ETS Domain Research Articles

Abstract B7: ETS1 overexpression in prostate cancer cells

Abstract The ETS1 transcription factor is overexpressed in human prostate cancers in comparison to nonmalignant prostate and exhibits nuclear, in addition to aberrant cytoplasmic localisation. A previous report has identified direct interaction between ETS1 and the androgen receptor (AR) in the prostate cancer cell line, LNCaP and we have demonstrated that this ligand-dependent interaction is mediated via the ETS (DNA binding) domain of ETS1 and the ligand-binding domain of the AR. In a cohort of 44 prostate cancer cases, mRNA for the full-length ETS1 isoform, p54ETS1 was detected in all prostate tumour and adjacent nonmalignant prostate tissues, while expression of the internally truncated p42ETS1 isoform was very low or undetectable. In protein lysates extracted from the same cases, p54ETS1 was detected in all samples, with expression of a lower molecular weight ETS1 corresponding in size to the dominant negative p27ETS1 isoform also observed. Increased expression of one or both of the ETS1 isoforms was identified in 38 (86%) cases, supporting the elevated expression of ETS1 in human prostate tumours. Both ETS1 and the AR upregulate expression of the prostatic tumour suppressor, NKX3.1 via sequences located in the 5′ promoter and 3′ untranslated region (3′UTR), respectively. Although unresponsive to androgens, the proximal ∼2kb of the NKX3.1 promoter, and in particular a 76bp sequence surrounding the ETS binding site (EBS) become androgen responsive following ETS1 overexpression. In addition, in LNCaP cells that stably overexpress p54ETS1, the biphasic proliferative responses to androgens, where low (≤10−10M) DHT concentrations stimulate proliferation and higher (>10−10M) DHT levels inhibit proliferation, were altered and DHT concentrations as low as 10−12M inhibited proliferation. The findings indicate that the increased expression of ETS1 commonly observed in human prostate tumours may modify the androgen responsiveness of the cancer cells, augmenting AR-mediated regulation of androgen target genes and potentially sensitising prostate cancer cells to the very low androgen levels in men treated with androgen ablation therapies. Citation Format: Jacqueline M. Bentel, Jennet Harvey, Marc A. Thomas, Jamie J. Rodgers, Ebony J. Rouse, Darren M. Preece, Siaw M. Chai, Michael R. Epis, Ronald J. Cohen, Peter J. Leedman, Kimberley Roehrig. ETS1 overexpression in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B7.

The microRNA miR-34 modulates ageing and neurodegeneration in Drosophila

Human neurodegenerative diseases possess the temporal hallmark of afflicting the elderly population. Hence, aging is among the most significant factors to impinge on disease onset and progression1, yet little is known of molecular pathways that connect these processes. Central to understanding this connection is to unmask the nature of pathways that functionally integrate aging, chronic maintenance of the brain and modulation of neurodegenerative disease. microRNAs (miRNA) are emerging as critical players in gene regulation during development, yet their role in adult-onset, age-associated processes are only beginning to be revealed. Here we report that the conserved miRNA miR-34 regulates age-associated events and long-term brain integrity in Drosophila, presenting such a molecular link between aging and neurodegeneration. Fly miR-34 expression is adult-onset, brain-enriched and age-modulated. Whereas miR-34 loss triggers a gene profile of accelerated brain aging, late-onset brain degeneration and a catastrophic decline in survival, miR-34 upregulation extends median lifespan and mitigates neurodegeneration induced by human pathogenic polyglutamine (polyQ) disease protein. Some of the age-associated effects of miR-34 require adult-onset translational repression of Eip74EF, an essential ETS domain transcription factor involved in steroid hormone pathways. These studies indicate that miRNA-dependent pathways may impact adult-onset, age-associated events by silencing developmental genes that later have a deleterious influence on adult life cycle and disease, and highlight fly miR-34 as a key miRNA with a role in this process

