Transcriptional Repressor Gfi1 Research Articles

The orphan nuclear receptor Ear-2 (Nr2f6) is a novel negative regulator of T cell development

We describe a novel role for the orphan nuclear receptor Ear-2 in regulating T cell development. Retrovirus-mediated overexpression of Ear-2 (EAR-2++) in a bone marrow (BM) transplantation assay resulted in limited T cell development and a greater than tenfold decrease in thymus size and cellularity relative to controls. Ear-2-transduced murine BM hematopoietic stem cells (HSCs) in OP9-DL1 cultures showed a proliferation deficit during days 1-5 after induction of differentiation, which corresponded to increased expression of the cell cycle regulators p21 (cdkn1a) and p27 (cdkn1b), as well as increased expression of Hes1, Notch3, Egr1, and Scl (Tal1) and decreased expression of Gli1, Gfi-1, HoxA9, PU.1, Nrarp, and Tcf1. In addition, there was a block in differentiation at the DN4 to double-positive (DP) transition accompanied by an increase in apoptosis, similar to the deficit seen in the RORγt null mouse. Gene expression profiling revealed that, like the RORγt-deficient mouse, EAR-2++ DP cells had decreased expression of BclXL and increased expression of the proapoptosis gene Bad. In addition, EAR-2++ DP cells had decreased expression of Bcl11b, PU.1, and HoxA9, and increased expression of Id2. Based on these findings, we conclude that EAR-2++ cells were able to migrate to, but not fully repopulate, the thymus because of a cell-intrinsic defect in the proliferation of DN1 cells followed by a block in differentiation from the DN4 to DP stage of T cell development. We conclude that Ear-2 is a novel negative regulator of T-cell development and that downregulation of Ear-2 is indispensable for the proliferation of DN1 cells and the survival of DN4-DP cells.

Inhibitory role of the transcription repressor Gfi1 in the generation of thymus-derived regulatory T cells

Foxp3(+) regulatory T (T(reg)) cells are essential for the maintenance of self-tolerance and immune homeostasis. The majority of T(reg) cells is generated in the thymus as a specific subset of CD4(+) T cells, known as thymus-derived or natural T(reg) (nT(reg)) cells, in response to signals from T-cell receptors, costimulatory molecules, and cytokines. Recent studies have identified intracellular signaling and transcriptional pathways that link these signals to Foxp3 induction, but how the production of these extrinsic factors is controlled remains poorly understood. Here, we report that the transcription repressor growth factor independent 1 (Gfi1) has a key inhibitory role in the generation of nT(reg) cells by a noncell-autonomous mechanism. T cell-specific deletion of Gfi1 results in aberrant expansion of thymic nT(reg) cells and increased production of cytokines. In particular, IL-2 overproduction plays an important role in driving the expansion of nT(reg) cells. In contrast, although Gfi1 deficiency elevated thymocyte apoptosis, Gfi1 repressed nT(reg) generation independently of its prosurvival effect. Consistent with an inhibitory role of Gfi1 in this process, loss of Gfi1 dampens antitumor immunity. These data point to a previously unrecognized extrinsic control mechanism that negatively shapes thymic generation of nT(reg) cells.

Gfi1 as a regulator of p53 and a therapeutic target for ALL

The transcriptional repressor Gfi1 can be a so-called "oncorequisite" factor that is required for the development and maintenance of lymphoid neoplasia, such as Acute Lymphoblastic Leukemia (ALL), but does not have a direct role in the ontogeny of the disease. The study supporting this role of Gfi1 (Khandanpour C, Phelan J, et al., Cancer Cell, 2013, 23:200-214) shows that inhibition of Gfi1 cannot only cure mice from ALL but also blocks the expansion of human primary ALL cells. The study concludes that this feature of Gfi1 can be exploited to improve current ALL therapies.

