-cell activation in both mature T cells in vitro and in thymocytes in vivo (6). While GFI1B transgenic mice display normal thymic cellularity, they share with G®1de®cient mice the characteristic of peripheral T lymphopenia. In addition, GFI1B transgenics display thymic abnormalities,

*To whom correspondence should be addressed. Tel: +1 502 852 2080; Fax: +1 502 852 2079; Email: [email protected]

Nucleic Acids Research, Vol. 32 No. 8 ã Oxford University Press 2004; all rights reserved

Nucleic Acids Research, 2004, Vol. 32, No. 8 including a defect in the formation of CD8 SP cells that can be partially corrected by simultaneous transgenic expression of GFI1 (6). Therefore, some of the observed GFI1B-mediated effects on T lymphopoiesis may result from the disruption of normal functions of GFI1 (6). The antagonistic roles of GFI1 and GFI1B in T-cell biology might have been predicted from the frequency with which these two factors are activated in Moloney Murine Leukemia Virus (MoMLV)-induced T-cell tumors. G®1 is the second most frequently activated target of MoMLV insertion mutagenesis in T-cell malignancies, leading to overexpression of wild-type G®1; however, G®1B is rarely activated (12). Because overexpression of G®1 is tumorigenic, we decided to examine the normal regulation of the G®1locus. Here we show that homologous regions of the rat, mouse and human G®1 loci contain GFI1 recognition sequences that mediate autoregulation in both primary and transformed T lymphocytes. Moreover, such binding sites may also be targeted by GFI1B to repress G®1 expression directly. MATERIALS AND METHODS Mice The GFI1 and GFI1B transgenic mice were as described previously (6). All mice were housed in the Donald Baxter Barrier Facility at the University of Louisville and used in accordance with protocols approved by the university's Institutional Animal Care and Use Committee. Plasmids Chloramphenicol acetyl transferase (CAT) reporter constructs and GFI1 and GFI1B expression plasmids used in transient transcription assays were as described previously (8). The luciferase reporters were generated by cloning the rat G®1 promoter and intron from the CAT reporter into pCBG99basic (Promega, Madison, WI). Site-directed mutagenesis was performed with Quickchange II (Stratagene, Valencia, CA). Plasmids for in vitro transcription and translation were generated by cloning rat G®1 or mouse G®1B into the pcDNA3 vector (Invitrogen, Carlsbad, CA). Retroviral vector expression constructs were generated by cloning triple Flag-epitope-tagged GFI1 into the MIEV vector (13). Transfections, transduction, and transcription assays Jurkat T cells were maintained in RPMI 1640 with 10% fetal bovine serum (FBS), 1% L-Gln and 1% Pen/Strep (all from Invitrogen). For stable transfections, Jurkat cells were electroporated as described previously (8). The transgene constructs encoding GFI1 or GFI1B were cotransfected with empty pcDNA3.1 vector DNA at a ratio of 22:1. Transfected cells were selected in the presence of 1 mg/ml Geneticin (Invitrogen) and cloned by limiting dilution. Transient transfections of Jurkat cells were performed with DMRIE-C (Invitrogen) according to the manufacturer's protocol. Brie¯y, 4 3 105 cells per well were transfected in 24-well plates. Six wells were transfected for each condition, and two wells of transfected cells were combined for a single data point. Therefore, luciferase assays were performed in triplicate. Click Beetle Luciferase assays (Promega) were performed according to the manufacturer's protocol.

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293T cells were maintained in Dulbecco's modi®ed Eagle's medium with 10% FBS, 1% L-Gln and 1% Pen/Strep (Invitrogen). Cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol with Opti-MEM low serum medium (Invitrogen) and 2.5 3 105 cells/well in 24-well plates that were pre-coated with polyL-Lysine (0.1 mg/ml; Sigma, St Louis, MO). Transfections were performed in triplicate. CAT assays were performed as described previously (8). For transduction experiments, Phoenix cells (14) were transiently transfected with retroviral constructs by means of CaPO4 (15), then co-cultured with Jurkat cells overnight. GFP+ cells were analyzed 4 days later, sorted on a FACSVantage (Beckton Dickinson) and then cloned by limiting dilution. Northern and western blot For northern blots, total RNA was extracted with Ultraspec RNA Isolation Solution (Biotecx Laboratories, Inc., Houston, TX) according to the manufacturer's protocol, and poly-A+ RNA was obtained using the Oligotex Direct mRNA puri®cation protocol (Qiagen Inc., Valencia, CA). Total RNA (20 mg) or Poly A+ RNA (5 mg) was electrophoresed in a 1% agarose-formaldehyde gel and transferred to MagnaGraph nylon membrane (Micron Separations, Inc., Westboro, MA). Membranes were probed in UltraHyb solution (Ambion, Austin, TX) according to the manufacturer's protocol. Radioactive probes were generated by Prime-a-Gene random priming kit (Promega). Western blot detection of GFI1 and GFI1B was performed as described previously (6). Nuclear extracts of U937 cells were made with NE-PER (Pierce, Rockford, IL) according to manufacturer's instruction, and protein concentration was determined using BCA Protein Assay Reagent (Pierce). Primary antibody was a commercially available antiserum to the last 20 amino acids of GFI1 (sc-6357; Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-Flag-HRP (Sigma) or our mouse monoclonal speci®c for GFI1 (2.5D.17). RNase and DNase For resolution of the rat locus, primer extension was performed with an end-labeled oligonucleotide complementary to exon 1 sequence, hybridized to 50 mg total RNA from rat Nb-2 lymphoma cells or normal rat thymus, and the reaction products were analyzed by electrophoresis on a 7% polyacrylamide/7 M urea sequencing gel. To characterize the exon±intron boundaries and genomic organization of G®1, an F344 rat genomic lDash clone (16) was digested with EcoR1 and the resulting fragments were subcloned in pBluescript SK (Stratagene). Next, oligonucleotide primers based on the G®1 cDNA sequence were used to probe Southern blots of the genomic subclones, for PCR ampli®cation and for sequencing. Cultures were harvested, and nuclei were isolated and subjected to incremental DNase I digestions as described previously (17). Electrophoretic mobility shift assays Shifts were performed essentially as described previously (18). In vitro transcription and translation (IVT) was performed with the TNT T7-coupled reticulocyte lysate system

