Dennis K. Watson

Ets target genes: past, present and future.

Ets is a family of transcription factors present in species ranging from sponges to human. All family members contain an approximately 85 amino acid DNA binding domain, designated the Ets domain. Ets proteins bind to specific purine-rich DNA sequences with a core motif of GGAA/T, and transcriptionally regulate a number of viral and cellular genes. Thus, Ets proteins are an important family of transcription factors that control the expression of genes that are critical for several biological processes, including cellular proliferation, differentiation, development, transformation, and apoptosis. Here, we tabulate genes that are regulated by Ets factors and describe past, present and future strategies for the identification and validation of Ets target genes. Through definition of authentic target genes, we will begin to understand the mechanisms by which Ets factors control normal and abnormal cellular processes.

Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor.

ABSTRACT The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.

Ets proteins in biological control and cancer

The Ets family consists of a large number of evolutionarily conserved transcription factors, many of which have been implicated in tumor progression. Extensive studies on this family of proteins have focused so far mainly on the biochemical properties and cellular functions of individual factors. Since most of the Ets factors can bind to the core consensus DNA sequence GGAA/T in vitro, it has been a challenge to differentiate redundant from specific functions of various Ets proteins in vivo. Recent findings, however, suggest that such apparent redundancy may in fact be a central component of a network of differentially regulated specific Ets factors, resulting in distinct biological and pathological consequences. The programmed “Ets conversion” appears to play a critical role during tumor progression, especially in control of cellular changes during epithelial–mesenchymal transition and metastasis. Coordination of multiple Ets gene functions also mediates interactions between tumor and stromal cells. As such, these new insights may provide a novel view of the Ets gene family as well as a focal point for studying the complex biological control involved in tumor progression.

Regulation of Ets function by protein - protein interactions.

Ets proteins are a family of transcription factors that share an 85 amino acid conserved DNA binding domain, the ETS domain. Over 25 mammalian Ets family members control important biological processes, including cellular proliferation, differentiation, lymphocyte development and activation, transformation and apoptosis by recognizing the GGA core motif in the promoter or enhancer of their target genes. Protein–protein interactions regulates DNA binding, subcellular localization, target gene selection and transcriptional activity of Ets proteins. Combinatorial control is a characteristic property of Ets family members, involving interaction between Ets and other key transcriptional factors such as AP-1, NFκB and Pax family members. Specific domains of Ets proteins interact with many protein motifs such as bHLH, bZipper and Paired domain. Such interactions coordinate cellular processes in response to diverse signals including cytokines, growth factors, antigen and cellular stresses.

EAP1/Daxx interacts with ETS1 and represses transcriptional activation of ETS1 target genes

ETS1 is a member of the evolutionarily conserved family of ets genes, which are transcription factors that bind to unique DNA sequences, either alone or by association with other proteins. In this study, we have used the yeast two-hybrid system to identify an ETS1 interacting protein. The ETS1 N-terminal amino acid region was used as bait and an interaction was identified with the Daxx protein, referred to as EAP1 (ETS1 Associated Protein 1)/Daxx. This interactin has been shown to exist in yeast and in vitro. EAP1/Daxx and ETS1 are co-localized in the nucleus of mammalian cells. The region in EAP1/Daxx which specifically binds to ETS1 is located within its carboxy terminal 173 amino acid region. The ETS1 interaction region is located within its N-terminal 139 amino acids and is referred as the Daxx Interaction Domain (DID). The DID appears to be conserved in several other ets family members, as well as in other proteins known to interact with Daxx. The EAP1/Daxx interacts with both isoforms of ETS1, p51-ETS1 and p42-ETS1. Interaction of EAP1/Daxx with ETS1 causes the repression of transcriptional activation of the MMP1 and BCL2 genes. The interaction domains of both ETS1 and EAP1/Daxx are required for this repression and deletion of either domain abolishes this activity.

