ETS DNA-binding Domain Research Articles

Identification of a monopartite sequence in PU.1 essential for nuclear import, DNA‐binding and transcription of myeloid‐specific genes

The Ets transcription factor PU.1 is an essential regulator of normal hematopoiesis, especially within the myeloid lineage. As such, endogenous PU.1 predominantly localizes to the nucleus of mammalian cells to facilitate gene regulation. However, to date, little is known regarding the mechanisms of PU.1 nuclear transport. We found, using HeLa and RAW 264.7 macrophage cells, that PU.1 enters the nucleus via passive diffusion and active transport. The latter can be facilitated by: (i) the classical pathway requiring importin alpha and beta; (ii) the non-classical pathway requiring only importin beta; or (iii) direct interaction with nucleoporins. A group of six positively charged lysine or arginine residues within the Ets DNA-binding domain was determined to be crucial in active nuclear import. These residues directly interact with importin beta to facilitate a predominantly non-classical import pathway. Furthermore, luciferase reporter assays demonstrated that these same six amino acids are crucial for PU.1-mediated transcriptional activation of myeloid-specific genes. Indeed, these residues may represent a consensus sequence vital for nuclear import, DNA-binding and transcriptional activity of Ets family members. By identifying and characterizing the mechanisms of PU.1 nuclear import and the specific amino acids involved, this report may provide insights into the molecular basis of diseases.

Single-chain Antibodies to the EWS NH2 Terminus Structurally Discriminate between Intact and Chimeric EWS in Ewing's Sarcoma and Interfere with the Transcriptional Activity of EWS In vivo

The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.

Expression, purification, and structural prediction of the Ets transcription factor ERM

The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors.Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an α-helix. In contrast, the 250-residue central domain seems to have very little structure.

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor

We investigated nuclear localization signal (NLS) determinants within the AT-hook and ETS DNA-binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium-specific ETS transcription factors. Deletion mutants containing the AT-hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT-hook domain, four basic residues ( 244KRKR 247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236–252 and 249–267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues ( 318KKK 320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Does a truncated form of the transcription factor Ets1 exist in breast cancer cells?

Does a truncated form of the transcription factor Ets1 exist in breast cancer cells?

Conserved ETS Domain Arginines Mediate DNA Binding, Nuclear Localization, and a Novel Mode of bZIP Interaction

The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1beta. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPbeta carboxyl-terminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.

Oncogenic TLS-ERG Fusion Protein Promotes Differentiation of Myeloid Progenitors into Myeloblasts but Blocks Their Terminal Differentiation.

In a subset of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) in blast crisis, the TLS (translocation liposarcoma) gene is fused to the ERG (ets-related gene) gene through a recurrent t(16;21) translocation. This chromosomal translocation results in the generation of the TLS-ERG fusion protein. More recently, the same TLS-ERG fusion protein has also been identified in Ewing's sarcoma in children. To investigate the oncogenic mechanism of TLS-ERG, we stably expressed TLS-ERG or its mutant TLS-ERGΔets in mouse L-G myeloid progenitor cells through retroviral transduction. L-G cells normally proliferate in the presence of IL-3 and undergo terminal differentiation into hypersegmented neutrophils when treated with G-CSF. In contrast, expression of TLS-ERG in L-G cells led to spontaneous differentiation into immature myeloblasts but G-CSF treatment of these cells failed to induce terminal differentiation. L-G myeloblasts harboring TLS-ERG were characterized by high levels of myeloperoxidase (MPO), a phenomenon also observed in immature myeloblasts isolated from human leukemia patients with the t(16;21) translocation. As deletion of the ets DNA-binding domain from TLS-ERG abolished its ability to promote spontaneous myeloblast differentiation or the ability to up-regulate the MPO gene, we further examined how TLS-ERG fusion protein might affect the functions of other regulators such as PU.1, an ets transcription factor known to play a critical role in the differentiation of myeloid progenitors. Though both proteins have similar DNA-binding property, TLS-ERG differs from PU.1 through binding directly to RNA polymerase II as well as through escaping repression by histone modification enzymes. Together, these findings suggest that the TLS-ERG fusion protein can cause blockage of myeloid differentiation by interfering with the gene expression program normally controlled by key regulators.

