Mutations in a newly identified gene, pag-3, cause ectopic expression of touch neuron genes meo-7, mec-7lacZ and mec-4lacZ in the lineal sisters of the ALM touch neurons, the BDU neurons. pag-3 mutants also show a reverse kinker uncoordinated phenotype. The first pag-3 allele was isolated in a screen for mutants with altered immunofluorescence staining patterns. Two additional pag-3 alleles have been identified in a noncomplementation screen of 38,000 haploid genomes. All of the pag-3 alleles are recessive to wild type and cause the same phenotypes. Two-factor crosses, deficiency mapping and three-factor crosses locate pag-3 to the right arm of the X chromosome between unc-3 and unc-7. Because recessive mutations in pag-3 result in expression of several touch cell specific genes in the BDU neurons, wild-type pag-3 must directly or indirectly suppress expression of these genes in the BDU neurons. Although pag-3 mutants do not show mec-3lacZ expression in their BDU neurons, expression of mec-7lacZ and mec-4lacZ in the BDU neurons of pag-3 mutants requires mec-3 (Jia, 1996).
Mutations in the C. elegans gene pag-3 result in misexpression of touch receptor-specific genes in the BDU interneurons and in motility defects. pag-3 encodes a C2H2-type zinc finger protein related to the mammalian GFI-1 protein. Sequencing of the three pag-3 alleles shows that two apparent null alleles encode a nonsense mutation before the zinc fingers and a missense mutation in the fourth zinc finger that changes a coordinating histidine to a tyrosine. The third allele contains a nonsense mutation in the N-terminal region but is not a null allele. Northern analysis shows that a single pag-3 transcript of about 1.6 kb is present in embryos and L1, L2 and L3 larvae. pag-3 message levels are about twofold higher in pag-3 mutants than in wild-type animals, which suggests that pag-3 may negatively regulate its own expression. pag-3lacZ fusion genes are expressed in the BDU interneurons, the touch neurons, 11 VA and 11 VB ventral cord motor neurons, two AVF interneurons and in unidentified neurons of the retrovesicular ganglion. The BDU neurons and the ALM touch neurons are lineal sister cells in the AB.a lineage and the VA and VB motor neurons are lineal sister cells in the AB.p lineage. The VA motor neurons are required for backward movement and the VB motor neurons are required for forward movement. Mosaic analysis has shown that the wild-type pag-3 gene is required in the AB.p lineage for coordinated movement and in the AB.a lineage to suppress touch neuron gene expression in the BDU neurons. Because pag-3 is expressed in both the BDU neurons and in the touch neurons, another protein(s) not expressed in the touch neurons may interact with pag-3 to repress touch neuron gene expression in the BDU neurons. Alternatively, another protein in the touch receptor cells may inactivate PAG-3 and allow expression of the touch receptor program. These results show that pag-3 is a temporally regulated gene that is expressed early in development and functions in multiple types of neurons. They also strongly suggest that the PAG3 protein is a DNA-binding protein with properties similar to the mammalian proto-oncogene product GFI-1 (Jia, 1997).
The Caenorhabditis briggsae homolog of the C. elegans pag-3 gene was cloned and sequenced. When transformed into a C. elegans pag-3 mutant, the C. briggsae pag-3 gene rescues the pag-3 reverse kinker and lethargic phenotypes. The C. elegans pag-3 gene fused to lacZ is expressed in the same pattern in C. elegans and C. briggsae. Unlike many gene homologs compared between C. elegans and C. briggsae, extensive sequence conservation is found in the non-coding regions upstream of the pag-3 exons, in several of the introns and in the downstream non-coding region. Furthermore, the splice acceptor and splice donor sites are conserved, and the size of the introns and exons is surprisingly similar. The predicted protein sequence of C. briggsae PAG-3 is 85% identical to the protein sequence of C. elegans PAG-3. Because so much of the non-coding region of pag-3 is conserved, the control of pag-3 may be quite complex, involving the binding of many trans-acting factors. These results suggest the evolutionary conservation of the pag-3 gene sequence, its expression and function (Aamodt, 2000).