Lymphoid Progenitor Cells from Childhood Acute Lymphoblastic Leukemia Are Functionally Deficient and Express High Levels of the Transcriptional Repressor Gfi-1

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34+ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

CD8 Lineage-specific Regulation of Interleukin-7 Receptor Expression by the Transcriptional Repressor Gfi1

Interleukin-7 receptor α (IL-7Rα) is essential for T cell survival and differentiation. Glucocorticoids are potent enhancers of IL-7Rα expression with diverse roles in T cell biology. Here we identify the transcriptional repressor, growth factor independent-1 (Gfi1), as a novel intermediary in glucocorticoid-induced IL-7Rα up-regulation. We found Gfi1 to be a major inhibitory target of dexamethasone by microarray expression profiling of 3B4.15 T-hybridoma cells. Concordantly, retroviral transduction of Gfi1 significantly blunted IL-7Rα up-regulation by dexamethasone. To further assess the role of Gfi1 in vivo, we generated bacterial artificial chromosome (BAC) transgenic mice, in which a modified Il7r locus expresses GFP to report Il7r gene transcription. By introducing this BAC reporter transgene into either Gfi1-deficient or Gfi1-transgenic mice, we document in vivo that IL-7Rα transcription is up-regulated in the absence of Gfi1 and down-regulated when Gfi1 is overexpressed. Strikingly, the in vivo regulatory role of Gfi1 was specific for CD8(+), and not CD4(+) T cells or immature thymocytes. These results identify Gfi1 as a specific transcriptional repressor of the Il7r gene in CD8 T lymphocytes in vivo.

Gfi-1 inhibits the expression of eosinophil major basic protein (MBP) during G-CSF-induced neutrophilic differentiation

The zinc finger transcriptional repressor Gfi-1 has been shown to play a critical role in early granulopoiesis; however, its role in late neutrophilic development is poorly understood. We report here that forced expression of a dominant negative Gfi-1 mutant, N382S, resulted in augmented mRNA levels of eosinophil major basic protein (MBP) in myeloid cells induced with G-CSF to undergo terminal neutrophilic differentiation. MBP is a cytotoxic protein that is abundantly expressed in eosinophils, but not in neutrophils. Ectopic expression of MBP inhibited the proliferation and survival of differentiating myeloid cells in response to G-CSF. Significantly, while GFI-1 is upregulated during neutrophilic differentiation, it is rapidly downregulated upon induction of eosinophilic differentiation, which was associated with increased MBP expression. Knockdown of GFI-1 in eosinophilic cells also led to increased level of MBP mRNA. These results indicate that Gfi-1 functions to inhibit the expression of MBP and aberrant expression of MBP as a result of loss of Gfi-1 function may cause premature apoptosis of differentiating neutrophils. In contrast, the rapid downregulation of Gfi-1 during eosinophilic development may allow for abundant expression of MBP, a hallmark of eosinophilic differentiation.

Gfi-1 inhibits proliferation and colony formation of p210BCR/ABL-expressing cells via transcriptional repression of STAT 5 and Mcl-1

Expression of the transcription repressor Gfi-1 is required for the maintenance of murine hematopoietic stem cells. In human cells, ectopic expression of Gfi-1 inhibits and RNA interference-mediated Gfi-1 downregulation enhances proliferation and colony formation of p210BCR/ABL expressing cells. To investigate the molecular mechanisms that may explain the effects of perturbing Gfi-1 expression in human cells, Gfi-1-regulated genes were identified by microarray analysis in K562 cells expressing the tamoxifen-regulated Gfi-1-ER protein. STAT 5B and Mcl-1, two genes important for the proliferation and survival of hematopoietic stem cells, were identified as direct and functionally relevant Gfi-1 targets in p210BCR/ABL-transformed cells because: (i) their expression and promoter activity was repressed by Gfi-1 and (ii) when constitutively expressed blocked the proliferation and colony formation inhibitory effects of Gfi-1. Consistent with these findings, genetic or pharmacological inhibition of STAT 5 and/or Mcl-1 markedly suppressed proliferation and colony formation of K562 and CD34+ chronic myelogenous leukemia (CML) cells. Together, these studies suggest that the Gfi-1STAT 5B/Mcl-1 regulatory pathway identified here can be modulated to suppress the proliferation and survival of p210BCR/ABL-transformed cells including CD34+ CML cells.

