Putative Ets Research Articles

A negative regulatory element within the proximal promoter region of the rat ornithine decarboxylase gene.

A putative Ets site with a core of GGAA located at nt -88 to -85 of the rat ornithine decarboxylase (ODC) gene was characterized by site-directed mutagenesis and transient expression assays. Mutation of this site, when in pODClux2m, which contains a cluster of four Sp1-binding sites, resulted in a 2.6-fold increase in basal promoter activity in untreated cells, whereas the ratio of activity in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells relative to the ratio in untreated cells (the induction ratio) remained largely unchanged. However, when the mutation was in pODClux168, which contains only a single Sp1-binding site (GC box V), it caused little alteration to either basal promoter activity or TPA induction ratio. A protein of 55-60 kDa was found specifically bound to this site, as shown by ultraviolet cross-linking assay. In competition assay and methylation interference assay, this protein was shown to occupy the GGAA core, although it showed no antigenic relation to c-Ets-1 in an supershift assay. We suggest that this protein binds specifically to the GGAA core and functions to inhibit activation of the ODC promoter by distal elements, including the upstream Sp1 sites.

Functional analysis of the human RAG 2 promoter

Recombination activating genes RAG1 and RAG2 are essential components of V(D)J recombination, a process that generates the specific antigen receptors in lymphocytes. To understand the mechanisms underlying the lineage and developmental regulation of transcription of RAG2, we have characterized the human RAG2 exon 1A promoter. In this study, a series of deletion constructs were used to isolate the promoter while a linker scanning approach was taken to assess functionally relevant cis elements within the promoter. Two regulatory domains were identified. The −140 to −123 region is critical for promoter activity in all cell lines tested. Mutations to the putative Ets (−122 to −118) or to the C/EBP (−137 to −129) consensus core sequences did abrogate promoter activity, although specific DNA/protein interactions remained, as determined by EMSA. The −69 to −48 region demonstrates lineage specific promoter activity. Mutations to an overlapping, BSAP-myb-Ikaros-myb site (−65 to −39) resulted in differential promoter activity in human B and T cells. EMSA analysis of this region showed a B cell specific protein complex. Transfection of BSAP into cell lines trans-activates the human RAG2 promoter. We conclude that transcriptional regulation of the human RAG2 gene is complex, involving both tissue specific and ubiquitous factors, and both proximal and distal regulatory elements.

The Definition of Cell Type

What do we mean by “cell type?” Two articles in this issue of Circulation Research address this question in different ways. The first study, by Dube et al,1 uses very well-defined tools to define endothelial differentiation at the level of transcriptional control. The second study, by Kowal et al,2 looks for novel genes that distinguish two cell types. These studies, taken together, illustrate a major change in how we define cell type, a change that is about to be accelerated by the power of systematic genomics. Dube et al1 use in vitro systems to explore the promoter structure for the endothelial cell-specific receptor tyrosine kinase Tie2. Tie2 is a receptor for both angiopoetin-1 and angiopoetin-2. Like vascular endothelial growth factor, angiopoetin-1 is essential for normal vascular development whereas angiopoetin-2 is a naturally occurring antagonist for angiotensin I and Tie2. Thus, regulation of expression of the Tie2 receptor is likely a key issue in the formation of blood vessels.3 4 Dube et al1 identify a putative Ets transcription site in the Tie2 promoter. One of the proteins binding to this site, NERF2, can be shown to promote transcription in endothelial cells but not in the other cells studied. Related NERFs and other Ets binding factors either did not have this activity or were not found in the cultured cells used by these authors. Although mechanistically solid for transfected endothelial cell lines in vitro, the studies presented by Dube et al1 provide no evidence that NERF2 is expressed in endothelial cells in vivo, and the in vitro studies only use Chinese hamster ovary, 293, and CV-1 cell lines as comparison cells. These aneuploid, heterogeneously derived cell lines may or may not provide a representative comparison with the endothelial cell strains used in the present study, much …

TFEC Is a Macrophage-Restricted Member of the Microphthalmia-TFE Subfamily of Basic Helix-Loop-Helix Leucine Zipper Transcription Factors

The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed with all other known MiT subfamily members (Mitf, TFE3, TFEB). Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU.1 under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage.