During C. elegans development, the patterns of cell divisions, cell fates and programmed cell deaths are reproducible from animal to animal. In a search for mutants with abnormal patterns of programmed cell deaths in the ventral nerve cord, mutations were identified in the gene pag-3, which encodes a zinc-finger transcription factor similar to the mammalian Gfi-1 and Drosophila Senseless proteins. In pag-3 mutants, specific neuroblasts express the pattern of divisions normally associated with their mother cells, producing with each reiteration an abnormal anterior daughter neuroblast and an extra posterior daughter cell that either terminally differentiates or undergoes programmed cell death, which accounts for the extra cell corpses seen in pag-3 mutants. In addition, some neurons do not adopt their normal fates in pag-3 mutants. The phenotype of pag-3 mutants and the expression pattern of the PAG-3 protein suggest that in some lineages pag-3 couples the determination of neuroblast cell fate to subsequent neuronal differentiation. It is proposed that pag-3 counterparts in other organisms determine blast cell identity and for this reason may lead to cell lineage defects and cell proliferation when mutated (Cameron, 2002).
The PVQ and BDU neurons in pag-3 mutants are abnormal, despite being generated by apparently normal cell lineages, suggesting that these neurons fail to differentiate properly in pag-3 mutants. The VA motoneurons also differentiate abnormally in pag-3 mutants, as demonstrated by the defective backwards movement of these animals and by abnormal expression of an unc-4 reporter gene. A role for pag-3 in differentiating and differentiated neurons is supported by the expression pattern of the protein. In the Q neuroblast lineages PAG-3 protein is detected only after the generation of the terminally differentiating AVM and PVM mechanosensory neurons. In many neurons, including the PVQ, BDU and touch neurons, PAG-3 protein is present throughout the life of the animal. Some of these cells are abnormal in pag-3 mutants (the BDU and PVQ neurons), while others have no obvious defect. These data suggest that pag-3 functions in diverse contexts within the developing nervous system, with an essential role in the development of some neuroblasts and neurons and a currently unapparent role in other neurons (Cameron, 2002).
PAG-3 does not appear to be associated with any obvious single characteristic of differentiated neurons. In the ventral nerve cord, PAG-3 is expressed in cells that live and in cells that undergo programmed cell death; in cells that express lin-11 and in cells that do not; and transiently in the VA2-10 motoneurons but continuously in VA11 and VA12. PAG-3 is required for cell-fate determination by the Pn.aa neuroblasts but not by the Pn.ap neuroblasts. PAG-3 was expressed in adult animals in neurons of several different types, including motoneurons (VA11, VA12), sensory neurons (ALM, PLM, AVM and PVM) and interneurons (BDU, RIG and AVF). Given its extensive similarity to mammalian Gfi-1 transcription factors, PAG-3 most probably affects cell fates by regulating transcription. Rather than affecting expression of a single common set of genes in all neuronal lineages, pag-3 is expressed in many neuronal subtypes at different points in neuronal development suggesting that pag-3 cooperates with other factors to regulate the expression of cell type- and developmental stage-specific sets of genes to generate the complex pattern of neuronal subtypes seen in C. elegans (Cameron, 2002).
pag-3 expression is specifically activated in the Pn.aa neuroblasts, the descendants of which are abnormal in pag-3 mutants. By contrast, expression is not activated to a detectable level in the sisters of these cells, the Pn.ap neuroblasts, the descendants of which appeared normal in pag-3 mutants. These observations suggest that pag-3 acts specifically in the Pn.aa neuroblasts and their descendants. PAG-3 protein was present at the time of generation of neurons descended from each Pn.aa cell. It is shortly after the generation of the terminal cells in these lineages that these neurons begin to adopt identifying characteristics, such as class-specific patterns of axonal projections or the morphological characteristics of a dying cell. PAG-3 thus may well be expressed in response to Pn.aa lineage-specific signals to determine blast cell fates and then act later to induce distinctive differentiated features characteristic of the neurons produced by those lineages (Cameron, 2002).