Sequence-Specific Control of ETS Transcription Factors

The ETS family of transcription factors participate in a broad array of developmental and disease processes. ETS proteins are united by a conserved DNA-binding domain that targets 10-bp sequences harboring a central 5’-GGAA/T-3’ consensus. A primary recognition helix confers specificity for the consensus by direct readout of the major groove while adjoining mobile elements contact backbone contacts of flanking 5' and 3' sequences. Although flanking contacts play a secondary role in specifying the ETS binding site, they profoundly influence its affinity and transactivational activity. Elucidating the biophysical basis for the intrinsic selectivity of the ETS domain is therefore essential to understanding other interactions within the transcription regulatory complex. We have characterized the structure and thermodynamics of the ETS complexes with a high-, low-affinity, and nonspecific sequence. Calorimetric measurements reveal that the unbound ETS domain is a noncovalent, entropically-driven dimer. DNA binding induces ETS dissociation with sequence-dependent thermodynamics, suggesting distinct conformational arrangements accompanying the binding of each sequence variant. Footprinting titrations show that hypersensitivity to DNase I at the consensus is an experimental hallmark of sequence-specific ETS binding, regardless of affinity, indicating the importance of DNA distortion triggered by indirect readout. High- and low-affinity ETS cognates can be readily differentiated in terms of sensitivity to methylation in the latter. Based on this data, we present a model for ETS-DNA interactions in which indirect and direct readout are structurally linked to achieve sequence-specific binding. Complex formation in turn induces rheostatic rearrangements of ETS conformation (as manifest in terms of complex stability) that represent specific permissive or inhibitory states for protein-protein interactions (as manifest in ETS dimerization). Thus, the sequence identity of an ETS binding site may serve a functional role in initiating regulatory functions as well as an address for localization in the genome.

Ets-1 Activates Overexpression of JunB and CD30 in Hodgkin's Lymphoma and Anaplastic Large-Cell Lymphoma

Overexpression of CD30 and JunB is a hallmark of tumor cells in Hodgkin's lymphoma (HL) and anaplastic large-cell lymphoma (ALCL). We reported that CD30-extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signaling induces JunB, which maintains constitutive activation of the CD30 promoter. Herein, we localize a cis-acting enhancer in the JunB promoter that is regulated by Ets-1. We show that E26 transformation-specific-1 (Ets-1) (-146 to -137) enhances JunB promoter activation in a manner that is dependent on CD30 or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-ERK1/2 MAPK pathway. Ets-1 knockdown reduces the expression of both JunB and CD30, and CD30 knockdown significantly reduces JunB expression in HL and ALCL cell lines. NPM-ALK knockdown also reduces JunB expression in ALCL cell lines expressing NPM-ALK. Collectively, these results indicate that CD30 and NPM-ALK cooperate to activate the ERK1/2 MAPK-Ets-1 pathway. Ets-1, constitutively activated by ERK1/2-MAPK, plays a central role in the overexpression of JunB and CD30, which are both involved in the pathogenesis of HL and ALCL.

Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Abstract 1284GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. Disclosures:No relevant conflicts of interest to declare.

Molecular dynamics simulations and in silico peptide ligand screening of the Elk-1 ETS domain.

BackgroundThe Elk-1 transcription factor is a member of a group of proteins called ternary complex factors, which serve as a paradigm for gene regulation in response to extracellular signals. Its deregulation has been linked to multiple human diseases including the development of tumours. The work herein aims to inform the design of potential peptidomimetic compounds that can inhibit the formation of the Elk-1 dimer, which is key to Elk-1 stability. We have conducted molecular dynamics simulations of the Elk-1 ETS domain followed by virtual screening.ResultsWe show the ETS dimerisation site undergoes conformational reorganisation at the α1β1 loop. Through exhaustive screening of di- and tri-peptide libraries against a collection of ETS domain conformations representing the dynamics of the loop, we identified a series of potential binders for the Elk-1 dimer interface. The di-peptides showed no particular preference toward the binding site; however, the tri-peptides made specific interactions with residues: Glu17, Gln18 and Arg49 that are pivotal to the dimer interface.ConclusionsWe have shown molecular dynamics simulations can be combined with virtual peptide screening to obtain an exhaustive docking protocol that incorporates dynamic fluctuations in a receptor. Based on our findings, we suggest experimental binding studies to be performed on the 12 SILE ranked tri-peptides as possible compounds for the design of inhibitors of Elk-1 dimerisation. It would also be reasonable to consider the score-ranked tri-peptides as a comparative test to establish whether peptide size is a determinant factor of binding to the ETS domain.

Vascular endothelial and endocardial progenitors differentiate as cardiomyocytes in the absence of Etsrp/Etv2 function