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(Promega) according to the manufacturer's protocol. Jurkat nuclear extracts were made using a standard Dignam protocol (19). The sequences and identities of the four probes used are as follows: (i) probe B30 is a transcriptionally active, synthetic GFI1 recognition site in place of a site at ±550, with rat promoter sequence surrounding. The sequence is 5¢GGGATCGCCACACCAAATCACTGCACCCCGGT-3¢ and the mutant probe is 5¢-GGGATCGCCACACCAGGTCACTGCACCCCGGT-3¢. (ii) Probe 2 is the GFI1 binding site at ±701 of the rat promoter and surrounding promoter sequence. The sequence is 5¢-GGAGCAAACCTCTGGGATTGGGTGTCAAGGTA-3¢ and the mutant probe is 5¢-GGAGCAAACCTCAGGGACCGGGTGTCAAGGTA-3¢. (iii) Probe 4 contains two GFI1 binding sites at ±434 and ±421 of the rat promoter. The sequence is 5¢-GGTCCCGTCAATCTGTGTCCTGAATCTGTGAC-3¢, and the mutant probes are 5¢-GGTCCCGTCGGTCTGTGTCCTGAATCTGTGAC3¢ (M1), 5¢-GGTCCCGTCAATCTGTGTCCTGGGTCTGTGAC-3¢ (M2) and 5¢-GGTCCCGTCGGTCTGTGTCCTGGGTCTGTGAC-3¢ (M3). (iv) Probe I contains two GFI1 binding sites in the rat intron1 that are conserved in mouse but not human. The sequence is 5¢-GGAACCCCCAATCAGTTCACCTAATCTCGGGT-3¢, and the mutant probes are 5¢-GGAACCCCCGGTCAGTTCACCTAATCTCGGGT-3¢ (M1), 5¢-GGAACCCCCAATCAGTTCACCTGGTCTCGGGT-3¢ (M2) and 5¢-GGAACCCCCGGTCAGTTCACCTGGTCTCGGGT-3¢ (M3). Bolded nucleotides are GFI1 core recognition sequences that were changed to generate mutant probes (AATC to GGTC). The underlined bases were added for labeling with Klenow (New England Biolabs, Beverly, MA) and [a-32P]dCTP (Amersham, Piscataway, NJ). Jurkat nuclear extracts (2.5 mg) or IVT protein (3±5 ml) were pre-incubated in binding buffer (18) at room temperature for 20 min, with cold oligonucleotide or antibodies [anti-GFI1 (sc-8558) or normal goat antiserum (sc-2028)] for competition and super-shift assays. Labeled probe (50 000 c.p.m.) was added, and the reaction continued at room temperature for 30 min. Samples were electrophoresed through a 6% non-denaturing polyacrylamide gel, which was dried and exposed to ®lm. Chromatin immunoprecipitation analysis Chromatin immunoprecipitation analysis (ChIP) assays were performed as described previously (20). Antibodies used for immunoprecipitation were rabbit anti-GFI1 (Pharmingen, catalogue no. 559680) and normal rabbit control, or goat anti-GFI1 and normal goat control (sc-2027, sc-8558 and sc2028, respectively; Santa Cruz Biotechnology, Inc.). Each experiment was performed at least twice with similar results, and representative data are shown.

examine the rat G®1 locus for regions of transcriptional control. Genomic clones from the rat locus were analyzed by restriction endonuclease digestion, followed by Southern blotting with G®1 cDNA probes and sequencing. We determined both the exon±intron boundaries of the gene (Fig. 1A) and the organization of the rat G®1 locus (Fig. 1B). Speci®cally, examination of the sequence of exon±intron boundaries revealed the presence of seven exons and six introns. The exon±intron boundary sequences are homologous to the previously published mouse locus (21), with minor exceptions. The genomic organization of rat G®1 is shown as a diagram in Figure 1B and spans 9.5 kb of sequence. These data were corroborated by the recently available rat genomic sequence in DDBJ/EMBL/GenBank (data not shown). To determine potential regulatory regions, DNase hypersensitivity assays were performed (Fig. 1C) with two different genomic probes (Fig. 1B, I and II). Analysis of the G®1 locus in rat Nb2 and A2 leukemia cell lines with probe I revealed hypersensitive sites clustering around exon 1 (Fig. 1B, arrows). Many of these sites occur within the ®rst intron, but the major site of DNase hypersensitivity is just upstream of the ®rst exon (Fig. 1B, longer arrow, and C, arrow). In LE3Spl cells, two downstream hypersensitive sites were also identi®ed with probe II (Fig. 1C) and are indicated by arrows in Figure 1B. With either probe, the pattern of DNase hypersensitivity was similar with or without prolactin addition to prolactin-dependent Nb2 cells, and with or without IL-2 addition to the IL-2-dependent LE3Spl cell line (data not shown). Thus, these regulatory regions are unlikely to be growth-factor dependent. We next mapped the start site of transcription of the rat G®1 gene. The mouse G®1 locus has both a major transcription start site 5¢ of exon 1a, and a minor one in the ®rst intron (21). We therefore performed RNase protection analysis to locate the transcriptional start site(s) in the rat G®1 gene. In RNA from rat Nb2 lymphoma cells, a sequence proximal to exon 1 was identi®ed as the single start site of transcription in the rat G®1 gene (Fig. 2D). This sequence (Fig. 1D, TCAGAGC) corresponds to the consensus sequence of an initiator element (Inr) (22). Inr elements are commonly utilized as transcription start sites in TATA-less lymphoid-speci®c genes such as terminal deoxynucleotidyl transferase (23). In agreement with this, we found no evidence of a canonical TATA box in the sequence of this region. Moreover, the relative position of the Inr corresponds to the major site of DNaseI hypersensitivity. In contrast to both mouse and human G®1, which generate multiple sized transcripts, the rat locus generates a single transcript (16). Unlike murine G®1, no start sites were detected in the ®rst intron of the rat gene (data not shown). However, the rat G®1 start site is very similar to that of the major start site of the mouse gene.