The function of PAG-3 may be similar to that of UNC-86 (Drosophila homolog: Acj6), a POU-homeodomain protein that couples cell lineage cues to aspects of terminal differentiation. Like mutations in pag-3, mutations in unc-86 result in the reiteration of some neuroblast lineages. unc-86 is the only other C. elegans gene known to be able to mutate to cause reiterative cell lineage defects that specifically affect development of the nervous system. In addition to being required for neuroblast determination, unc-86 also specifies characteristics of the mechanosensory neurons generated by those neuroblasts. pag-3 may function similarly in the ventral nerve cord lineages. In these lineages, pag-3 determines Pn.aa neuroblast fate and may also establish the fate of the VA and VB motoneurons generated by those neuroblasts (Cameron, 2002).
X-MyT1 is a C2HC-type zinc finger protein involved in the primary selection of neuronal precursor cells in Xenopus. Expression of this gene is positively regulated by the bHLH protein X-NGNR-1 and negatively regulated by the Notch/Delta signal transduction pathway. X-MyT1 is able to promote ectopic neuronal differentiation and to confer insensitivity to lateral inhibition, but only in cooperation with bHLH transcription factors. Inhibition of X-MyT1 function inhibits normal neurogenesis as well as ectopic neurogenesis caused by overexpression of X-NGNR-1. On the basis of these findings, it is suggested that X-MyT1 is a novel, essential element in the cascade of events that allows cells to escape lateral inhibition and to enter the pathway that leads to terminal neuronal differentiation (Bellefroid, 1996).
Zebrafish growth factor independent 1 (gfi1) expression is detected in the ganglion cells of the neural retina and in developing hair cells of the ear. In keeping with a role in the development of sensory hair cells, gfi1 is also expressed in neuromasts of the anterior and posterior lateral line system. Finally, gfi1 is expressed in the developing epithalamus in the dorsal diencephalon where its transcription is restricted to the parapineal (Dufourcq, 2004).
The proliferation of cultured rat Nb2 lymphoma cells is dependent on prolactin (PRL) acting as the principal growth factor. Previously, PRL-independent Nb2 sublines were obtained by PRL starvation of the parent line and cloning of surviving cells. Development of PRL independence has been, in some cases, associated with a reciprocal translocation involving chromosome 14 at breakpoint 14p22. A novel 14p22 zinc finger protein-encoding gene, Gfi-1, has been examined for a role in Nb2 cell proliferation. PRL-dependent Nb2 cells express the gene during active growth; in comparison, in stationary, early G1-arrested cells obtained by an 18 hr lactogen starvation, Gfi-1 gene expression is markedly decreased. Addition of PRL to such stationary cells leads to induction of Gfi-1 gene expression within a few hr with a maximum in late G1. Actively growing cells from 5 different PRL-independent Nb2 sublines, cultured in chemically defined, mitogen-free medium, express the gene constitutively. In two sublines, carrying the 14p22 rearrangement, the gene is markedly overexpressed. The results suggest the Gfi-1 gene product has a regulatory role in Nb2 cell mitogenesis and that unscheduled activation could contribute to loss of PRL dependency (Gilks, 1995).
The Gfi-1 proto-oncogene encodes a zinc finger protein with six C2H2-type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/T)GCA, has been defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site has been confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oliginucleotides have defined the relative importance of specific bases at the consensus binding site. Deletion of individual zinc fingers demonstrates that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites are detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, are repressed by Gfi-1, and the repression is abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes (Zweidler-Mckay, 1996).
The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, it is concluded that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation (Grimes, 1996a).
The rat and mouse Growth Factor Independence (Gfi-1) genes allow cells in culture to overcome the depletion of growth factors in the culture medium and maintain their proliferative potential. As part of a cloning strategy to isolated genes from human chromosome 1p22 that are associated with a constitutional chromosome translocation from a patient with stage 4S neuroblastoma, the human homolog of the Gfi gene has been identified and a 50 Kb map position has been identified within a well characterised YAC contig from the region. The full length cDNA sequence is 81% homologous with the rodent counterparts and, at the protein level, is even more highly conserved (Roberts, 1997).