Persistent down-regulation of Fli1, a suppressor of collagen transcription, in fibrotic scleroderma skin

The molecular and cellular mechanisms that maintain proper collagen homeostasis in healthy human skin and are responsible for the dysregulated collagen synthesis in scleroderma remain primarily unknown. This study demonstrates that Fli1 is a physiological negative regulator of collagen gene expression in dermal fibroblasts in vitro and in human skin in vivo. This conclusion is supported by the analyses of mouse embryonic fibroblasts from Fli1(-/-), Fli1(+/-), and Fli1(+/+) mice. In cultured human and mouse fibroblasts Fli1 expression levels are inversely correlated with the collagen type I expression levels. These in vitro observations were validated in vivo. In healthy human skin Fli1 protein is expressed in fibroblasts and endothelial cells. Significantly, absence of Fli1 expression in individual fibroblasts correlates with elevated collagen synthesis. In contrast to healthy skin, Fli1 protein is consistently absent from fibroblasts and significantly reduced in endothelial cells in clinically involved scleroderma skin, which correlates with enhanced collagen synthesis in systemic sclerosis skin. This study supports the role of Fli1 as a suppressor of collagen transcription in human skin in vivo. Persistent down-regulation of Fli1 in scleroderma fibroblasts in vivo may directly contribute to uncontrolled matrix deposition in scleroderma skin.

Endothelial Fli1 Deficiency Impairs Vascular Homeostasis : A Role in Scleroderma Vasculopathy

Systemic sclerosis or scleroderma (SSc) is a complex autoimmune connective tissue disease characterized by obliterative vasculopathy and tissue fibrosis. The molecular mechanisms underlying SSc vasculopathy are largely unknown. Friend leukemia integration factor 1 (Fli1), an important regulator of immune function and collagen fibrillogenesis, is expressed at reduced levels in endothelial cells in affected skin of patients with SSc. To develop a disease model and to investigate the function of Fli1 in the vasculature, we generated mice with a conditional deletion of Fli1 in endothelial cells (Fli1 CKO). Fli1 CKO mice showed a disorganized dermal vascular network with greatly compromised vessel integrity and markedly increased vessel permeability. We show that Fli1 regulates expression of genes involved in maintaining vascular homeostasis including VE-cadherin, platelet endothelial cell adhesion molecule 1, type IV collagen, matrix metalloproteinase 9, platelet-derived growth factor B, and S1P(1) receptor. Accordingly, Fli1 CKO mice are characterized by down-regulation of VE-cadherin and platelet endothelial cell adhesion molecule 1, impaired development of basement membrane, and a decreased presence of alpha-smooth muscle actin-positive cells in dermal microvessels. This phenotype is consistent with a role of Fli1 as a regulator of vessel maturation and stabilization. Importantly, vascular characteristics of Fli1 CKO mice are recapitulated by SSc microvasculature. Thus, persistently reduced levels of Fli1 in endothelial cells may play a critical role in the development of SSc vasculopathy.

Ets1 Is an Effector of the Transforming Growth Factor β (TGF-β) Signaling Pathway and an Antagonist of the Profibrotic Effects of TGF-β

Extracellular matrix (ECM) production and turnover are tightly controlled under normal physiological conditions. Ets factors regulate matrix turnover by activating transcription of several metalloproteinases (MMPs) and are frequently overexpressed in aggressive tumors and arthritis. Because of the prominent role of transforming growth factor β (TGF-β) in ECM synthesis, this study was undertaken to determine the possible interactions between Ets1 and the TGF-β pathway. Experiments using adenoviral delivery of Ets1 in human fibroblasts have established that Ets1 strongly suppresses TGF-β induction of collagen type I and other matrix-related genes and reverses TGF-β-dependent inhibition of MMP-1. Subsequent experiments utilizing COL1A2 promoter demonstrated that Ets1 in the presence of TGF-β signaling interferes with the stimulatory role of p300. To gain further insight into the mechanism of Ets1 inhibition of the TGF-β signaling, the protein levels and post-translational modifications of Ets1 after TGF-β treatment were analyzed. The level of total Ets1 protein was not affected after 24 h of TGF-β stimulation. Moreover, TGF-β did not affect either serine or threonine phosphorylation levels of Ets1. However, TGF-β induced rapid and prolonged lysine acetylation of Ets1. In addition, analyses of endogenous p300·Ets1 complexes revealed that acetylated Ets1 is preferentially associated with the p300/CBP complexes. TGF-β treatment leads to dissociation of Ets1 from the CBP/p300 complexes. Together, these findings suggest that elevated expression of Ets1 in fibroblasts fundamentally alters their responses to TGF-β in favor of matrix degradation and away from matrix deposition as exemplified by arthritis and cancer.