Identi®cation of the G®1 promoter

Mouse and human G®1 loci contain sequences homologous to the rat G®1 promoter containing putative GFI1 binding sites

We cloned G®1 in an insertion mutagenesis screen for targets of MoMLV that could mediate progression of rat T-cell leukemias from IL-2-dependent to -independent growth (16). The identi®cation of G®1 as an oncogene in rat T-cell leukemia lines provided a biological impetus for studying the transcriptional regulation of rat G®1. We therefore sought to

The sequence around the transcription start site of the rat G®1 locus was compared with the sequences of the mouse and human G®1 loci. This analysis revealed a great deal of homology among rat, mouse and human sequences up to 808 bp upstream of the putative Inr of the rat locus (Fig. 2A, gray areas). The extent of sequence homology is unusual given

RESULTS


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that the nearest protein coding sequences are at least 1.6 kb distal, emphasizing the probable importance of this region in the transcriptional control of G®1. The Inr sequence is conserved in the mouse locus, and is located near the previously mapped start site of transcription for mouse G®1. In contrast, the Inr sequence is not conserved in the human sequences we identi®ed. However, the reported major start site of the human G®1 promoter (24) is just upstream of the sequences shown and contains a consensus Inr element according to our analysis (data not shown). Thus, mouse, rat and human G®1 are likely to have similar cis-acting transcriptional control elements. We examined the rat promoter sequence for transcription factor binding sites by computer-assisted matrix similarity analysis (MatInspector) (25). Transcription factor binding sites conserved among all three species are illustrated (Fig. 2). These include nuclear factor of activated T cells (NFAT), interferon response factor 2 (IRF2) and GATA, all of which respond to exogenous signals in T cells (26±28). Additional putative regulatory sites include recognition sequences for E4BP4/NFIL3, HoxA9, SP-1, and the Ets factors ETS1 and ELK1. Strikingly, several putative GFI1 binding sites that are conserved between mouse, rat and human loci were identi®ed (Fig. 2). Alignment of sequences in the ®rst intron of the rat locus with mouse and human G®1 revealed extensive homology in rodents and two potential GFI1 binding sites (data not shown). Endogenous G®1 is repressed by ectopic expression of GFI1 in murine primary thymocytes and in human Jurkat T cells

Figure 1. Characterization of the rat G®1 locus and identi®cation of the transcription start site. (A) Exon±intron boundaries of rat G®1. Shown are the splice donor and acceptor sites as determined by sequencing. (B) Schematic representation of the organization of the rat G®1 locus. Exons 1±7 are depicted as gray boxes. Arrows identify DNase hypersensitive sites, with the longest arrow representing the start site of transcription. Roman numerals indicate probes used for DNase hypersensitivity analysis. (C) Southern blot analysis of DNase hypersensitivity assays. Nuclei from Nb2 (lanes 1±4 of left panel), A2 (lanes 5±8 of left panel) or LE3Spl (right panel) rat cell lines were digested with increasing concentrations of DNase I, the DNA was digested with HindIII restriction endonuclease, and Southern blots were hybridized with probes I and II. l/HindIII molecular weight markers are indicated on the left of each blot. The arrow indicates the major site of hypersensitivity, corresponding to the longest arrow in (B). Minor bands represent other regions of putative transcriptional control corresponding to smaller arrows in (B). (D) RNase protection assay identi®es the start site of transcription. The left panel shows representative results of RNase protection analysis from rat Nb-2 lymphoma cells (1) and normal rat thymus (2). Arrows indicate the major protected fragments and the corresponding nucleotides on a sequencing gel. The right panel depicts the sequence of the start site and the consensus sequence of an Inr.

The presence of GFI1 recognition sequences in the G®1 promoter suggested autoregulation. We previously reported low-level expressing GFI1-transgenic mice constructed with the Lck proximal promoter, and a rat G®1 cDNA that lacks the 3¢ untranslated region (Fig. 3A). We therefore examined the levels of endogenous G®1 transcript in these GFI1-transgenic thymocytes. Northern analysis with a murine G®1 3¢untranslated-region probe on Poly-A+-selected RNA from three GFI1-transgenic mouse thymi revealed an average 40% reduction in the level of endogenous G®1 transcript (Fig. 3B). The modest effect on endogenous G®1 levels is in accord with the low level of transgene expression. As expected, western blot analysis of thymocyte whole-cell lysates revealed similar levels of overall GFI1 protein in GFI1-transgenic and littermate control thymocytes (Fig. 3C). Analysis of CD2 promoter-driven GFI1 transgenic thymocytes revealed similar results (data not shown). To determine if human G®1 could be autoregulated, we transfected a human T-cell line (Jurkat) with a selectable marker and the Lck-promoter GFI1-transgene construct (Fig. 3A), and selected stable clones. Northern analysis of Poly-A+-selected RNA with a human G®1-speci®c probe revealed profoundly reduced levels of endogenous G®1 message in multiple independent GFI1-transfected clones, two of which are shown (Fig. 3D). Western analysis con®rmed high levels of GFI1 protein in the clones compared with control Jurkat cells (Fig. 3E). To ensure that the observed repression is not the result of artifact induced by integration of the transgene or antisense generated by transgene concatemers, we repeated our analysis with independent clones of

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Figure 2. Mouse and human G®1 loci contain sequences highly homologous to the rat G®1 promoter, and contain conserved GFI1 binding sites. Sequence alignment of mouse and human G®1 loci to the rat G®1 promoter. Regions in gray represent areas of identity among all three species. Putative transcription factor binding sites and the Inr are underlined and annotated. The numbers next to GFI1 binding sites were assigned based on the EMSA probe that contains them (see Materials and Methods). Numbering of nucleotides is according to the rat sequence, with +1 as the ®rst nucleotide in the putative Inr, which is the mapped start site of transcription.