After rearrangement of the T-cell receptor (TCR) ßlocus, early CD4(-)/CD8(-) double negative (DN) thymic T-cells undergo a process termed 'ßselection' that allows the preferential expansion of cells with a functional TCR ßchain. This process leads to the formation of a rapidly cycling subset of DN cells that subsequently develop into CD4(+)/CD8(+) double positive (DP) cells. Using transgenic mice that constitutively express the zinc finger protein Gfi-1 and the serine/threonine kinase Pim-1, it was found that the levels of both proteins are important for the correct development of DP cells from DN precursors at the stage where 'ßselection' occurs. Analysis of the CD25(+)/CD44(-,lo) DN subpopulation from these animals reveals that Gfi-1 inhibits and Pim-1 promotes the development of larger ßselected cycling cells ('L subset') from smaller resting cells ('E subset') within this subpopulation. It is concluded that both proteins, Pim-1 and Gfi-1, participate in the regulation of ßselection-associated pre-T-cell differentiation in opposite directions and that the ratio of both proteins is important for pre-T-cells to pass the 'E' to 'L' transition correctly during ßselection (Schmidt, 1998a).
Gfi-1 is a cellular proto-oncogene that has been identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. Both IL-6-induced G1 arrest and differentiation of the myelomonocytic cell line M1 are linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21WAF1. Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes reveal that Gfi-1B represses p21WAF1 directly by binding to a high-affinity site at -1518 to -1530 in the p21WAF1 promoter. Forced expression of Gfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocks the IL-6-mediated induction of p21WAF1 and inhibits G1 arrest and differentiation. It is concluded that Gfi-1B is a direct repressor of the p21WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G1 arrest and differentiation of M1 cells perhaps because it prevents p21WAF1 induction by IL-6 (Tong, 1998).
STAT factors act as signal transducers of cytokine receptors and transcriptionally activate specific target genes. The recently discovered protein PIAS3 binds directly to STAT3 and blocks transcriptional activation. Experimental evidence is presented implementing the zinc finger protein Gfi-1 as a new regulatory factor in STAT3-mediated signal transduction. The interaction between the two proteins first became evident in a yeast two-hybrid screen but is also seen in coprecipitation experiments from eukaryotic cells. Moreover, both Gfi-1 and PIAS3 colocalize in a characteristic nuclear dot structure. While PIAS3 exerts a profound inhibitory effect on STAT3-mediated transcription of target promoters, Gfi-1 can overcome the PIAS3 block and significantly enhances STAT3-mediated transcriptional activation. In primary T cells, Gfi-1 is able to amplify IL-6-dependent T-cell activation. Since Gfi-1 is a known, dominant proto-oncogene, these findings bear particular importance for the recently described ability of STAT3 to transform cells malignantly and offers an explanation of the oncogenic potential of Gfi-1 in T lymphocytes (Rodel, 2000).
Gfi-1 is a nuclear zinc finger protein with the activity of a transcriptional repressor and the ability to predispose for the development of T-cell lymphoma when expressed constitutively at high levels. Whereas thymic T-cell precursors express endogenous Gfi-1, mature peripheral T-cells lack Gfi-1 but upregulate its expression transiently after antigenic stimulation and activation of Erk1/2 demonstrating a role of Gfi-1 in T-cell activation. Constitutive expression of Gfi-1 accelerates S phase entry of primary, resting T-cells upon antigenic stimulation. In addition, high level Gfi-1 expression inhibits phorbol ester induced G1 arrest and activation induced cell death in Jurkat T-cells. These effects of Gfi-1 concur with lower absolute levels and hyperphosphorylation of the pocket protein pRb. Moreover, phorbol ester induced expression of the negative cell cycle regulator p21(WAF1) is blocked in the presence of Gfi-1. These findings suggest that Gfi-1 contributes to T-cell lymphomagenesis by overriding a late G1 cell cycle checkpoint that controls activation induced death and S phase entry of T-cells (Karsunky, 2002).