Wnt-4 and Ets-1 signaling pathways for regeneration after acute renal failure

Ischemic acute renal failure (ARF) is the most common form of ARF in the adult population. The molecular mechanisms of tubular regeneration after ischemic renal injury remain largely unknown. An understanding of the mechanisms that lead to renal cell proliferation and regeneration will be necessary for the exploration of novel therapeutic strategies for the treatment of ARF. It has been suggested that regeneration processes may recapitulate developmental processes in order to restore organ or tissue function. The adult tubular epithelial cells have a potent ability of regenerate after cellular damage. We examined functional role of two developmental genes, Wnt-4 and Ets-1, in renal tubular regeneration in ARF. The Wnt-β-catenin pathway plays key roles in embryogenesis. Wnt-4 is known to be expressed in the mesonephric duct in the embryonic development. To clarify the significance of the Wnt-4-β-catenin pathway in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. After clamping left rat renal artery for 1 hour, we examined whole kidney homogenate and total RNA extracted at 3, 6, 12, 24, 48, and 72 hours after reperfusion by Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR). Wnt-4 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after ischemia, respectively. In immunohistologic examination, Wnt-4 was expressed in the proximal tubules and coexpressed with aquaporin 1 and proliferating cell nuclear antigen (PCNA). Cyclin D1 and cyclin A were expressed at 12 to 48 hours after reperfusion. Furthermore, overexpression of Wnt-4 and β-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 and cyclin A in LLC-PK1 cells. These data suggest that the Wnt-4-β-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. To clarify the significance of Ets-1 in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. Ets-1 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after the ischemia, respectively. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with PCNA. Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 hours and 6 hours in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell cycle progression of renal tubules in ARF. Our data suggest that Wnt-4-β-catenin and Ets-1 pathways regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF. These developmental genes may play key roles in dedifferentiation and regeneration of the renal tubular cells after ARF.

Synergistic action of E74B and ecdysteroid receptor in activating a 20-hydroxyecdysone effector gene

A number of insect effector genes activated by the steroid hormone 20-hydroxyecdysone (20E) are dually controlled by the ecdysteroid receptor (EcR/USP) and products of ecdysteroid early responsive genes (E74, E75, and Broad). However, the molecular mechanism of this dual action is poorly understood. Here we examined transcriptional activation of the vitellogenin (Vg) gene in the yellow fever mosquito, Aedes aegypti, by EcR/USP and E74 in response to an elevation of 20E titers. There are two isoforms of the Aedes E74 gene, AaE74A and AaE74B, which have a common C-terminal Ets DNA-binding domain and isoform-specific N termini in the female mosquito. Inhibiting expression of AaE74B but not AaE74A by RNA interference led to substantial reduction in the Vg gene expression. AaE74B and the ecdysteroid receptor synergistically enhanced 20E-induced transcription of the Vg promoter. This action required the E74-binding sites and the ecdysone response elements in the Vg 5' regulatory region. Two-hybrid assays and coimmunoprecipitation analyses demonstrated direct interaction between AaE74B and AaEcR/AaUSP. Moreover, disruption of this interaction by a dominant negative E74 mutant abolished the enhanced activation of Vg. Therefore, the cooperative interaction between AaE74B and the ecdysteroid receptor is required for high-level expression of the Vg gene in vivo. The synergistic activation is accomplished through their 20E-dependent protein-protein interaction on the gene promoter. This study reveals how the 20E direct-indirect regulation of an effector gene is achieved at the molecular level.