Jurkat T cells that were transduced with a GFI1-expressing retroviral vector (Fig. 3F). Again, northern analysis revealed a profound decrease in endogenous G®1 expression (Fig. 3G). Western analysis revealed increased expression of GFI1 in the transduced cell lines (Fig. 3H). Therefore, both mouse and human G®1 respond to the level of GFI1 in a manner consistent with autoregulation. GFI1 binds to the G®1 promoter in living Jurkat T cells To determine a molecular basis for the regulation of G®1 by forced GFI1 expression, we performed ChIP in Jurkat T cells. Cross-linked chromatin was immunoprecipitated with two independent antisera that are speci®c for GFI1. In templates

generated from either antiserum immunoprecipitation, products of the ampli®cation reaction were seen with primers directed to the rat/human homologous sequences in Figure 2 (Fig. 3I, top and middle panels). In contrast, no product was seen in ampli®cation reactions from control-antiserum templates (Fig. 3I, top and middle panels), emphasizing the speci®city of the immunoprecipitation. Furthermore, primers speci®c for a region of the G®1 locus that does not contain GFI1 binding sites by our analyses did not amplify a product (Fig. 3I, bottom panel). GFI1 is expressed in U937 myeloid monocytic cells and ChIP analyses have shown that it is bound to several promoters in this cell line (20). Nonetheless, primers directed to the promoter of G®1 failed to amplify a product


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Figure 3. Endogenous G®1 is repressed by ectopic expression of GFI1 in thymocytes and a T-cell line. (A) Diagram of the transgene construct used in creating GFI1 transgenic mice. The transgene vector (34) is under the control of the Lck proximal promoter and the human CD2 enhancer, ensuring expression in both developing and mature T cells. (B) Northern blot analysis of Poly-A+ RNA (5 mg) from wild type (WT) and GFI1 transgenic (GFI1) mouse thymocytes. Endogenous G®1 was detected with a probe speci®c to the 3¢ untranslated region of mouse G®1 (top panel). Blots were stripped and probed with a loading control (MDM2, lower panel). (C) Western blot analysis of GFI1 in whole-cell lysates of WT and GFI1 transgenic thymocytes. (D) Northern blot analysis of Poly-A+ RNA (5 mg) from independent Jurkat clones transfected either without (WT) or with (GFI1) the GFI1 transgene construct using a probe speci®c for human G®1 (top panel). Ethidium bromide staining of the Poly-A+ RNA is shown for equal loading (lower panel). (E) Western analysis of GFI1 expression in the same transfected Jurkat T cells. Similar results were obtained with other clones. (F) Map of FLAG-G®1-encoding (GFI1) retrovirus vector. (G) Northern blot analysis of total RNA (20 mg) from independent Jurkat T-cell clones transduced with either empty vector (WT) or a GFI1-expressing retrovirus (top panel). Ethidium bromide staining shows equal loading of RNA (bottom panel). (H) Western blot analysis with either anti-FLAG (top panel) or anti-GFI1 (bottom panel) antibody reveals relative expression levels of total and ectopic GFI1 in the same cells. (I) Chromatin immunoprecipitation analysis of wild-type Jurkat T cells with PCR primers speci®c for human G®1 sequences identi®ed in Figure 2. (Top panel) ChIP performed with GFI1-speci®c rabbit polyclonal antiserum (GFI1 Ab) or non-speci®c rabbit serum as control (Control Ab). (Middle panel) ChIP performed with GFI1-speci®c goat polyclonal antiserum (GFI1 Ab) or non-speci®c goat serum (Control Ab). (Bottom panel) ChIP performed with goat antiserum and primers speci®c for a 3¢ region of G®1. (J) ChIP performed on wild-type U937 cells with primers speci®c for human G®1 promoter sequences identi®ed in Figure 2, and goat antiserum. In (I) and (J), input = 0.005±0.01% of input chromatin.

from U937 chromatin that had been immunoprecipitated with goat anti-GFI1 antibody (Fig. 3J), and forced expression of GFI1 in U937 cells did not lead to silencing of G®1 transcription (data not shown). Therefore, in living Jurkat T cells, but not in U937 myeloid cells, GFI1 is speci®cally bound to the human G®1 sequences shown in Figure 2. GFI1 binds to sequences in the G®1 promoter in vitro The GFI1/GFI1B consensus binding site is de®ned as TAAATCAC(A/T)GCA, with an absolute requirement for the AATC core (7,9). Mutation of AATC to GGTC ablates DNA binding (7,9). To determine which of the putative sites illustrated in Figure 2 can be bound by GFI1, we designed 30 bp oligonucleotide probes for use in electrophoretic mobility shift assay (EMSA) with nuclear extracts from human Jurkat or rat NB2 cell lines (data not shown). Both extracts shifted the same probes. A synthetic GFI1 binding site that has been shown to be active in transient transcription assays (B30) (8) was included for comparison. Incubation of Jurkat nuclear extract with the B30 probe resulted in the formation of two prominent protein±DNA complexes (Fig. 4A, lane 2). As reported previously (9), excess unlabeled B30 oligonucleotide, but not excess `AATC to GGTC' mutant B30, was able to compete for binding to the radiolabeled B30 oligonucleotide (Fig. 4A, lanes 3 and 4). Several different protein±DNA complexes were seen with individual probes from the mouse/rat/human homologous G®1 sequences in Figure 2, most of which were relatively minor in intensity and did not display GFI1-speci®c interaction. For