SOCS proteins take part in a classical negative feedback loop to attenuate cytokine signaling. Although STAT family members positively modulate Socs gene expression, little else is known about Socs gene regulation. This study identifies functional binding sites for GFI-1B, a proto-oncogenic transcriptional repressor, in the promoters of murine Socs1 and Socs3. Thus, mutating these sites relieved transcriptional repression, as determined by luciferase reporter assays of transiently transfected erythropoietin-responsive 32D-EpoR and HCD57 cells. Furthermore, cotransfection of Gfi-1B expression plasmid repressed reporter activity of wild-type (but not mutagenized) Socs1 and Socs3 promoters, strongly suggestive of direct GFI-1B binding to these promoters. In addition, overexpression of Gfi-1B resulted in reduced transcript levels of Socs1 and Socs3, but not Socs2 or Cis. Upon stimulation with erythropoietin, Socs transcripts are rapidly induced, whereas Gfi-1B mRNA is down-regulated. Interestingly, the latter effect appears to rely on STAT5 activity, but not on phosphoinositide 3-kinase or MAPK pathways. Thus, cytokine-mediated STAT5 activation allows relief of direct repression by GFI-1B of the Socs1 and Socs3 promoters, but apparently not of the Socs2 and Cis promoters. This constitutes a previously undescribed mode of controlling cytokine responsiveness, through the direct repression of a tumor suppressor (SOCS1) by a proto-oncoprotein (GFI-1B) (Jegalian, 2002).
Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. Human beta1 sGC transcriptional regulation was investigated in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, this study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression (Sharina, 2003).
Granulocyte-colony-stimulating factor (G-CSF) stimulates the activation of multiple signaling pathways, leading to alterations in the activities of transcription factors. Gfi-1 is a zinc finger transcriptional repressor that is required for granulopoiesis. How Gfi-1 acts in myeloid cells is poorly understood. The expression of Gfi-1 is up-regulated during G-CSF-induced granulocytic differentiation in myeloid 32D cells. Truncation of the carboxyl terminus of the G-CSF receptor, as seen in patients with acute myeloid leukemia evolving from severe congenital neutropenia, disrupts Gfi-1 up-regulation by G-CSF. Ectopic expression of a dominant negative Gfi-1 mutant, N382S, which is associated with severe congenital neutropenia, results in premature apoptosis and reduces proliferation of cells induced to differentiate with G-CSF. The expression of neutrophil elastase (NE) and CCAAT enhancer-binding protein epsilon (C/EBPepsilon) is significantly increased in 32D cells expressing N382S. In contrast, overexpression of wild type Gfi-1 abolishes G-CSF-induced up-regulation of C/EBPepsilon but has no apparent effect on NE up-regulation by G-CSF. Notably, G-CSF-dependent proliferation and survival are inhibited upon overexpression of C/EBPepsilon but not NE. These data indicate that Gfi-1 down-regulates C/EBPepsilon expression and suggest that increased expression of C/EBPepsilon as a consequence of loss of Gfi-1 function may be deleterious to the proliferation and survival of early myeloid cells (Zhuang, 2006).
Gfi-1 and Gfi-1b, homologs of Drosophila senseless, are novel proto-oncogenes identified by retroviral insertional mutagenesis. By gene targeting, it has been established that Gfi-1b is required for the development of two related blood lineages, erythroid and megakaryocytic, in mice. Gfi-1b-/- embryonic stem cells fail to contribute to red cells of adult chimeras. Gfi-1b-/- embryos exhibit delayed maturation of primitive erythrocytes and subsequently die with failure to produce definitive enucleated erythrocytes. The fetal liver of mutant mice contains erythroid and megakaryocytic precursors arrested in their development. Myelopoiesis is normal. Therefore, Gfi-1b is an essential transcriptional regulator of erythroid and megakaryocyte development (Saleque, 2002).
The mammalian protein Gfi-1b, like its orthologs in Drosophila and C. elegans, Senseless and PAG-3 respectively, regulates the development of specific cellular lineages. The three proteins also show similar DNA-binding specificities consistent with >80% sequence identity between their DNA-binding zinc fingers, and presumably have similar target sites in vivo. However, whether they regulate analogous target genes and pathways in vivo remains to be elucidated. Notably, both PAG-3 and Senseless lack the SNAG repression domain, and this structural variation could lead to mechanistic differences between them in regulating their targets. The control of sensory organ development in Drosophila by an autoregulatory loop comprised of senseless and the basic-helix-loop-helix (bHLH) proneural genes daughterless, achaete-scute, and atonal raises the possibility that Gfi-1b may also interact in a transcriptional network with bHLH factors, within or outside the hematopoietic system. A likely candidate within the hematopoietic system is the bHLH factor SCL/tal-1, a gene required for development of all hematopoietic lineages. Because loss of Gfi-1b does not affect SCL/tal-1 expression in fetal liver colonies, it is concluded that Gfi-1b is not required for SCL expression. Whether SCL regulates Gfi-1b expression is unknown (Saleque, 2002).
Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosoophila and pag-3 in C. elegans are expressed and required in the nervous system. This study shows that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies: they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death (Wallis, 2003).
Gfi1 is a transcriptional repressor implicated in lymphomagenesis, neutropenia, and hematopoietic development, as well as ear and lung development. This study demonstrates that Gfi1 functions downstream of Math1 in intestinal secretory lineage differentiation. Gfi1-/- mice lack Paneth cells, have fewer goblet cells, and supernumerary enteroendocrine cells. Gfi1-/- mice show gene expression changes consistent with this altered cell allocation. These data suggest that Gfi1 functions to select goblet/Paneth versus enteroendocrine progenitors. A model of intestinal cell fate choice is proposed in which beta-catenin and Cdx function upstream of Math1, and lineage-specific genes such as Ngn3 act downstream of Gfi1 (Shroyer, 2005).
Gfi-1 is a zinc finger transcriptional repressor originally recognized for its role in T cell differentiation and lymphomas. Recent experiments reveal that gene-targeted Gfi-1-deficient mice are neutropenic and that Gfi-1 mutations cause human neutropenia. In both cases, myeloid progenitor cells lose the ability to distinctly differentiate granulocytes from monocytes. The molecular mechanism of the hematopoietic abnormalities caused by Gfi-1 deficiency remains undetermined because of a lack of known Gfi-1 target genes. To identify Gfi-1 targets in vivo, large-scale chromatin immunoprecipitation analysis was performed on a set of 34 candidate genes in myeloblast (KG-1 and HL-60), monoblast (U937), and T lymphocyte cell lines (Jurkat), in concert with RT-PCR-based expression profiling. Thirty-two Gfi-1 binding sites were identified in a functionally variable set of 16 genes, including complements of cell-cycle regulators, transcription factors, and granulocyte-specific markers. Cluster analysis of expression patterns and chromatin immunoprecipitation data reveals that Gfi-1 targets a subset of genes differentiating hematopoietic lineages and therefore plays a relatively superior role in the hierarchy of factors governing stem cell differentiation (Duan, 2003).
Expression of Gfi (growth factor-independence)-1B, a Gfi-1-related transcriptional repressor, is restricted to erythroid lineage cells and is essential for erythropoiesis. The transcription start site of the human Gfi-1B gene has been determined and its first non-coding exon has been located approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, it has been demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of the Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of the Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, these results have indicated that this GATA-1-mediated trans-activation is dependent on NF-Y binding to the CCAAT site. It is concluded that functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter (Huang, 2004)
Gfi1b is a 37 kDa transcriptional repressor with six zinc-finger domains that is differentially expressed during hemato- and lymphopoiesis. Transcription from the Gfi1b gene locus is silenced in the spleen but not in the bone marrow of transgenic mice that constitutively express Gfi1b under the control of the pan-hematopoietic vav promoter. Sequence analysis of the Gfi1b promoter showed the presence of potential Gfi1/Gfi1b-binding sites close to the mRNA start site. The expression of reporter gene constructs containing the Gfi1b core promoter appended to the luciferase gene were strongly repressed in the presence of exogenous Gfi1b. Moreover, analysis of combinatorial mutant mice that carry the vav-Gfi1b transgene and a green fluorescent protein-tagged Gfi1 gene locus demonstrated that the Gfi1 gene can be repressed by Gfi1b. Direct binding of Gfi1b and Gfi1 to the potential binding sites in the Gfi1b promoter could be demonstrated by gel-shift analyses in vitro. Chromatin-immunoprecipitation experiments showed that both the Gfi1b and the Gfi1 promoter are indeed occupied by Gfi1b in vivo. Hence, it is conclude that Gfi1b can auto-repress its own expression, but, in addition, is also able to cross-repress expression of the Gfi1 gene most likely in a cell type specific manner (Vassen, 2005).