Previous studies have suggested that embryonic vascular endothelial, endocardial and myocardial lineages originate from multipotential cardiovascular progenitors. However, their existence in vivo has been debated and molecular mechanisms that regulate specification of different cardiovascular lineages are poorly understood. An ETS domain transcription factor Etv2/Etsrp/ER71 has been recently established as a crucial regulator of vascular endothelial differentiation in zebrafish and mouse embryos. In this study, we show that etsrp-expressing vascular endothelial/endocardial progenitors differentiate as cardiomyocytes in the absence of Etsrp function during zebrafish embryonic development. Expression of multiple endocardial specific markers is absent or greatly reduced in Etsrp knockdown or mutant embryos. We show that Etsrp regulates endocardial differentiation by directly inducing endocardial nfatc1 expression. In addition, Etsrp function is required to inhibit myocardial differentiation. In the absence of Etsrp function, etsrp-expressing endothelial and endocardial progenitors initiate myocardial marker hand2 and cmlc2 expression. Furthermore, Foxc1a function and interaction between Foxc1a and Etsrp is required to initiate endocardial development, but is dispensable for the inhibition of myocardial differentiation. These results argue that Etsrp initiates endothelial and endocardial, and inhibits myocardial, differentiation by two distinct mechanisms. Our findings are important for the understanding of genetic pathways that control cardiovascular differentiation during normal vertebrate development and will also greatly contribute to the stem cell research aimed at regenerating heart tissues.

Elk-1 interacts with dynein upon serum stimulation but independent of serine 383 phosphorylation.

Elk-1 belongs to the Ternary Complex Factor (TCF) subfamily of the ETS (from E26 viral oncogene) domain superfamily of transcription factors, and has been known as a regulator of mitogen-induced immediate early gene transcription upon Mitogen Activated Protein Kinase (MAPK) activation. Elk-1 has been previously shown to interact with neuronal microtubules, and here we show that P-S383-Elk-1, in addition to co-localizing with motor proteins kinesin, Eg5 and Mitotik Kinesin-Like Protein (MKLP) at the mitotic spindles, it physically interacts with dynein in a serum induction-dependent but Ser383 phosphorylation-independent manner.

Resection of Non–Small Cell Lung Cancers Reverses Tumor-Induced Gene Expression Changes in the Peripheral Immune System

To characterize the interactions of non-small cell lung cancer (NSCLC) tumors with the immune system at the level of mRNA and microRNA (miRNA) expression and to define expression signatures that characterize the presence of a malignant tumor versus a nonmalignant nodule. We have examined the changes of both mRNA and miRNA expression levels in peripheral blood mononuclear cells (PBMC) between paired samples collected from NSCLC patients before and after tumor removal using Illumina gene expression arrays. We found that malignant tumor removal significantly changes expression of more than 3,000 protein-coding genes, especially genes in pathways associated with suppression of the innate immune response, including natural killer cell signaling and apoptosis-associated ceramide signaling. Binding sites for the ETS domain transcription factors ELK1, ELK4, and SPI1 were enriched in promoter regions of genes upregulated in the presence of a tumor. Additional important regulators included five miRNAs expressed at significantly higher levels before tumor removal. Repressed protein-coding targets of those miRNAs included many transcription factors, several involved in immunologically important pathways. Although there was a significant overlap in the effects of malignant tumors and benign lung nodules on PBMC gene expression, we identified one gene panel which indicates a tumor or nodule presence and a second panel that can distinguish malignant from nonmalignant nodules. A tumor presence in the lung influences mRNA and miRNA expression in PBMC and this influence is reversed by tumor removal. These results suggest that PBMC gene expression signatures could be used for lung cancer diagnosis.

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.

Protein expression of the Ets transcription factor Elf-1 in breast cancer cells is negatively correlated with histological grading, but not with clinical outcome

Several members of the Ets (E26 transformation specific) transcription factor family are involved in tumor progression, e.g. by activating matrix metalloproteases. Ets proteins share a unique DNA-binding domain, the Ets domain, which specifically recognizes GGAA/T-containing sequences common in many promoters. While the roles of quite a number of Ets proteins in carcinogenesis have been well established, little is known about the importance of the Ets protein Elf-1 (E74-like factor 1) in cancer. Herein, we analyzed the expression of Elf-1 in breast cancer. We found that, like T-cells, breast cancer cells express both the 80 and 98 kDa isoforms of the Elf-1 protein with the 98 kDa isoform only be present in the nucleus. Immunohistochemical analysis of 119 breast cancer biopsies showed anti-Elf-1 immunoreactivity exclusively in the nucleus. Elf-1 expression varied largely among the breast cancer samples showing a negative correlation with histological grading. However, no association of Elf-1 expression with clinical outcome was observed, even when sub-cohorts of patients who received either only adjuvant endocrine treatment or only chemotherapy were separately analyzed. These data suggest that Elf-1 may modulate breast cancer progression to some extent without having an impact on survival of breast cancer patients.