Gfi1 expressed in bone marrow stromal cells is a novel osteoblast suppressor in patients with multiple myeloma bone disease

AbstractProtracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti–TNF-α and anti–IL-7 antibodies. Importantly, BMSCs isolated from Gfi1−/− mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.

GABP transcription factor is required for myeloid differentiation, in part, through its control of Gfi-1 expression

AbstractGABP is an ets transcription factor that regulates genes that are required for myeloid differentiation. The tetrameric GABP complex includes GABPα, which binds DNA via its ets domain, and GABPβ, which contains the transcription activation domain. To examine the role of GABP in myeloid differentiation, we generated mice in which Gabpa can be conditionally deleted in hematopoietic tissues. Gabpa knockout mice rapidly lost myeloid cells, and residual myeloid cells were dysplastic and immunophenotypically abnormal. Bone marrow transplantation demonstrated that Gabpα null cells could not contribute to the myeloid compartment because of cell intrinsic defects. Disruption of Gabpa was associated with a marked reduction in myeloid progenitor cells, and Gabpα null myeloid cells express reduced levels of the transcriptional repressor, Gfi-1. Gabp bound and activated the Gfi1 promoter, and transduction of Gabpa knockout bone marrow with Gfi1 partially rescued defects in myeloid colony formation and myeloid differentiation. We conclude that Gabp is required for myeloid differentiation due, in part, to its regulation of the tran-scriptional repressor Gfi-1.

Genetic Defects in Severe Congenital Neutropenia: Emerging Insights into Life and Death of Human Neutrophil Granulocytes

The discovery of genetic defects causing congenital neutropenia has illuminated mechanisms controlling differentiation, circulation, and decay of neutrophil granulocytes. Deficiency of the mitochondrial proteins HAX1 and AK2 cause premature apoptosis of myeloid progenitor cells associated with dissipation of the mitochondrial membrane potential, whereas mutations in ELA2/ELANE and G6PC3 are associated with signs of increased endoplasmic reticulum stress. Mutations in the transcriptional repressor GFI1 and the cytoskeletal regulator WASP also lead to defective neutrophil production. This unexpected diversity of factors suggests that multiple pathways are involved in the pathogenesis of congenital neutropenia.

The p150 subunit of the histone chaperone Caf-1 interacts with the transcriptional repressor Gfi1

Modification of histones is critically involved in regulating chromatin structure and gene expression. The zinc finger protein Gfi1 silences transcription by recruiting a complex of histone modifying enzymes such as LSD-1/CoRest and HDAC-1 to target gene promoters. Here we present evidence that Gfi1 forms a complex with the p150 subunit of the histone chaperone chromatin assembly factor-1 (Caf-1). Gfi1 and p150 interact at endogenous expression levels and co-localize in distinct sub-nuclear structures. We show that p150 enhances Gfi1-mediated transcriptional repression and that it occupies Gfi1 target gene promoters in transfected cells and primary murine T cells only in the presence of Gfi1. Finally, size exclusion chromatography shows a fraction of p150 to coelute with Gfi1, LSD-1 and HDAC-1 and thus provides evidence that p150 is part of the Gfi1 repression complex. Since p150 binds directly to histones H3 and H4, our findings suggest that p150 may link the DNA-bound Gfi1 repressor complex to histones enabling modifications required for transcriptional silencing.

Expression of the Transcriptional Repressor Gfi-1 Is Regulated by C/EBPα and Is Involved in Its Proliferation and Colony Formation–Inhibitory Effects in p210BCR/ABL-Expressing Cells

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1, but not of a transcriptional repressor mutant (Gfi-1P2A), inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells.