Regulation of the CD13/Aminopeptidase N Gene by DMP1, a Transcription Factor Antagonized by D-Type Cyclins

The binding of the Myb-like DMP1 transcription factor to DNA consensus sequences [CCCG(G/T)ATGT] in artificial promoters is antagonized by D-type cyclins with no requirement for their catalytic partners, cyclin-dependent kinase (CDK) 4 and CDK6. The subset of DMP1 binding sites containing the GGA core can bind Ets family transcription factors Ets-1 and Ets-2. Screening of a series of natural promoters revealed that the CD13/aminopeptidase N (APN; EC 3.4.11.2) promoter could bind and be activated by DMP1. Activation of CD13/APN required both the intact DNA binding and transactivation domains of DMP1 and was inhibited by D-type cyclins, but not by cyclins A, B, C, or H, in a CDK-independent manner. CD13/APN is transactivated by a cooperative interaction between c-Myb bound to its cognate site and Ets-1 tethered to one of three GGA core-containing sites located 30-50 base pairs downstream. DMP1 binds to one of the Ets binding sites (designated Ets C) and synergizes with c-Myb in activating CD13/APN expression. Analysis of nuclear lysates from KG1a early myeloid cells using an oligonucleotide probe containing only the DMP1/Ets C binding site indicated that endogenous DMP1 and a putative Ets family member bind this element in vivo. DMP1-DNA complexes were significantly more stable than those containing the Ets factor. These data indicate that two different Myb family proteins collaborate in regulating APN gene expression and point to a role for DMP1 in normal myeloid cell development.

Analysis of differentiation-induced expression mechanisms of thyrotropin receptor gene in adipocytes.

Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between -89 to -68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between -146 and -90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from -146 to -127 bp, including a cAMP response element-like sequence and from -112 to -106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5'-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.

Analysis of Differentiation-Induced Expression Mechanisms of Thyrotropin Receptor Gene in Adipocytes

Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between −89 to −68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between −146 and −90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from− 146 to −127 bp, including a cAMP response element-like sequence and from −112 to −106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5′-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.

An NFAT-Dependent Enhancer Is Necessary for Anti-IgM-Mediated Induction of Murine CD5 Expression in Primary Splenic B Cells

CD5 is a 67-kDa membrane glycoprotein the expression of which in murine splenic B cells is induced by surface IgM cross-linking. To analyze this induction, we transiently transfected primary splenic B cells with luciferase reporter constructs driven by various wild-type and mutated CD5 5'-flanking sequences. The transfected cells were subsequently cultured in medium with or without F(ab')2 anti-IgM (anti-IgM), and luciferase expression was assayed. Using this approach, we identified a 122-bp enhancer element necessary for anti-IgM-mediated induction of the CD5 promoter. Electrophoretic mobility shift assays indicated that four inducible and four constitutive complexes form on the enhancer fragment in nuclear extracts of primary B cells. Supershift assays revealed that two of the inducible complexes contained NFATc. Point mutations that abolished NFAT binding severely impaired enhancer function. Thus, CD5 is a target of NFAT in B cells. A third inducible complex required an intact H4TF-1 site. One of several constitutive complexes required an intact Ebox site while a second required an intact putative ets binding site. Mutation of the H4TF-1, Ebox, and Ets sites, in the presence of wild-type NFAT sites, significantly reduced the activity of the enhancer. Therefore, the induction of B cell CD5 expression requires NFAT binding and binding to at least one of three additional sites in the CD5 enhancer.

Integrin αvPromoter Activity in Keratinocytes

Background.During reepithelialization keratinocytes show increased expression of the integrin subunit αv. We have investigated the promoter region of the αvintegrin subunit to learn more about its regulation.Methods.The promoter region of the human integrin αvgene was cloned into a luciferase reporter vector. Deletional mutants were created using PCR. Computerized sequence analysis was performed using the Wisconsin Package. Gel-shift analysis was performed using keratinocyte nuclear extracts and oligonucleotides spanning the regions of interest.Results.Deletion from −522 bp to −235 resulted in no discernible effect on promoter activity. In contrast deletion of the next 22 bp, which included a putative ets binding site, reduced activity by approximately half. Further deletion to −139 bp essentially abolished promoter activity. Computer searching of this region of the integrin αvpromoter revealed two tandemly repeated motifs, TCCTCCTCC, that had previously been implicated in the function of the epidermal growth factor receptor (EGFR) promoter. Comparison of the αvintegrin promoter to the EGFR promoter revealed an area of high homology in this region. Gel-shift analysis revealed binding of a single-strand specific DNA binding protein to single-stranded oligos comprising these motifs, but no binding of factors to the double-stranded oligo containing the ets binding site.Conclusions.In keratinocytes αvintegrin expression is controlled by a region of the promoter with high homology to the epidermal growth factor receptor promoter. This region binds single-strand specific DNA binding proteins that are likely to be important in controlling transcription.

Identification of a neuregulin and protein-tyrosine phosphatase response element in the nicotinic acetylcholine receptor epsilon subunit gene: regulatory role of an Rts transcription factor.