simplicity, these minor GFI1-non-speci®c sites are not shown. However, T-cell nuclear extracts retarded the mobility of probes containing sites labeled 2 and 4 in Figure 2, and the sites in the ®rst intron (I) in a GFI1-dependent manner (Fig. 4A, lanes 6, 11 and 16). Binding of both radiolabeled probes 2 and I was competed by adding excess unlabeled self or excess unlabeled B30, but not by adding excess unlabeled `AATC to GGTC' mutant B30 (Fig. 4A, lanes 7±9 and 17±19). Surprisingly, binding of Jurkat nuclear extract to radiolabeled probe 4 was competed only by adding excess unlabeled probe 4, and not by adding either excess unlabeled B30 or mutant B30 (Fig. 4A, lanes 12±14). We therefore conclude that the probes bind to three distinct protein complexes, two of which recognize sequences in the B30 probe, the other of which does not. To determine whether these complexes contain GFI1, we performed supershift and competition analysis on each of the three probes (2, 4 and I). Figure 4B shows the results of these analyses performed with probe 2, which contains a single GFI1 binding site corresponding to ±701 of the rat promoter (relative to the Inr). This probe shifts in a doublet pattern in the presence of Jurkat nuclear extract (lane 2), and the shift was ablated when the nuclear extract was pre-incubated with antiGFI1 antibody (lane 3), whereas a control antibody had no effect (lane 4). Furthermore, while binding was competed away in the presence of excess cold probe 2 (lane 5), there was no competition in the presence of excess cold mutant 2 (lane 6). When the mutant probe 2 was radiolabeled and used alone, the doublet was not seen (lane 7). To con®rm that GFI1

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Figure 4. GFI1 binds directly to sequences of the rat G®1 promoter containing conserved GFI1/GFI1B binding sites. (A) EMSA analysis of a probe containing a control GFI1 consensus site and three probes containing putative GFI1 recognition sequences in regulatory regions of rat G®1. NE = Jurkat nuclear extract; B = probe B30 (synthetic GFI1 binding site); 2 = probe 2 (GFI1 binding site 2 annotated in Fig. 2); 4 = probe 4 (containing two GFI1 binding sites annotated as 4a and 4b in Fig. 2); I = intronic probe (containing two GFI1 binding sites in the ®rst intron of rat G®1); M = mutant probe B30 (AATC core of the GFI1 binding site mutated to GGTC). (B) Supershift and cold competition analysis of probe 2. NE = Jurkat nuclear extract; IVTGFI1 = in vitrotranscribed and -translated FLAG-epitope-tagged GFI1; G = anti-GFI1 antibody; F = anti-FLAG antibody; C = control IgG for anti-GFI1 or anti-FLAG; M = mutant probe 2 (AATC®GGTC). (C) Supershift and cold competition analysis of probe 4. NE, IVTGFI1, G, F and C are as de®ned in (B). M1 = probe 4 mutant 1 (site 4a mutated from AATC®GGTC); M2 = probe 4 mutant 2 (site 4b AATC®GGTC); M3 = probe 4 mutant 3 (4a and 4b AATC®GGTC). (D) Supershift and cold competition analysis of the intronic probe. All abbreviations are as described for probe 4, with each of two GFI1 binding sites being mutated individually (M1 and M2) or simultaneously (M3).

binds directly to probe 2, we performed EMSA with IVT FLAG-epitope-tagged GFI1 (Fig. 4B), or DNA-bindingde®cient GFI1 (data not shown). IVT GFI1 formed a complex with probe 2 that migrated at a signi®cantly higher rate than

that of complexes formed with Jurkat nuclear extract (compare lanes 2 and 9). Furthermore, pre-incubation of IVT GFI1 with anti-FLAG antibody resulted in the formation of a supershift, while a control antibody had no effect (lanes 10 and 11,

Nucleic Acids Research, 2004, Vol. 32, No. 8 respectively). DNA-binding-defective IVT GFI1 did not form a complex with probe 2 (data not shown). Taken together, these data suggest that GFI1 in Jurkat nuclear extract binds directly to probe 2 in the context of a larger complex, rather than as a monomer. The same analysis was performed with probe 4, which contains two GFI1 binding sites at ±434 and ±421 of the rat promoter. Pre-incubation of Jurkat nuclear extract with antiGFI1 antibody abated complex formation, whereas a control antibody did not (Fig. 4C, lanes 3 and 4), and pre-incubation of IVT GFI1 with anti-FLAG antibody resulted in a supershift, whereas a control antibody did not (lanes 14 and 15). These results are similar to those observed with probe 2. Further analysis revealed that only one of the two GFI1 binding sites is necessary for GFI1 from Jurkat nuclear extract to bind to probe 4. Each site was mutated independently (4a mutant = M1 and 4b mutant = M2) or both sites were mutated together (M3), and these mutants were used in competition analyses. Both wild-type and M2 oligonucleotides effectively competed away the complex (lanes 5 and 7), whereas M1 and M3 had no effect (lanes 6 and 8). Furthermore, when the mutant oligonucleotides were labeled and used as probes, M1 and M3 did not form a complex, while M2 formed the same complex as the wild-type probe 4. These analyses clearly demonstrate that only site 4a binds to GFI1 in Jurkat nuclear extract, and that site 4b is dispensable for this binding. The same analysis was performed with probe I, which contains two GFI1 binding sites from the ®rst intron of rat G®1 (Fig. 4D). These sequences are conserved between mouse and rat genes. The results of this analysis were similar to those obtained with probe 4. Pre-incubation with antibodies showed ablation of binding to Jurkat nuclear extract and supershift of IVT GFI1 (lanes 3 and 14, respectively), and competition analysis showed that only one of two putative GFI1 binding sites is necessary for the formation of complexes (lanes 5±11). We therefore conclude that GFI1 in protein complexes can bind to cognate binding sites that are present in mouse/rat/ human conserved sequences shown in Figure 2, and in the ®rst intron that is conserved in rodent genomes. GFI1 directly represses G®1 To determine if the GFI1-binding sites in Figure 4 mediate GFI1-responsive transcriptional repression, we performed transient transcription analyses. We constructed a CAT reporter driven by the rat G®1 promoter and ®rst intron (±808 bp up to the beginning of exon 2), containing all of the DNase hypersensitive sites identi®ed in the 5¢ end of the gene (Fig. 1). Co-transfection of 293T cells with this reporter and a GFI1-expression construct resulted in repression of reporter activity (Fig. 5A). However, co-transfection with a plasmid encoding a SNAG-repression-domain mutant of GFI1 (P2A) had no effect (Fig. 5A). Furthermore, co-transfection of this reporter with increasing amounts of a plasmid encoding a fusion protein that consists of the herpes simplex virus VP16 transactivator and the zinc ®ngers of GFI1 results in dosedependent activation of the G®1 reporter (Fig. 5B). Therefore, the region of rat G®1 containing the proximal promoter and ®rst intron is capable of binding and being repressed by GFI1 in 293T cells. To permit similar experiments in Jurkat T cells, we changed to a luciferase reporter. We constructed a Click Beetle