Cell Lineage metastability in Gfi1-deficient mouse intestinal epithelium

Elucidating the mechanisms determining multipotent progenitor cell fate remains a fundamental project of contemporary biology. Various tissues of mice and men with defects in the zinc-finger transcriptional repressor Gfi1 have dramatic perturbations in the proportions of their differentiated cell types. In Gfi1-deficient intestinal epithelium there is a shift from mucous and Paneth towards enteroendocrine cells, leading to the proposal that Gfi1 functions in the allocation of the progeny derived from a hypothetical common granulocytic progenitor. However, studies of clones have yielded no evidence of such a common progenitor prompting us to investigate alternate mechanisms explaining the Gfi1-deficient phenotype. We report that mucous and Paneth but not enteroendocrine lineage cells normally express Gfi1. Sporadic mucous and Paneth lineage cells in the crypts of Gfi1-deficient mice aberrantly express the pro-enteroendocrine transcription factor Neurog3, indicating that stable repression of Neurog3 in these lineages requires Gfi1. Importantly, we also find mucous and Paneth lineage cells in various stages of cellular reprogramming into the enteroendocrine lineage in Gfi1-deficient mice. We propose that mucous and Paneth cell lineage metastability, rather than reallocation at the level of a hypothetical common granulocytic progenitor, is responsible for the shifts in cell type proportions observed in Gfi1-deficient intestinal epithelium.

Abstract 4731: Resequencing of the MYC-335 enhancer in the colorectal cancer associated 8q24 region

Abstract Genome-wide association studies have identified multiple different regions at 8q24 associated with risk of colorectal, prostate, breast and bladder cancers. It has been suggested that the cancer predisposition could be mediated through altered MYC expression. One of the regions, marked by SNP rs6983267, has been consistently shown to confer susceptibility to colorectal and prostate cancers. In our previous work, we aimed to understand the biological basis of the colorectal cancer (CRC) predisposition associated with the G allele of rs6983267. By utilizing a computer program, Enhancer Element Locator, we showed that rs6983267 affects a TCF4 binding site in an evolutionary conserved enhancer element (MYC-335). Enhancer activity of this element was confirmed in vitro and in vivo. The G allele of rs6983267 was shown to enhance the activity of the MYC-335, and in the presence of active Wnt signaling potentially lead to increased expression of a target gene, possibly MYC. To examine the contribution of other variants in the MYC-335 to CRC predisposition, we determined the genetic variation within MYC-335 in different ethnic groups by resequencing the 1271-bp genomic fragment in Caucasian, African and African American samples. This effort identified eight variants which showed population-specific allele frequencies. One variant, a 2-bp deletion, affected a putative transcription factor binding site and was present only in individuals with African origin. The deletion affected GA nucleotides at the fifth and sixth position of the putative binding sequence for transcriptional repressor GFI1 (CAGAGATTGC). The identified GFI1 binding sequence is evolutionary highly conserved and the deletion is predicted to cause a 5.5-fold lower binding affinity. Both the SNP rs6983267 and the GA deletion affect transcription factor binding sites and are differentially distributed in different ethnic populations, which suggests that both alleles are functional and under selective pressure from environmental exposure. We hypothesized that the deletion could contribute to CRC predisposition by abolishing the binding of the repressor, which in turn would lead to enhanced transcriptional activity. Here, we show preliminary results that GFI1 has preferential binding affinity for the wild type allele in the heterozygous cell line HeLa. Also, we investigated the role of the deletion in CRC predisposition in African American individuals. The 2-bp deletion identified here might be a novel population-specific colorectal cancer risk variant. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4731.