Up-regulation of ERM/ETV5 correlates with the degree of myometrial infiltration in endometrioid endometrial carcinoma

To elucidate alterations in gene expression in endometrioid endometrial carcinoma (EEC), differential gene expression profiling was previously described in both tumour and non-tumour contexts, and the up-regulation of the RUNX1/AML1 proto-oncogene in EEC was characterized. Among the set of genes found to be up-regulated significantly in EEC, the most relevant, ERM/ETV5, corresponds to the PEA3 subfamily and is a member of the Ets family of transcription factors that contain the Ets DNA-binding domain and are involved in matrix remodelling. In the present work, an attempt was made to characterize the expression of ERM/ETV5 in EEC throughout the process of tumourigenesis. Gene expression levels of ERM/ETV5 were quantified by real-time quantitative PCR (RT-Q-PCR) using a large panel of samples ranging from non-invasive IA to metastatic IIIA stages, and protein expression was characterized by tissue array immunohistochemistry (TMA). RT-Q-PCR validated ERM/ETV5 up-regulation in EEC and demonstrated a specific and significant increase restricted to those tumour stages associated with myometrial invasion. TMA showed that ERM/ETV5 up-regulation correlated mainly with the transition from atrophic endometrium to hyperplasia and carcinoma during tumour progression. Furthermore, ERM/ETV5 gene and protein expression levels were associated with low tumour grade. Finally, ERM/ETV5 up-regulation correlated with that of RUNX1/AML1. All of these results lead to the proposal of a co-operative role between ERM/ETV5 and RUNX1/AML1 during the early events of endometrial tumourigenesis, which may be associated with a switch to myometrial infiltration.

Ewing's Sarcoma Oncoprotein EWS–FLI1: The Perfect Target Without a Therapeutic Agent

Ewing's sarcoma family of tumors (ESFT) affect patients between the ages of 3 and 40 years, with most cases occurring in the second decade of life. ESFTs are characterized by a translocation that occurs in 95% of tumors. This translocation joins the Ewing's sarcoma gene (EWS) located on chromosome 22 to an ets family gene; either friend leukemia insertion (FLI)1 located on chromosome 11, t(11;22), or ets-related gene (ERG) located on chromosome 21, t(21;22). The EWS-FLI1 fusion transcript encodes a 68 kDa protein with two primary domains. The EWS domain is a potent transcriptional activator, while the FLI1 domain contains a highly conserved ets DNA binding domain. ESFT presents a clinical challenge, especially in patients with metastatic disease in which dose-intensifying chemotherapy with bone-marrow transplantation does not improve survival. EWS-FLI1 is only present in ESFT cells and does not exist in any normal cell of the body. Experiments using ESFT cell lines or animal xenograft models have proven that EWS-FLI1 is required for tumor survival. Therefore, ESFT contains a unique protein generated by a tumor-specific translocation that has great potential as a molecular target for therapy. However, therapeutic applications directed towards eliminating or inactivating EWS-FLI1 have not reached the clinic. EWS-FLI1 has been a very difficult molecule to directly analyze in vitro due to poor solubility. Recent advances in generating recombinant EWS-FLI1 and novel data on the cellular functions of EWS-FLI1 should enhance progress towards understanding and application.

Mutations of the PU.1 Ets domain are specifically associated with murine radiation-induced, but not human therapy-related, acute myeloid leukaemia