At the neuromuscular synapse, innervation induces endplate-specific expression of adult-type nicotinic acetylcholine receptors by selective expression of their subunit-encoding genes (alpha2betaepsilondelta) in endplate-associated myonuclei. These genes are specifically regulated by protein-tyrosine phosphatase (PTPase) activity. In addition, neuregulin/acetylcholine-receptor-inducing activity, a nerve-derived factor that stimulates nicotinic acetylcholine receptor synthesis, induces adult-type specific epsilon subunit gene expression via activation of a Ras/mitogen-activated protein kinase pathway. However, the DNA regulatory elements and the binding proteins that mediate PTPase and neuregulin-dependent gene expression remain unknown. Herein we report that PTPase, neuregulin, and Ras-dependent regulation of the epsilon subunit gene map to a 15-bp promoter sequence. Interestingly, this same 15-bp sequence appears to be necessary for low epsilon subunit gene expression in extrajunctional regions of the muscle fiber. Site-directed mutagenesis of a putative Ets binding site located within this 15-bp sequence, reduced PTPase, neuregulin, and Ras-dependent regulation. Overexpression of the rat muscle Ets-2 transcription factor resulted in a sequence-specific induction of epsilon subunit promoter activity. Further, a dominant negative mutant of Ets-2 abolished neuregulin-dependent induction of epsilon subunit gene expression. Thus, these results indicate a crucial role for the 15-bp element in determining synapse-specific and neuregulin-mediated motor neuron control of epsilon subunit gene expression and suggest the participation of Ets transcription factor(s) in this control.

CHARACTERIZATION OF THE GENE ENCODING MOUSE PLATELET GLYCOPROTEIN Ibβ

Platelet glycoprotein (GP) Ib/IX/V complex is a major receptor for von Willebrand factor (vWF), which mediates platelet adhesion and aggregation under high shear stress conditions. It is composed of GPIbα, GPIbβ, GPIX, and GPV. All subunits for the human receptor have been cloned and characterized. However, the function of GPIbβ is still elusive. To obtain further information of GPIbβ, we have determined the genomic sequence of mouse GPIbβ (1466 bp). The deduced amino acid sequence (206aa) was 88% identical to the human GPIbβ protein. All cysteine residues, putative N-linked glycosylation site (Asn41), and putative phosphorylation site (Ser166) were conserved. The promoter region contained putative GATA and ets binding motif implicated in megakaryocytic expression. Mouse GPIbβ also contained a leucine-rich glycoprotein (LRG) sequence of 24 amino acids same as human GPIbβ. © 1997 Elsevier Science Ltd

An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.

Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.

The human I alpha 1 region contains a TGF-beta 1 responsive enhancer and a putative recombination hotspot.

It appears that the switch recombination machinery of a B lymphocyte targets preferentially unrearranged heavy chain genes that have been rendered transcriptionally active. Transcriptional activation of the 'germline' human C alpha 1 and C alpha 2 genes is triggered by TGF-beta 1 and is controlled by proximal positive and distal negative regulatory elements residing upstream of the alpha 1 and alpha 2 switch regions respectively. In this report we characterize the positive proximal regulatory elements and analyse their interaction with DNA binding proteins. Our data demonstrate that a 100 bp fragment that contains a cAMP responsive element (CRE)/activating transcription factor (ATF) motif, a putative Ets binding site and an element that is created by two previously described neighbouring direct repeats (DRE), can increase the basal level of transcription and confer TGF-beta 1 inducibility to a heterologous promoter in an orientation- and position-independent manner. Ubiquitously expressed DNA binding proteins interact specifically with the CRE/ATF, the Ets site and the DRE element. Additionally, nuclear proteins interact with sequences which are located downstream of this enhancer are not essential for transcription in the transient expression assays utilized; however, they contain motifs that have been previously implicated in regulating DNA recombination events. These motifs include a Chi motif and a Chi-like element previously found in the recombination hotspot region of the Bcl-2 proto-oncogene and close to chromosomal breakpoints in T-ALL lines. Our findings raise the possibility that the intervening region associated regulatory elements in addition to regulating the transcriptional activation of the Ig heavy chain genes could also facilitate the physical interaction of transcription and recombination controlling molecular mechanisms.

Immortalization-susceptible elements and their binding factors mediate rejuvenation of regulation of the type I collagenase gene in simian virus 40 large T antigen-transformed immortal human fibroblasts.

Dramatic changes occur in expression of the type I collagenase gene during the process of immortalization in simian virus 40 large T antigen-transformed human fibroblasts (S. Imai and T. Takano, Biochem. Biophys. Res. Commun. 189:148-153, 1992). From transient transfection assays, it was determined that these changes involved the functions of two immortalization-susceptible cis-acting elements, ISE1 and ISE2, located in a 100-bp region about 1.7 kb upstream. The profiles of binding of an activator, Proserpine, to the enhancer ISE1 were similar in the extracts of young, senescent preimmortalized and immortalized cells. ISE2 contained both negative and positive regulatory elements located adjacent to each other. The positive regulatory element consisted of a tandem array of putative Ets family- and AP-1-binding sites. An activator, Pluto, interacted with this positive regulatory element and had an AP-1-related component as a complex. The binding activity of Pluto was predominantly detected only in the extract from senescent preimmortalized cells. In contrast, a repressor, Orpheus, which bound to the ATG-rich negative regulatory element of ISE2, was prominently detected in extracts from both young preimmortalized and immortalized cells and appeared to suppress transcription in an orientation-dependent manner. Thus, the interplay of Pluto and Orpheus was suggested to be crucial for regulation of the collagenase gene accompanying in vitro aging and immortalization. Proserpine seemed to interact with Pluto to mediate strong expression of the collagenase gene in cellular senescence. On the basis of these results, we propose a model for regulation of the collagenase gene during in vitro aging and immortalization.

Immortalization-susceptible elements and their binding factors mediate rejuvenation of regulation of the type I collagenase gene in simian virus 40 large T antigen-transformed immortal human fibroblasts.

Dramatic changes occur in expression of the type I collagenase gene during the process of immortalization in simian virus 40 large T antigen-transformed human fibroblasts (S. Imai and T. Takano, Biochem. Biophys. Res. Commun. 189:148-153, 1992). From transient transfection assays, it was determined that these changes involved the functions of two immortalization-susceptible cis-acting elements, ISE1 and ISE2, located in a 100-bp region about 1.7 kb upstream. The profiles of binding of an activator, Proserpine, to the enhancer ISE1 were similar in the extracts of young, senescent preimmortalized and immortalized cells. ISE2 contained both negative and positive regulatory elements located adjacent to each other. The positive regulatory element consisted of a tandem array of putative Ets family- and AP-1-binding sites. An activator, Pluto, interacted with this positive regulatory element and had an AP-1-related component as a complex. The binding activity of Pluto was predominantly detected only in the extract from senescent preimmortalized cells. In contrast, a repressor, Orpheus, which bound to the ATG-rich negative regulatory element of ISE2, was prominently detected in extracts from both young preimmortalized and immortalized cells and appeared to suppress transcription in an orientation-dependent manner. Thus, the interplay of Pluto and Orpheus was suggested to be crucial for regulation of the collagenase gene accompanying in vitro aging and immortalization. Proserpine seemed to interact with Pluto to mediate strong expression of the collagenase gene in cellular senescence. On the basis of these results, we propose a model for regulation of the collagenase gene during in vitro aging and immortalization.

Identification of two ets related genes in a marine worm, the polychaete annelid Nereis diversicolor

The ETS family includes a growing number of transcription factors with a highly conserved DNA-binding domain, the ETS domain. We have used PCR amplification with degenerated oligonucleotides to isolate two putative ETS DNA-binding coding domains in a primitive form of cœlomate, the polychaete annelid Nereis diversicolor. These sequences are highly related to the ETS and ERG groups of the ets gene family. For the erg sequence an adjacent region encoding for 91 amino acids has been characterized after library screening, and we show an expression in cells isolated from the cœlomic cavity of the animal. A phylogenic analysis confirms that the ets-1/ets-2 and the erg/fli dichotomy arose specifically in the vertebrate lineage.

Identification of a transcriptional initiator element in the cytochrome c oxidase subunit Vb promoter which binds to transcription factors NF-E1 (YY-1, delta) and Sp1.

We have mapped the basal promoter activity of the mouse cytochrome c oxidase (COX) subunit Vb gene to the -17 to +20 region which contains two putative ets binding sites flanking an NF-E1 site fused to an Sp1 site. A 17-nucleotide sequence flanking the major transcription start site (-8 to +9), referred to as 17Inr (initiator sequence) was able to drive CAT activity in 3T3 cells to a level comparable to the construct containing sequences -17 to +20. This suggests that the 17Inr sequence contains the initiator activity. The 17Inr contains a pyrimidine-rich sequence, commencing with a CA that corresponds to the major transcription start site. Primer extension of RNA from transfected cells demonstrated that transcription initiation with the 17Inr template occurs at a site identical to the endogenous gene. DNA-protein binding by gel mobility shift and methylation interference analyses indicated that the pyrimidine-rich sequence immediately flanking the transcription start site consists of an NF-E1 factor binding motif with an overlapping upstream Sp1 binding site. A 13-nucleotide sequence, 13Inr (-4 to +9), which retains the NF-E1 binding activity but does not bind Sp1, was able to promote chloramphenicol acetyltransferase gene expression at levels similar to the 17Inr sequence, suggesting that NF-E1 factor binding is critical for initiator function. Finally, using an in vitro transcription system from Drosophila embryos we demonstrate that NF-E1 is necessary for transcription activation of both the 17Inr and the 13Inr initiator templates. Thus NF-E1 binding appears to be important for basal promoter function of the mouse COXVb gene.