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Luciferase reporter with the rat G®1 sequences (±808 bp up to the beginning of exon 2). Co-transfection of this reporter with an expression plasmid encoding GFI1 resulted in a decrease of reporter activity to an average of 55% of control. We next examined the requirement for sites 2, 4 and I, which bound GFI1-containing complexes during EMSA (Fig. 4). Surprisingly, mutation of either site 2 or site 4 individually had little or no effect on GFI1 repression. In contrast, mutation of both sites 2 and 4 decreased the GFI1-mediated repression of the reporter, such that the activity was an average of 75% of the control. Furthermore, mutation of GFI1 binding site I also decreased GFI1-mediated repression to a level comparable to that seen in the site 2 and 4 double mutant. Finally, mutation of GFI1 binding sites 2, 4 and I resulted in a complete lack of exogenous GFI1-mediated repression of this luciferase reporter. We conclude that promoter or intronic sites are suf®cient for GFI1-mediated repression, but all sites are necessary for ef®cient GFI1-mediated repression. GFI1B binds directly to the G®1 promoter and represses G®1 in T cells Transgenic expression of GFI1B (Fig. 6A) engenders defects in T lymphopoiesis, some of which can be rescued by simultaneous forced expression of GFI1 (6). Because GFI1 and GFI1B have nearly identical DNA binding domains (7), we previously reasoned that GFI1B may alter T lymphopoiesis by competing with endogenous GFI1 for DNA binding on speci®c promoters (6). We therefore sought to determine the extent to which transgenic expression of GFI1B disturbs the stoichiometry of GFI1/GFI1B in thymocytes. Dramatically, northern (Fig. 6B) and western (Fig. 6C) analysis of GFI1Btransgenic thymocytes revealed undetectable levels of GFI1. Thus, instead of competing with GFI1 for binding sites of target genes, transgenic GFI1B may occupy all of these binding sites in the absence of GFI1. We next determined whether GFI1B directly represses G®1 in a manner similar to GFI1. First, we examined Jurkat T cells cotransfected with the GFI1B transgene construct and a selectable marker. Northern analysis of independent clones revealed that ectopic expression of GFI1B in Jurkat cells resulted in repression of endogenous G®1 (Fig. 6D). Notably, higher levels of GFI1B protein correlated with lower levels of G®1 message (Fig. 6E). Next, we examined the ability of GFI1B to bind sites 2, 4 and I. In fact, IVT GFI1B binds ef®ciently to all three probes, as shown by EMSA analysis with supershift (Fig. 6F). Moreover, like GFI1, GFI1B expression constructs are capable of repressing the G®1CAT reporter in 293T cells (Fig. 6G) and in Jurkat T cells (Fig. 6H). Finally, this repression is abrogated upon mutation of the GFI1 binding sites 2, 4 and I (Fig. 6H). Therefore GFI1B may also regulate G®1 through the same sequences used by GFI1. DISCUSSION GFI1 is a transcriptional repressor protein that plays important biological roles in hematopoietic and neuronal cell development (1±3,29). G®1 was originally identi®ed in a screen for genes that, upon deregulated expression by MoMLV insertion, engender progression of T-cell leukemias to IL-2-independent growth (16). While the frequency of MoMLV insertion in the

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Figure 5. GFI1-mediated repression of rat G®1 promoter in Jurkat T cells is dependent upon speci®c GFI1 binding sites. (A) The G®1-promoter-driven CAT reporter construct was cotransfected into 293T cells with CMV14 expression vector as control (CMV), or CMV14 expression plasmids encoding GFI1 or the SNAG-repression-domain mutant P2A-GFI1. Transfection ef®ciency was controlled by cotransfection of a betagalactosidase expression construct. Relative CAT activity was derived by normalization to betagalactosidase levels within individual transfections. The plot represents the averaged normalized CAT activity 6 SEM from triplicate transfections. Similar results were obtained in three independent experiments. (B) The VP16-Zn mutant encodes the herpes simplex virus transcriptional activator VP16 fused to the zinc ®ngers of GFI1. VP16-Zn activates transcription of GFI1/GFI1B target genes (7). The G®1-promoter CAT reporter was cotransfected with CMV5 expression vector, or a CMV5 expression plasmid encoding VP16-Zn. Normalization was performed as in (A). Fold activation was derived by dividing the normalized CAT activity in the presence of VP16-Zn to CMV5 vector controls. (C) Rat G®1-promoter driven luciferase construct, or the same construct with speci®c AATC®GGTC mutations in GFI1 binding sites, was co-transfected into Jurkat T cells with either CMV14 expression plasmid or CMV14 expressing GFI1. A control luciferase plasmid was also transfected, and experimental values were normalized to the control as per the manufacturer's protocol (Promega Click Beetle Dual Luciferase assay). 2, 4 and I represent sites identi®ed in EMSA analysis as GFI1-binding sites. X indicates mutation of the site from AATC®GGTC.