The Ets Transcription Factor ELF5 Functions as a Tumor Suppressor in the Kidney

Renal cell carcinoma is an important clinical disease with poorly understood etiology. ELF5 is an epithelial-specific member of the Ets family of transcription factors, characterized by the 80 amino acid Ets domain that binds the purine-rich GGAA/T Ets motif found in the promoter regions of a variety of genes. Since ELF5 is highly expressed in kidney and has been postulated to function as a tumor suppressor, at least in the context of the breast, we investigated its role in kidney cancer. In renal cell carcinoma ELF5 expression was consistently decreased in tumor samples versus normal. ELF5 mRNA was decreased in 94% of lesions tested and ELF5 protein was undetectable in 40/40 kidney-derived carcinomas. Re-expression of the ELF5 gene in 786-O renal carcinoma cells suppressed their tumorigenic capacity in vitro and in vivo. This work is the first to suggest that ELF5 has tumor suppressor activity in the kidney.

Genomic and Biochemical Insights into the Specificity of ETS Transcription Factors

ETS proteins are a group of evolutionarily related, DNA-binding transcriptional factors. These proteins direct gene expression in diverse normal and disease states by binding to specific promoters and enhancers and facilitating assembly of other components of the transcriptional machinery. The highly conserved DNA-binding ETS domain defines the family and is responsible for specific recognition of a common sequence motif, 5'-GGA(A/T)-3'. Attaining specificity for biological regulation in such a family is thus a conundrum. We present the current knowledge of routes to functional diversity and DNA binding specificity, including divergent properties of the conserved ETS and PNT domains, the involvement of flanking structured and unstructured regions appended to these dynamic domains, posttranslational modifications, and protein partnerships with other DNA-binding proteins and coregulators. The review emphasizes recent advances from biochemical and biophysical approaches, as well as insights from genomic studies that detect ETS-factor occupancy in living cells.

Oncogenic Fusion Protein EWS/FLI1 Down-regulates Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms

Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.

Dimer formation and conformational flexibility ensure cytoplasmic stability and nuclear accumulation of Elk-1

The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin–proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.

ETS-domain transcription factor Elk-1 mediates neuronal survival: SMN as a potential target

Elk-1 belongs to the ternary complex factors (TCFs) subfamily of the ETS domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with serum response elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAi-mediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-of-motor neuron-1 ( SMN1) gene as a novel target of Elk-1, and show that the ets motifs in the SMN1 promoter are involved in this regulation.

Probing the Conformational Dynamics of Protein-DNA Complexes Through Hydration

The DNA-binding domain of the ETS family of transcription factors interacts with DNA in a sequence-selective manner, yet it tolerates remarkable variation in the recognition sequence. The biophysical basis for this sequence selectivity is currently unknown. Previous investigations have found significant differences in the thermodynamics of strong and weak ETS-DNA complexes. However, the ETS domain is structurally similar in both DNA-bound and unbound states. In the absence of significant coupled folding by either protein or DNA, selectivity may be linked to differences in conformational dynamics in the protein-DNA complex. Specifically, sequence selectivity may arise from differential reductions in conformational dynamics upon complex formation. We hypothesize that time-averaged differences in conformation dynamics can be resolved through their equilibrium hydration properties. The rationale for our hypothesis is that folded but conformationally mobile segments should be more solvent-accessible than segments that are relatively immobilized. To test this hypothesis, we are probing the hydration of various ETS-DNA complexes by high-resolution DNA and protein footprinting. We expect that this approach will provide a quantitative measure of a complex's overall conformational dynamics as well as identify structural elements that become conformationally stabilized upon complex formation.

Gabp Transcription Factor Is Required for Myeloid Differentiation, In Part, through Its Control of Gfi-1 Expression

AbstractAbstract 740GABP is an ets transcription factor which 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. In order to examine the role of Gabp in myeloid differentiation, we created mice in which exons that encode the ets DNA-binding domain are flanked by LoxP recombination sites (Gabpa fl/fl or floxed mice), and bred them to mice that bear Cre recombinase under the control of the pIC-inducible promoter, Mx1. Injection of these mice with pIC induces conditional deletion of Gabpa in hematopoietic tissues, and these mice are referred to as Gabpa knock-out mice (KO). Control mice that carry floxed Gabpa but lack Mx1-Cre were also treated with pIC. KO mice rapidly lost myeloid cells in peripheral blood, bone marrow, and spleen. Bone marrow cells from control and KO mice were sorted for CD11b and Gr1 to distinguish mature granulocytes from immature granulocytes and monocytes. KO mice had few mature myeloid cells, and the residual cells exhibited a dysplastic phenotypic appearance that did not resemble either normal maturing monocytes or granulocytes; instead they exhibited morphologic characteristics of both lineages. These KO myeloid cells simultaneously expressed differentiation-associated monocyte-restricted and granulocyte-restricted genes and markers of immaturity (Ly6C and c-kit). Thus, the patterns of gene and antigen expression by these dysplastic KO myeloid cells did not correspond to either normal monocytes or granulocytes. We recognized that these KO myeloid cells resemble those seen in mice with disruption of the transcriptional repressor, Gfi-1. We isolated granulocyte-monocyte progenitor (GMP) cells from control and KO bone mice, and found that expression of Gfi-1 was significantly reduced in KO GMPs, compared to controls. Chromatin immunoprecipitation demonstrated that Gabp bound the Gfi-1 promoter in vivo and transfection analysis showed that Gabp activated the Gfi-1 promoter. To test the hypothesis that Gabp controls myeloid differentiation through its regulation of Gfi-1, we grew bone marrow cells from both control and KO mice in liquid culture with SCF, Il-3 and Il-6, or GM-CSF. We transduced these cells with retrovirus bearing Gfi-1, and analyzed myeloid colony formation, and expression of CD11b and Gr1. Transduction of KO bone marrow cells with Gfi-1 partially restored myeloid colony formation in semi-solid media, and the percentage of Gr1+CD11b+ cells in liquid culture. We conclude that GABP plays a non-redundant role in myeloid development, and it controls differentiation of myeloid cells, at least in part, through its regulation of the transcriptional repressor, Gfi-1. Disclosures:No relevant conflicts of interest to declare.