Murine radiation-induced acute myeloid leukaemia (AML) is characterized by loss of one copy of chromosome 2. Previously, we positioned the critical haematopoietic-specific transcription factor PU.1 within a minimally deleted region. We now report a high frequency (>65%) of missense mutation at codon 235 in the DNA-binding Ets domain of PU.1 in murine AML. Earlier studies, outside the context of malignancy, determined that conversion of arginine 235 (R235) to any other amino-acid residue leads to ablation of DNA-binding function and loss of expression of downstream targets. We show that mutation of R235 does not lead to protein loss, and occurs specifically in those AMLs showing loss of one copy of PU.1 (P=0.001, Fisher's exact test). PU.1 mutations were not found in the coding region, UTRs or promoter of human therapy-related AMLs. Potentially regulatory elements upstream of PU.1 were located but no mutations found. In conclusion, we have identified the cause of murine radiation-induced AML and have shown that loss of one copy of PU.1, as a consequence of flanking radiation-sensitive fragile domains on chromosome 2, and subsequent R235 conversion are highly specific to this mouse model. Such a mechanism does not operate, or is extremely rare, in human AML.

Carrier-independent Nuclear Import of the Transcription Factor PU.1 via RanGTP-stimulated Binding to Nup153

PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using green fluorescent protein-PU.1 fusions, we show that the Ets DNA binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted directly with two nucleoporins, Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, increased dramatically in the presence of RanGMPPNP, indicating the formation of a PU.1.RanGTP.Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Because Nup62 is located close to the midplane of the nuclear pore complex whereas Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 toward the nuclear side of the nuclear pore complex by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.

The Structural and Dynamic Basis of Ets-1 DNA Binding Autoinhibition

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an alpha-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1DeltaN301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1DeltaN301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1DeltaN301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1DeltaN300 dimer, confirming that the latter represents a domain-swapped species. (15)N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only approximately 15- and approximately 75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.

Expression and Function of Ets-1 during Experimental Acute Renal Failure in Rats

The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. The hypothesis in this study was that Ets-1 is re-expressed during regeneration after acute renal failure (ARF) and plays a key role in the transcriptional regulation of cyclin D1 and the cell cycle progression in renal tubular cells. For clarifying the significance of Ets-1 in ARF, a rat ARF model in vivo and LLC-PK1 cells as an in vitro model were used. After the left rat renal artery was clamped for 1 h, the whole kidney homogenate was examined and total RNA was extracted at 6, 12, 24, 48, and 72 h after reperfusion by Western blot analysis and real-time reverse transcription-PCR. Ets-1 mRNA and protein expression were strongly increased at 6 to 24 h after the ischemia, respectively. The expression of hypoxia-inducible factor-1alpha was increased dramatically as early as 6 h after ischemia-reperfusion and decreased at 48 and 72 h after ischemia-reperfusion. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with proliferating cell nuclear antigen (PCNA). Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 and 6 h in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. The Ets-1 induction by hypoxia was abolished by the transfection of dominant-negative hypoxia-inducible factor-1alpha. A gel shift assay demonstrated that Ets-1 binds to the ets-1 binding site of the cyclin D1 promoter in the ischemia-reperfusion condition. Overexpression of Ets-1 did not significantly change the caspase 3 activity or the value of cell death ELISA in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell-cycle progression of renal tubules in ARF. The Ets-1 pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.

Cloning and Characterization of a Novel Leukemogenetic Gene TEL-PTPRR.