G®1 locus indicates some selective advantage of forced GFI1 expression (12), validation of the oncogenic potential of GFI1 came from studies in transgenic mice. Transgenic overexpression of GFI1 is poorly oncogenic alone, but potently collaborates with either MoMLV infection or transgenic expression of MYC or PIM oncoproteins to cause leukemia (30). Therefore, cellular context may determine GFI1 oncogenic function. These data implicate GFI1 in T-cell biology. Indeed, mutation of G®1in mice and humans leads to lymphopenia (1,3,4). Since G®1 expression is critical for normal hematopoiesis, and overcoming regulatory control of G®1 accelerates oncogenesis, the transcriptional control of

G®1 is of great interest. Here we have shown that GFI1, and the closely related GFI1B, both repress transcription of G®1. This is the ®rst example of a gene targeted directly by GFI1, and the ®rst example of a gene targeted by both GFI1 and GFI1B. We present several lines of evidence supporting a direct mechanism of G®1 repression by GFI1. First, GFI1 binding sites in the rat G®1 promoter are conserved in the mouse and human G®1 loci. The conservation of these sequences is striking because the nearest coding sequence is at least 1.6 kb 3¢ of the Inr. Secondly, in primary thymocytes, transgenic expression of GFI1 correlates with lower levels of endogenous


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Figure 6. GFI1B represses G®1 and can bind to con®rmed GFI1-binding sites. (A) Diagram of the transgene construct used in creating GFI1B transgenic mice. The transgene vector (34) is identical to that utilized to create GFI1 transgenic animals. (B) Northern blot analysis of Poly-A+ RNA (5 mg) from wildtype (WT) and GFI1B transgenic (GFI1B) mouse thymocytes. Endogenous G®1 was detected with a probe speci®c to the 3¢ untranslated region of mouse G®1 (top panel). Blots were stripped and probed with a loading control (MDM2) (lower panel). (C) Western analysis of wild type (WT) and GFI1B transgenic thymocyte whole-cell lysates with an antibody against the last 20 amino acids of GFI1 that is cross-reactive to GFI1B (sc-6375). (D) Northern blot analysis (top panel) of 5 mg of Poly-A+ RNA from Jurkat T cells stably transfected without (WT) or with (GFI1B) the GFI1B transgene using a probe speci®c for human G®1. Ethidium bromide staining is shown to con®rm equal loading of RNA (center panel). (E) Western analysis of GFI1B levels in the same Jurkat clones. (F) EMSA analysis of sites 2, 4 and I with Myc-epitope-tagged IVTGFI1B. M = anti-MYC antibody; C = control IgG. (G) Transient transcription assay with cotransfection of either GFI1B-encoding (GFI1B), or control empty expression construct (CMV) and the G®1-CAT reporter in 293T cells. (H) Transient transcription assay with cotransfection of either GFI1B-encoding (GFI1B), or control empty expression construct (CMV) and the G®1-luciferase reporter (wild-type = top, mutant for sites 2, 4 and I = bottom). X indicates AATC®GGTC mutant.

G®1 steady-state mRNA levels. Thirdly, in the human Jurkat T-cell line, forced overexpression of GFI1, either by the Lck promoter or retroviral LTR, correlates with reduced endogenous G®1 steady-state mRNA levels. Fourthly, in EMSA analysis, in vitro-transcribed/-translated GFI1 and endogenous GFI1-containing nuclear complexes bind directly to sequences in G®1. Fifthly, mutation of these binding sites in reporter constructs ablates transcriptional response to GFI1 expression. Finally, ChIP analyses with two different antisera indicate that GFI1 binds speci®cally to mouse/rat/human conserved sequences in living Jurkat T cells. These in vitro and in vivo analyses provide strong support for the hypothesis that G®1 is autoregulated. In fact, G®1 autoregulation is

evolutionarily conserved, as pag-3, the Caenorhabditis elegans ortholog of G®1, is autoregulated (31). GFI1 binds to mouse/rat/human conserved sequences in living Jurkat T cells, but not in U937 myeloid cells. Interestingly, G®1±/± mice, which completely lack GFI1, have severe lymphopenia and profound neutropenia (1±3), while humans that carry a heterozygous dominant-negative mutation in G®1 display severe congenital neutropenia (SCN), but only mild lymphopenia (4). It is possible that expression of mutant GFI1 proteins in SCN patient T cells might lead to derepression of the wild-type G®1 allele, providing compensatory expression of wild-type GFI1 and suf®cient GFI1 activity in T cells. In contrast, the myeloid progenitor cell line U937