The Drosophila jing gene is a downstream target in the Trachealess/Tango tracheal pathway

Primary branching in the Drosophila trachea is regulated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix-PAS (bHLH-PAS) heterodimers, the POU protein Drifter (Dfr)/Ventral Veinless (Vvl), and the Pointed (Pnt) ETS transcription factor. The jing gene encodes a zinc finger protein also required for tracheal development. Three Trh/Tgo DNA-binding sites, known as CNS midline elements, in 1.5 kb of jing 5′ cis-regulatory sequence (jing1.5) previously suggested a downstream role for jing in the pathway. Here, we show that jing is a direct downstream target of Trh/Tgo and that Vvl and Pnt are also involved in jing tracheal activation. In vivo lacZ enhancer detection assays were used to identify cis-regulatory elements mediating embryonic expression patterns of jing. A 2.8-kb jing enhancer (jing2.8) drove lacZ expression in all tracheal cell lineages, the CNS midline and Engrailed-positive segmental stripes, mimicking endogenous jing expression. A 1.3-kb element within jing2.8 drove expression that was restricted to Engrailed-positive CNS midline cells and segmental ectodermal stripes. Surprisingly, jing1.5-lacZ expression was restricted to tracheal fusion cells despite the presence of consensus DNA-binding sites for bHLH-PAS, ETS, and POU domain transcription factors. Given the absence of Trh/Tgo DNA-binding sites in the jing1.3 enhancer, these results are consistent with previous observations suggesting a combinatorial basis to Trh-/Tgo-mediated transcriptional regulation in the trachea.

Amino acid residues in the β3 strand and subsequent loop of the conserved ETS domain that mediate basic leucine zipper (bZIP) recruitment and potentially distinguish functional attributes of Ets proteins

Ets family members share a conserved DNA-binding ETS domain, and serve a variety of roles in development, differentiation and oncogenesis. Besides DNA binding, the ETS domain also participates in protein-protein interactions with other structurally unrelated transcription factors. Although this mechanism appears to confer tissue- or development stage-specific functions on individual Ets proteins, the biological significance of many of these interactions remains to be evaluated, because their molecular basis has been elusive. We previously demonstrated a direct interaction between the ETS domain of the widely expressed GABPalpha (GA-binding protein alpha) and the granulocyte inducer C/EBPalpha (CCAAT/enhancer-binding protein alpha), and suggested its involvement in co-operative transcriptional activation of myeloid-specific genes, such as human FCAR encoding FcalphaR [Fc receptor for IgA (CD89)]. By deletion analysis, we identified helix alpha3 and the beta3/beta4 region as the C/EBPalpha-interacting region. Domain-swapping of individual sub-domains with those of other Ets proteins allowed us to highlight beta-strand 3 and the subsequent loop, which when exchanged by those of Elf-1 (E74-like factor 1) reduced the ability to recruit C/EBPalpha. Further analysis identified a four-amino acid swap mutation of this region (I387L/C388A/K393Q/F395L) that reduces both physical interaction and co-operative transcriptional activation with C/EBPalpha without affecting its transactivation capacity by itself. Moreover, re-ChIP (re-chromatin immunoprecipitation) analysis demonstrated that GABPalpha recruits C/EBPalpha to the FCAR promoter, depending on these residues. The identified amino acid residues could confer the specificity of the action on the Ets proteins in diverse biological processes through mediating the recruitment of its partner factor.