Contributions to Neutropenia from PFAAP5 (N4BP2L2), a Novel Protein Mediating Transcriptional Repressor Cooperation between Gfi1 and Neutrophil Elastase

"Neutropenia" refers to deficient numbers of neutrophils, the most abundant type of white blood cell. Two main forms of inherited neutropenia are cyclic neutropenia, in which neutrophil counts oscillate with a 21-day frequency, and severe congenital neutropenia, in which static neutropenia may evolve at times into leukemia. Mutations of ELA2, encoding the protease neutrophil elastase, can cause both disorders. Among other genes, severe congenital neutropenia can also result from mutations affecting the transcriptional repressor Gfi1, one of whose genetic targets is ELA2, suggesting that the two act through similar mechanisms. In order to identify components of a common pathway regulating neutrophil production, we conducted yeast two-hybrid screens with Gfi1 and neutrophil elastase and detected a novel protein, PFAAP5 (also known as N4BP2L2), interacting with both. Expression of PFAAP5 allows neutrophil elastase to potentiate the repression of Gfi1 target genes, as determined by reporter assays, RNA interference, chromatin immunoprecipitation, and impairment of neutrophil differentiation in HSCs with PFAAP5 depletion, thus delineating a mechanism through which neutrophil elastase could regulate its own synthesis. Our findings are consistent with theoretical models of cyclic neutropenia proposing that its periodicity can be explained through disturbance of a feedback circuit in which mature neutrophils inhibit cell proliferation, thereby homeostatically regulating progenitor populations.

Gfi-1 Represses CDKN2B Encoding p15INK4B through Interaction with Miz-1

Zinc finger (ZF) transcriptional repressor Gfi-1 plays an important role in hematopoiesis and inner ear development, and also functions as an oncoprotein that cooperates with c-Myc 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 the cyclin-dependent kinase inhibitor p15INK4B. Gfi-1 did not directly bind to CDKN2B, but interacted with Miz-1 and, via Miz-1, was recruited to the core promoter of CDKN2B. The C-terminal zinc finger domains of Gfi-1 and Miz-1 are involved in the interaction. 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 repressed transcriptional activation of CDKN2B by Miz-1 and in response to TGFb. Notably, Gfi-1 and c-Myc formed a ternary complex with Miz-1 and were both recruited to the CDKN2B core promoter via Miz-1, and acted in collaboration to repress CDKN2B. Consistent with its role in repressing CDKN2B transcription, knockdown of Gfi-1 in human leukemic cells resulted in augmented levels of p15INK4B, which was associated with attenuated cell proliferation. The expression of p15INK4B was also significantly higher in Gfi-1−/− mouse bone marrow (BM) cells than in Gfi-1+/+ BM cells. Our data reveal a novel mechanism of transcriptional repression by Gfi-1 and also identify CDKN2B as a new Gfi-1 target gene. The findings may have important implications for understanding the role of Gfi-1 in normal development and the cooperation between Gfi-1 and c-Myc in lymphomagenesis.

Differential impact of the transcriptional repressor Gfi1 on mature CD4+ and CD8+ T lymphocyte function

The transcriptional repressor Gfi1 is a nuclear zinc-finger protein that is expressed in T cell precursors in the thymus, but is down-regulated in mature, resting T cells. Gfi1 expression rises transiently to levels seen in thymocytes upon antigenic activation. We show here that lack of Gfi1 causes delayed cell cycle entry and apoptosis after antigenic stimulation in both mature CD4+ and CD8+ T cells ex vivo. DNA micro-array analysis demonstrated that this correlated with an up-regulation of the death receptor CD95, the proapoptotic factors Bad and Apaf1 and the cell cycle inhibitor p21, and a down-regulation of Bcl-2 expression in Gfi1-/- T cells. Surprisingly, while Gfi1-deficient CD4+ T cells showed the same defective behavior in vivo, Gfi1-deficient CD8+ T cells showed no aberration in vivo and were fully able to mount an anti-viral immune response. This indicates that Gfi1 exerts different functions in CD4+ and CD8+ T cells very likely by maintaining different genetic programs in both cell types, and appears to be essential for the CD4 helper T cell immune response but dispensable for the function of cytotoxic CD8+ T cells.