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does not appear to exhibit G®1 autoregulation. If similar myeloid cells in SCN patients do not have compensation by the wild-type G®1 allele, then these cells would effectively lose most or all GFI1 function. This possibility warrants future investigation to determine if cell-type speci®c G®1 autoregulation provides an explanation for the differences in phenotype observed in G®1±/± mice when compared with SCN patients with a mutation in a single allele of G®1. GFI1B can control G®1 expression. The abilities of GFI1 and GFI1B to repress endogenous G®1 seem comparable. Similar expression levels of the two factors in stably transfected Jurkat T cells resulted in similar repression. Both factors bound to the same DNA binding sites in EMSA analyses. Co-transfection of equal amounts of GFI1 and GFI1B expression plasmids with a G®1 promoter-driven reporter resulted in nearly equal levels of repression in 293T cells, and the repression was dependent on the same binding sites in Jurkat T cells. Thus, GFI1B is capable of regulating G®1. The repression of G®1 by GFI1B potentially provides new insight into the transient expression of GFI1B in thymocytes that have been recently signaled for positive selection. Namely, GFI1B may be present to temporally repress G®1. Indeed, the expression of GFI1B during beta selection is followed by a decrease in GFI1 expression (32). In contrast, forced expression of GFI1 in immature double-positive thymocytes has been shown to be detrimental, resulting in a block to beta selection (32). The coincident expression of GFI1B at this stage of development may be necessary to maintain appropriate levels of G®1, thereby allowing the progression through this critical checkpoint. GFI1B antagonism to GFI1-mediated T-cell activation may be understood as follows. First, in normal thymocytes the expression level of GFI1B is suf®ciently low such that it does not completely repress G®1, and both GFI1 and GFI1B are expressed. This simultaneous expression results in competition for DNA-binding sites on target genes, which would allow proper signaling and selection of developing thymocytes. However, transgenic expression of GFI1B disrupts GFI1/GFI1B stoichiometry and results in the complete repression of G®1 transcription. Consequently, common target genes are occupied by GFI1B. Since GFI1 increases T-cell activation (6,10,11), GFI1B expression may indirectly inhibit T-cell activation through the repression of G®1. Additionally, GFI1B may have effects distinct from those of GFI1 on at least some target genes, as presumed occupancy by GFI1B does not necessarily result in the same effects as occupancy by GFI1. The demonstration of GFI1 and GFI1B regulation of G®1, together with promoter analyses, suggests a very complex regulatory network controlling the expression of G®1. The extent of sequence homology between the rat G®1 promoter and murine and human G®1 loci emphasizes the probable importance of this region in the transcriptional control of G®1. Indeed, C.elegans and Caenorhabditis briggsae display extensive sequence homology in the promoter regions for the G®1 ortholog pag-3, even though they diverged 20±50 million years ago (33). Thus, the GFI family may be conserved signal transduction proteins that mediate pathways initiated by other transcription factors. Computer-assisted matrix similarity analysis (MatInspector) (25) of the rat/mouse/human homologous sequences in Figure 2 suggests that such factors

may include NFAT, IRF2, GATA proteins, E4BP4/NFIL3, HoxA9, SP-1 and Ets proteins. Thus, in GFI1-expressing cells, a balance between positive regulators of G®1 expression, autoregulation and GFI1B repression should determine total G®1 levels. Future work should be directed towards identifying positive regulators of G®1 expression. ACKNOWLEDGEMENTS The authors would like to thank Stuart Orkin for providing mouse genomic G®1 clones for sequencing, and Morgan Jeffries for help with EMSA analyses. The authors are also grateful for excellent technical assistance from Rachel Rivoli and Natalie Claudio. This work was supported by Hope Street Kids, and in part by the Commonwealth of Kentucky Research Challenge Trust Fund and the Jewish Hospital Foundation. L.L.D. was supported by a National Science Foundation Graduate Research Fellowship. This work was supported in part by NIH PHS CA56110 (to P.N.T.), NIH PHS DK55820, NIH DK58161 and Borroughs-Welcome Fund SATR-1002189 (to M.H.) REFERENCES 1. Yucel,R., Karsunky,H., Klein-Hitpass,L. and Moroy,T. (2003) The transcriptional repressor G®1 affects development of early, uncommitted c-Kit+ T cell progenitors and CD4/CD8 lineage decision in the thymus. J. Exp. Med., 197, 831±844. 2. Karsunky,H., Zeng,H., Schmidt,T., Zevnik,B., Kluge,R., Schmid,K.W., Duhrsen,U. and Moroy,T. (2002) In¯ammatory reactions and severe neutropenia in mice lacking the transcriptional repressor G®1. Nature Genet., 30, 295±300. 3. Hock,H., Hamblen,M.J., Rooke,H.M., Traver,D., Bronson,R.T., Cameron,S. and Orkin,S.H. (2003) Intrinsic requirement for zinc ®nger transcription factor g®-1 in neutrophil differentiation. Immunity, 18, 109±120. 4. Person,R.E., Li,F.Q., Duan,Z., Benson,K.F., Wechsler,J., Papadaki,H.A., Eliopoulos,G., Kaufman,C.L., Bertolone,S.J., Nakamoto,B. et al. (2003) G®1 proto-oncogene mutation causes human neutropenia and targets neutrophil elastase. Nature Genet., 34, 308±312. 5. Saleque,S., Cameron,S. and Orkin,S.H. (2002) The zinc-®nger protooncogene G®-1b is essential for development of the erythroid and megakaryocytic lineages. Genes Dev., 16, 301±306. 6. Doan,L.L., Kitay,M.K., Yu,Q., Singer,A., Herblot,S., Hoang,T., Bear,S.E., Morse,H.C., III, Tsichlis,P.N. and Grimes,H.L. (2003) Growth factor independence-1B expression leads to defects in T cell activation, IL-7 receptor alpha expression, and T cell lineage commitment. J. Immunol., 170, 2356±2366. 7. Tong,B., Grimes,H.L., Yang,T.Y., Bear,S.E., Qin,Z., Du,K., ElDeiry,W.S. and Tsichlis,P.N. (1998) The G®-1B proto-oncoprotein represses p21WAF1 and inhibits myeloid cell differentiation. Mol. Cell. Biol., 18, 2462±2473. 8. Grimes,H.L., Chan,T.O., Zweidler-McKay,P.A., Tong,B. and Tsichlis,P.N. (1996) The G®-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal. Mol. Cell. Biol., 16, 6263±6272. 9. Zweidler-McKay,P.A., Grimes,H.L., Flubacher,M.M. and Tsichlis,P.N. (1996) G®-1 encodes a nuclear zinc ®nger protein that binds DNA and functions as a transcriptional repressor. Mol. Cell. Biol., 16, 4024±4034. 10. Rodel,B., Tavassoli,K., Karsunky,H., Schmidt,T., Bachmann,M., Schaper,F., Heinrich,P., Shuai,K., Elsasser,H.P. and Moroy,T. (2000) The zinc ®nger protein G®-1 can enhance STAT3 signaling by interacting with the STAT3 inhibitor PIAS3. EMBO J., 19, 5845±5855. 11. Karsunky,H., Mende,I., Schmidt,T. and Moroy,T. (2002) High levels of the onco-protein G®-1 accelerate T-cell proliferation and inhibit activation induced T-cell death in Jurkat T-cells. Oncogene, 21, 1571±1579.

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