Gfi-1 and Gfi-1B can repress transcription and play important roles in hematopoietic cell survival and differentiation. Although these proteins are known to bind DNA through a C-terminal zinc-finger domain and may require an N-terminal SNAG domain (SNAIL/Gfi-1) to repress transcription, the mechanism by which Gfi-1 and Gfi-1B act is unknown. A first step towards understanding the mechanism by which these proteins repress transcription is to identify interacting proteins that could contribute to transcriptional repression. ETO (also termed MTG8), was first identified through its involvement in the (8;21) translocation associated with acute myelogenous leukemia. It attaches to the nuclear matrix and associates with histone deacetylases and the co-repressors N-CoR, SMRT, and mSin3A, and may act as a co-repressor for site-specific transcriptions factors. Gfi-1 interacts with ETO and related proteins both in vitro and in vivo and with histone deacetylase proteins in vivo. A portion of Gfi-1 and Gfi-1B associates with the nuclear matrix, as is the case with ETO. Moreover, Gfi-1 and ETO co-localize to punctate subnuclear structures. When co-expressed in mammalian cells, Gfi-1 associates with histone deacetylse-1 (HDAC-1), HDAC-2, and HDAC-3. These data identify ETO as a partner for Gfi-1 and Gfi-1B, and suggest that Gfi-1 proteins repress transcription through recruitment of histone deacetylase-containing complexes (McGhee, 2003).
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 Gfi1 display lymphoid abnormalities. This study describes autoregulation of Gfi1 in primary mouse thymocytes and a human T-cell line. GFI1 binding to cis-element sequences conserved between rat, mouse and human Gfi1 mediates direct and potent transcriptional repression. In addition, dramatic regulation of Gfi1 can also be mediated by GFI1B. These data provide the first example of a gene directly targeted by GFI1 and GFI1B. Moreover, they support a role for auto- and trans-regulation of Gfi1 by GFI1 and GFI1B in maintaining the normal expression patterns of Gfi1, and suggest that GFI1B may indirectly affect T-cell activation through repression of Gfi1 (Doan, 2004).
In the search for genes expressed in hematopoietic stem cells, the expression of Gfi-1B (growth factor independence-1B) was found to be highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, its function was analyzed in human hematopoiesis. Enforced expression of Gfi-1B in human CD34(+) hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B does not promote erythroid commitment, but enhances proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biologic effects of Gfi-1B are mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription is dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression (Osawa, 2002).
During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, reveals that all mutants carry new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, has been found to be the target of provirus insertion in 1 additional IL-2i cell line, of the 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype (Gilks, 1993).
The clonality of lymphomas that originate in myc/pim-1 bitransgenic mice due to synergistic action of both oncogenes indicates the requirement of additional events for progression to full malignancy. To isolate genes that cooperate with both myc and pim-1, provirus tagging with E mu L-myc/pim-1 double transgenic mice were used. Accelerated tumor formation is found in infected animals and the gfi-1 gene and neighboring loci on mouse chromosome 5 are occupied by proviruses in about 53% of the tumors leading in all cases to high level gfi-1 expression. Forced expression of the gfi-1 encoded zinc finger protein in IL-2 dependent T-cells provokes increased survival upon IL-2 depletion and evidence is presented that this occurs at least in part through stimulation of proliferation. The data suggest that gfi-1 is a proto-oncogene cooperation with both myc and pim-1 genes in T-cell lymphomagenesis (Zornig, 1996).
The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis (Grimes, 1996b).
A prominent feature of retrovirus-induced immunodeficiency in mice (MAIDS) is early polyclonal activation of CD4+ T cells followed by the appearance of monoclonal lymphomas marked by clonal proviral integrations. These events appear to occur independent of interleukin-2 (IL-2), suggesting the activity of an alternative growth-promoting pathway. The possible contributions to T cell expansion of a gene, Gfi-1, previously shown to confer IL-2 independence to rat T cell lymphomas, was investigated. Seventeen mice with MAIDS that had clonal populations of T cells were studied. Proviral integrations at Gfi-1 were detected in two animals. These integrations were associated with enhanced transcription of Gfi-1. Unexpectedly, elevated levels of Gfi-1 transcripts were also observed in four T cell lymphomas without detectable integrations at this locus. This suggests that IL-2-independent T cell growth in MAIDS may be driven by transcriptional activation of Gfi-1 by proviral insertion or transactivation (Liao, 1997).