FLI1 and EWS-FLI1 function as ternary complex factors and ELK1 and SAP1a function as ternary and quaternary complex factors on the Egr1 promoter serum response elements

The ETS gene products are a family of transcriptional regulatory proteins that contain a highly conserved and structurally unique DNA binding domain, termed the ETS domain. Several ETS proteins bind to DNA as monomers, however it has been shown that the DNA binding activity is enhanced or modulated in the presence of other factors. By differential display and whole genome PCR techniques, we have recently shown that the Erg1 gene is a target for ETS proteins. The Egr1 promoter contains multiple ETS binding sites, three of which exist as parts of two serum response elements (SREI and SREII). The SRE is a cis-element that regulates the expression of many growth factor responsive genes. ELK1 and SAP1a have been shown to form ternary complexes with SRF on the SRE located in the c-fos promoter. Similarly, we examined whether the ELK1, SAP1a, FLI1, EWS-FLI1, ETS1, ETS2, PEA3 and PU.1 proteins can form ternary complexes with SRF on the Egr1 SREI and II. Our results demonstrate that indeed ELK1, SAP1a, FLI1 and EWS-FLI1 are able to form ternary complexes with SRF on Egr1 SREs. In addition, ELK1 and SAP1a can also form quarternary complexes on the Egr1 SREI. However, the proteins ETS1, ETS2, PEA3 and PU.1 were unable to form ternary complexes with SRF on either the Egr1 or c-fos SREs. Our data demonstrate that FLI1 and EWS-FLI1 constitute new members of a subgroup of ETS proteins that can function as ternary complex factors and further implicate a novel function for these ETS transcription factors in the regulation of the Egr1 gene. By amino acid sequence comparison we found that, in fact, 50% of the amino acids present in the B-box of SAP1a and ELK1, which are required for interaction with SRF, are identical to those present in both FLI1 (amino acids 231 – 248) and EWS-FLI1 proteins. This B-box is not present in ETS1, ETS2, PEA3 or PU.1 and these proteins were unable to form ternary complexes with SRF and Egr1-SREs or c-fos SRE. Furthermore, deletion of 194 amino terminal amino acids of FLI1 did not interfere with its ability to interact with SRF, in fact, this truncation increased the stability of the ternary complex. The FLI1 protein has a unique R-domain located next to the DNA binding region. This R-domain may modulate the interaction with SRF, providing a mechanism that would be unique to FLI1 and EWS-FLI1, thus implicating a novel function for these ETS transcription factors in the regulation of the Egr1 gene.

Ets transcription factors cooperate with Sp1 to activate the human Tenascin-C promoter

Tenascin-C (TN-C), an extracellular matrix glycoprotein is expressed during embryonic development, but is present only at low levels in normal adult tissues. TN-C is re-expressed during wound healing, fibrotic diseases and in cancer. To better understand the mechanisms that control TN-C gene expression, we examined the regulation of the human TN-C promoter in human fibroblasts. We demonstrate that a short segment of the TN-C promoter between bp −133 and −27 contains three evolutionarily conserved Ets binding sites (EBS). These three EBSs bind in vitro expressed Fli1 protein and mediate transactivation of the TN-C gene by Fli1. Furthermore, two proximal EBSs contribute significantly to basal activity of the TN-C promoter. GABP, which is present in human fibroblast nuclear extracts, interacts with the two proximal EBSs. In addition, several Sp1 and Sp3 binding sites have been located in close proximity to the EBSs within this promoter region. The studies performed in Drosophila cells demonstrate that either Fli1 or GABPα+β1 functionally interact with Sp1 resulting in a synergistic stimulation of the TN-C promoter activity. In conclusion, this study shows for the first time that the TN-C gene is regulated by Ets proteins, which together with Sp1 act as potent activators of TN-C expression.