The TEL gene mapped on 12p13 is rearranged by chromosomal translocations in a variety of human leukemias and is fused to various partner genes mainly encoding receptor type and non-receptor type tyrosine kinases and transcription factors. Using 3′ RACE method, we have cloned novel chimeric cDNAs TEL/protein tyrosine phosphatase receptor type R (PTPRR) that is generated by inv(12)(p13q13) found in an acute myeloid leukemia case. TEL is a member of ETS family transcription factors and contains the homodimerizing or heterodimerizing helix-loop-helix domain at the N-terminus and the DNA-binding ETS domain at the C-terminus. TEL works as a tumor suppressor. On the other hand, PTPRR is a brain-specific receptor type protein tyrosine phosphatase with a single intracellular catalytic domain. PTPRR is the first tyrosine phosphatase that was identified as a fusion partner for TEL. The chromosomal rearrangement joined exon 4 in the TEL gene and exon 7 in the PTPRR gene and produced ten isoforms of TEL/PTPRR cDNAs through alternative splicing. Among them, only one expresses TEL/PTPRR chimeric protein that consists of the HLH domain from TEL and the catalytic domain from PTPRR and the others express truncated forms of TEL. Because reciprocal PTPRR-TEL mRNA as well as wild-type-PTPRR mRNA was not detected by RT-PCR analysis, TEL-PTPRR is likely to be involved in leukemogenesis. To clarify molecular mechanisms by inv(12), we first molecularly analyzed the TEL/PTPRR chimeric cDNA and the N-terminally truncated TEL cDNA containing exons 1–4 (tTEL). When transiently expressed in NIH3T3 cells, both TEL/PTPRR and tTEL were equally contributed to the cytoplasm and the nucleus. Moreover, both isoforms were found to associate with wild-type-TEL by immunoprecipitation assays. Therefore, we speculated that TEL/PTPRR and tTEL affect wild-type-TEL's functions in the nucleus through heterodimerizing with it. As expected from their structures, both isoforms did not bind to the ETS binding consensus sequence (EBS). In reporter assays with (ETS)3tkLuc, they did not repress transcription through EBS, while wild-type-TEL did. Interestingly, both TEL/PTPRR and tTEL dominantly interfered with the wild-type-TEL-induced transcriptional repression in a dose-dependent manner. On in vitro phosphatase assays using p-NPP as a substrate, both isoforms were shown to completely lack phosphatase activities. To assess the biological properties of TEL/PTPRR, we employed human megakaryoblastic leukemia cell line UT7/GM that proliferate completely depending on granulocyte macrophage-colony stimulating factor (GM-CSF). Overexpression of TEL/PTPRR resulted in factor-independent proliferation in UT7/GM cells. Interestingly, phosphorylated STAT3 remained after GM-CSF withdrawal in the TEL/PTPRR-overexpressing cells, while it rapidly declined in the mock cells. All these data collectively suggest that appearance of inv(12) causes leukemic transformation in human myeloid cells, at least partly through inactivating tumor-suppressive functions of wild-type-TEL. Considering that STAT3-mediated signals are maintained even after the cytokine depletion when TEL/PTPRR is overexpressed, TEL/PTPRR may regulate JAK/STAT signals through unknown mechanisms.

TEL/AML1 Shows Dominant-Negative Effects over TEL as Well as AML1.