The gfi-1 gene encodes a zinc finger containing protein that is specifically expressed in T-lymphocytes and is a frequent target of proviral insertion in T-cell lymphoma provoked by infection with MoMuLV -- a non acute transforming retrovirus. Expression of a gfi-1 transgene targeted to T-cells by the lck proximal promoter provokes a reduction of peripheral CD4 and CD8 positive T-cells but nevertheless weakly predisposes transgenic animals for the development of T-cell lymphoma. Forced coexpression of the serine/threonine kinase Pim-1 can partially restore normal T-cell numbers in double pim-1/gfi-1 transgenic mice. Moreover, the combinatorial expression of Pim-1 and Gfi-1 leads to accelerated development of T-cell lymphoma with a mean latency period of 114 days. A similar accelerated rate of lymphoma development is observed when lck-gfi-1 mice were crossed with mice that carry a L-myc gene targeted to be expressed at high levels in T-cells. The results show that gfi-1 can act with low activity as a dominant oncogene when overexpressed but also demonstrate that it is most efficient only in the presence of a cooperative partner protein, as for example Pim-1 or L-Myc. In addition, the results suggest that Pim-1 and Gfi-1 are acting synergistically in both T-cell lymphomagenesis and T-cell development (Schmidt, 1998b).
This study reports essential roles of zinc finger transcription factor Gfi-1 in myeloid development. Gene-targeted Gfi-1-/- mice lack normal neutrophils and are highly susceptible to abscess formation by gram-positive bacteria. Arrested, morphologically atypical, Gr1+Mac1+ myeloid cells expand with age in the bone marrow. RNAs encoding primary but not secondary or tertiary neutrophil (granulocyte) granule proteins are expressed. The atypical Gr1+Mac1+ cell population shares characteristics of both the neutrophil and macrophage lineages and exhibits phagocytosis and respiratory burst activity. Reexpression of Gfi-1 in sorted Gfi-1-/- progenitors ex vivo rescues neutrophil differentiation in response to G-CSF. Thus, Gfi-1 not only promotes differentiation of neutrophils but also antagonizes traits of the alternate monocyte/macrophage program (Hock, 2003).
Haematopoietic stem cells (HSCs) sustain blood production throughout life. HSCs are capable of extensive proliferative expansion; a single HSC may reconstitute lethally irradiated hosts. In steady-state, HSCs remain largely quiescent and self-renew at a constant low rate, forestalling their exhaustion during adult life. Whereas nuclear regulatory factors promoting proliferative programmes of HSCs in vivo and ex vivo have been identified, transcription factors restricting their cycling have remained elusive. This study reports that the zinc-finger repressor Gfi-1 (growth factor independent 1), a cooperating oncogene in lymphoid cells, unexpectedly restricts proliferation of HSCs. After loss of Gfi-1, HSCs display elevated proliferation rates as assessed by 5-bromodeoxyuridine incorporation and cell-cycle analysis. Gfi-1-/- HSCs are functionally compromised in competitive repopulation and serial transplantation assays, and are rapidly out-competed in the bone marrow of mouse chimaeras generated with Gfi-1-/- embryonic stem cells. Thus, Gfi-1 is essential to restrict HSC proliferation and to preserve HSC functional integrity (Hock, 2004).
Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcription factor is critically required for the formation of pulmonary neuroendocrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH1 (Achaete and Scute) and the growth factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. This study shows that GFI1, as with ASH1, is expressed in neuroendocrine lung cancer cell lines and that GFI1 in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI1 expression strongly correlates with expression of ASH1, the neuroendocrine growth factor gastrin-releasing peptide, and neuroendocrine markers synaptophysin and chromogranin A. GFI1 colocalizes with chromogranin A and calcitonin-gene-related peptide in embryonic and adult murine pulmonary neuroendocrine cells. In addition, mice with a mutation in GFI1 display abnormal development of pulmonary neuroendocrine cells, indicating that GFI1 is important for neuroendocrine differentiation (Kazanjian, 2004).
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