Transcriptional Repressor Gfi1 Integrates Cytokine-Receptor Signals Controlling B-Cell Differentiation

Hematopoietic stem cell differentiation is specified by cytokines and transcription factors, but the mechanisms controlling instructive and permissive signalling networks are poorly understood. We provide evidence that CLP1-dependent IL7-receptor mediated B cell differentiation is critically controlled by the transcriptional repressor Gfi1. Gfi1-deficient progenitor B cells show global defects in IL7Rα-dependent signal cascades. Consequently, IL7-dependent trophic, proliferative and differentiation-inducing responses of progenitor B cells are perturbed. Gfi1 directly regulates expression levels of IL7Rα and indirectly controls STAT5 signalling via expression of SOCS3. Thus, Gfi1 selectively specifies IL7-dependent development of B cells from CLP1 progenitors, providing clues to the transcriptional networks integrating cytokine signals and lymphoid differentiation.

The Transcriptional Repressor GFI-1 Antagonizes PU.1 Activity through Protein-Protein Interaction

Mice lacking the zinc finger transcriptional repressor protein GFI-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Furthermore, Gfi-1(-/-) mice are highly susceptible to bacterial infection. Interestingly, Gfi-1(-/-) myeloid cells overexpress target genes of the PU.1 transcription factor such as the macrophage colony-stimulating factor receptor and PU.1 itself. We therefore determined whether GFI-1 modulates the transcriptional activity of PU.1. Our data demonstrate that GFI-1 physically interacts with PU.1, repressing PU.1-dependent transcription. This repression is functionally significant, as GFI-1 blocked PU.1-induced macrophage differentiation of a multipotential hematopoietic progenitor cell line. Retroviral expression of GFI-1 in primary murine hematopoietic progenitors increased granulocyte differentiation at the expense of macrophage differentiation. We interbred Gfi-1(+/-) and PU.1(+/-) mice and observed that heterozygosity at the PU.1 locus partially rescued the Gfi-1(-/-) mixed myeloid lineage phenotype, but failed to restore granulocyte differentiation. Our data demonstrate that GFI-1 represses PU.1 activity and that lack of this repression in Gfi-1(-/-) myeloid cells contributes to the observed mixed lineage phenotype.

Regulation of pulmonary Pseudomonas aeruginosa infection by the transcriptional repressor Gfi1

Pulmonary Pseudomonas aeruginosa infections are characterized by the release of proinflammatory mediators, focal induction of apoptosis in respiratory epithelial cells and internalization of the bacteria. Here, we demonstrate that the transcriptional regulator Gfi1 is critically involved in the regulation of proinflammatory cytokine release and the induction of apoptosis in respiratory epithelial cells and macrophages upon P. aeruginosa infection. Gfi1-deficient mice responded to a pulmonary P. aeruginosa infection with uncontrolled pulmonary release of interleukin (IL)-1 and tumour necrosis factor (TNF)-alpha, sepsis and death, which were delayed by injection of IL-1- and TNF-alpha-neutralizing antibodies. The uncontrolled release of cytokines seems to be caused by a failure of Gfi1-deficient respiratory epithelial cells in large to small bronchi and macrophages to respond to P. aeruginosa infection with an induction of apoptosis. Pharmacological inhibition of apoptosis in wild-type mice by intravenous injection of the broad-spectrum caspase inhibitor zVAD-cmk mimicked the phenotype of Gfi1-deficient mice and resulted in a profound sensitization of mice to P. aeruginosa, an increased release of cytokines, sepsis and death of the animals. Thus, Gfi1 controls apoptosis of respiratory epithelial cells and macrophages upon infection with P. aeruginosa. Inhibition of apoptosis by Gfi1 deficiency or caspase blockers sensitizes mice to P. aeruginosa infections, suggesting that apoptosis functions as a novel defence mechanisms in the regulation of the local innate immune response.