The TEL/AML1 chimeric gene is generated by the t(12;21) translocation in pre-B cell acute lymphoblastic leukemia. TEL/AML1 consists of the helix-loop-helix (HLH) dimerization domain from TEL and almost the entire of AML1, but loses the ETS DNA-binding domain from TEL. Dominant-negative effects of TEL/AML1 over wild-type-AML1 are believed to trigger the development of this type of leukemia. However, it could also be possible that TEL/AML1 affects wild-type-TEL's molecular and tumor suppressive functions through the HLH domain. To test this hypothesis, we investigated molecular and biological effects of TEL/AML1 on wild-type-TEL.Since TEL/AML1 contains the HLH domain from TEL, we first tested whether TEL/AML1 associates with wild-type-TEL by immunoprecipitation analysis. Wild-type-TEL was co-immunoprecipitated with TEL/AML1, but not with a TEL/AML1 mutant lacking the HLH domain. This suggests that TEL/AML1 associates with wild-type-TEL depending on the HLH domain and might affect its functions. To clarify functional alterations of TEL/AML1, we compared molecular function of wild-type-TEL and TEL/AML1 in DNA-binding and transcriptional reporter assays. Wild-type-TEL bound to ETS-binding site (EBS) and showed transcriptional repression on stromelysin-1 promoter containing EBS. On the other hand, TEL/AML1 failed to bind to EBS and showed no transcriptional repression on it. Moreover, TEL/AML1 showed a dominant-negative effect over the wild-type-TEL-mediated transcriptional repression. These data suggest that TEL/AML1 loses wild-type-TEL's molecular functions, but works as a dominant-negative molecule over wild-type-TEL. To further investigate TEL/AML1's biological effects over wild-type-TEL, we established NIH3T3 stable cell lines expressing wild-type-AML1, wild-type-TEL or TEL/AML1 and performed colony assays. TEL/AML1-expressing cells produced approximately the same numbers of colonies as the mock cells. Wild-type-AML1-expressing cells generated much more colonies, while wild-type-TEL-expressing cells generated less colonies. We further introduced TEL/AML1 cDNA into the wild-type-AML1- and the wild-type-TEL-expressing cells. Co-expression of TEL/AML1 releaved the wild-type-TEL's growth-suppression as well as the wild-type-AML1's growth-stimulation. Finally, we counted cell numbers of the same set of NIH3T3 stable cell lines in liquid culture and obtained the similar results as in soft agar culture.In this study we demonstrate that TEL/AML1 dominantly inhibits both molecular and biological functions of wild-type-TEL, possibly through heterodimerizing with it. Therefore, appearance of the t(12;21) translocation could lead to not only inactivation of wild-type-AML1 but also inactivation of wild-type-TEL. As TEL is a tumor suppressor, functional loss of wild-type-TEL seems to be one hit for the development of leukemia carrying the t(12;21) translocation. However, it has been reported that expression of TEL/AML1 is not sufficient to fully transform hematopoietic cells in vitro and in vivo and requires other hits to cause leukemia. Since deletion of the non-translocated TEL allele is associated in the vast majority of leukemia with the t(12;21) translocation, complete inactivation of wild-type-TEL by the allelic loss could be necessary to be the second hit that provides a further proliferative advantage to leukemic cells.

Carrier-Independent Nuclear Import of the Transcription Factor PU.1.

PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using GFP-PU.1 fusions, we show that the Ets DNA-binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted with the FG repeats of nucleoporins Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, dramatically increased in the presence of RanGMPPNP, indicating the formation of a PU.1/RanGTP/Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Since Nup62 is located close to the midplane of the nuclear pore complex (NPC) while Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 towards the nuclear side of the NPC by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.

TEL/AML1 shows dominant-negative effects over TEL as well as AML1

The TEL/AML1 chimeric gene is generated by the t(12;21) translocation in pre-B cell acute lymphoblastic leukemia. TEL/AML1 consists of the helix–loop–helix (HLH) dimerization domain from TEL and almost the entire of AML1, but loses the ETS DNA-binding domain from TEL. Dominant-negative effects of TEL/AML1 over wild-type-AML1 are believed to trigger the development of this type of leukemia. However, it could also be possible that TEL/AML1 affects wild-type-TEL’s molecular and tumor suppressive functions through the HLH domain. To test this possibility, we first confirmed that TEL/AML1 associates with wild-type-TEL. TEL/AML1 neither bound to the ETS-binding consensus site nor repressed transcription through it. Regardless, this prevented wild-type-TEL-induced transcriptional repression. Moreover, TEL/AML1 concomitantly inhibited wild-type-TEL-induced growth suppression and wild-type-AML1-mediated transforming activity in NIH3T3 cells. All these data indicate that TEL/AML1 exerts dominant-interfering effects on both AML1 and TEL, and that expression of TEL/AML1 could result in inactivation of TEL’s tumor suppressive functions in t(12;21)-carrying leukemia.

The ETS transcription factor GABPalpha is essential for early embryogenesis.

The ETS transcription factor complex GABP consists of the GABPalpha protein, containing an ETS DNA binding domain, and an unrelated GABPbeta protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpalpha gene. Embryos homozygous for the null Gabpalpha allele die prior to implantation, consistent with the broad expression of Gabpalpha throughout embryogenesis and in embryonic stem cells. Gabpalpha(+/-) mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpalpha protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpalpha function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo.