Prostate cancer, the most frequent solid cancer in older men, is a leading cause of cancer deaths. Although proliferation and differentiation of normal prostate epithelia and the initial growth of ...prostate cancer cells are androgen-dependent, prostate cancers ultimately become androgen-independent and refractory to hormone therapy. The prostate-specific antigen (PSA) gene has been widely used as a diagnostic indicator for androgen-dependent and -independent prostate cancer. Androgen-induced and prostate epithelium-specific PSA expression is regulated by a proximal promoter and an upstream enhancer via several androgen receptor binding sites. However, little progress has been made in identifying androgen-independent regulatory elements involved in PSA gene regulation. We report the isolation of a novel, prostate epithelium-specific Ets transcription factor, PDEF (prostate-derived Etsfactor), that among the Ets family uniquely prefers binding to a GGAT rather than a GGAA core. PDEF acts as an androgen-independent transcriptional activator of the PSA promoter. PDEF also directly interacts with the DNA binding domain of androgen receptor and enhances androgen-mediated activation of the PSA promoter. Our results, as well as the critical roles of other Ets factors in cellular differentiation and tumorigenesis, strongly suggest that PDEF is an important regulator of prostate gland and/or prostate cancer development.
Friend of GATA (FOG)-2 is a multi-zinc finger transcriptional corepressor protein that binds specifically to GATA4. Gene targeting
studies have demonstrated that FOG-2 is required for normal cardiac ...morphogenesis, including the development of the coronary
vasculature, left ventricular compact zone, and heart valves. To better understand the molecular mechanisms by which FOG-2
regulates these cardiac developmental programs, we screened a mouse day 11 embryo library using a yeast two-hybrid interaction
trap with the fifth and sixth zinc fingers of FOG-2 as bait. Using this approach, we isolated clones encoding the orphan nuclear
receptors chicken ovalbumin upstream promoter-transcription factor (COUP-TF) 2 and COUP-TF3. COUP-TF2-null embryos die during
embryonic development with defective angiogenesis and cardiac defects, a pattern that partly resembles the FOG-2-null phenotype.
The interaction between COUP-TF2 and FOG-2 in mammalian cells was confirmed by co-immunoprecipitation of these proteins from
transfected COS-7 cells. The sites of binding interaction between COUP-TF2 and FOG-2 were mapped to zinc fingers 5 and 6 and
fingers 7 and 8 of FOG-2 and to the carboxyl terminus of the COUP-TF proteins. Binding to COUP-TF2 was specific because FOG-2
did not interact with the ligand-binding domains of retinoid X receptor α, glucocorticoid receptor, and peroxisome proliferating
antigen receptor γ, which are related to the COUP-TF proteins. Full-length FOG-2 markedly enhanced transcriptional repression
by GAL4-COUP-TF2(117â414), but not by a COUP-TF2 repression domain mutant. Moreover, FOG-2 repressed COUP-TF2dependent synergistic
activation of the atrial natriuretic factor promoter by both GATA4 and the FOG-2-independent mutant GATA4-E215K. Taken together,
these findings suggest that FOG-2 functions as a corepressor for both GATA and COUP-TF proteins.
Most cancers originate as a result of aberrant gene expression in mainly glandular epithelial tissues leading to defects in epithelial cell differentiation. The latter is governed by distinct sets of ...transcriptional regulators. Here we report the characterization of epithelium-specific Ets factor, family member 3 (ESE-3), a novel member of the ESE subfamily of Ets transcription factors. ESE-3 shows highest homology to two other epithelium restricted Ets factors, ESE-1 and ESE-2. ESE-3, like ESE-1 and ESE-2, is exclusively expressed in a subset of epithelial cells with highest expression in glandular epithelium such as prostate, pancreas, salivary gland, and trachea. A potential role in branching morphogenesis is suggested, since ESE-3 transactivates the c-MET promoter via three high affinity binding sites. Additionally, ESE-3 binding to DNA sequences in the promoters of several glandular epithelium-specific genes suggests a role for ESE-3 in later stages of glandular epithelium differentiation. Although ESE-3 and ESE-1 bind with similar affinity to various Ets binding sites, ESE-3 and ESE-1 differ significantly in their ability to transactivate the promoters containing these sites. Our results support the notion that ESE-1, ESE-2, and ESE-3 represent a unique epithelium-specific subfamily of Ets factors that have critical but distinct functions in epithelial cell differentiation and proliferation.
Epithelial cell differentiation is tightly controlled by distinct sets of transcription factors that regulate the expression of stage-specific genes. We recently isolated the first ...epithelium-specific Ets transcription factor (ESE-1). Here we describe the characterization of ESE-2, a second epithelium-restricted ESE-1-related Ets factor. Like ESE-1, ESE-2 is induced during keratinocyte differentiation. However, whereas ESE-1 is expressed in the majority of epithelial cell types, ESE-2 expression is restricted to differentiated keratinocytes and glandular epithelium such as salivary gland, prostate, mammary gland, and kidney. In contrast to ESE-1, full-length ESE-2 binds poorly to DNA due to the presence of a negative regulatory domain at the amino terminus. Furthermore, although ESE-1 and the amino-terminally deleted ESE-2 bind with similar affinity to the canonical E74 Ets site, ESE-2 and ESE-1 differ strikingly in their relative affinity toward binding sites in the c-MET and PSMA promoters. Similarly, ESE-1 and ESE-2 drastically differ in their ability to transactivate epithelium-specific promoters. Thus, ESE-2, but not ESE-1, transactivates the parotid gland-specific PSP promoter and the prostate-specific PSA promoter. In contrast, ESE-1 transactivates the keratinocyte-specific SPRR2A promoter Ets site and the prostate-specific PSMA promoter significantly better than ESE-2. Our results demonstrate the existence of a unique class of related epithelium-specific Ets factors with distinct functions in epithelial cell gene regulation.
We previously isolated different isoforms of a new Ets transcription factor family member, NERF/ELF-2, NERF-2, NERF-1a, and
NERF-1b. In contrast to the inhibitory isoforms NERF-1a and NERF-1b, NERF-2 ...acts as a transactivator of the B cell-specific
blk promoter. We now report that NERF-2 and NERF-1 physically interact with AML1 (RUNX1), a frequent target for chromosomal translocations
in leukemia. NERF-2 bound to AML1 via an interaction site located in a basic region upstream of the Ets domain. This is in
contrast to most other Ets factors such as Ets-1 that bind to AML1 via the Ets domain, suggesting that different Ets factors
utilize different domains for interaction with AML1. The interaction between AML1 and NERF-2 led to cooperative transactivation
of the blk promoter, whereas the interaction between AML1 and NERF-1a led to repression of AML1-mediated transactivation. To delineate
the differences in function of the different NERF isoforms, we determined that the transactivation domain of NERF-2 is encoded
by the N-terminal 100 amino acids, which have been replaced in NERF-1a by a 19-amino acid transcriptionally inactive sequence.
Furthermore, acidic domains A and B, which are conserved in NERF-2 and the related proteins ELF-1 and MEF/ELF-4, but not in
NERF-1a, are largely responsible for NERF-2-mediated transactivation. Because translocation of the Ets factor Tel to AML1
is a frequent event in childhood pre-B leukemia, understanding the interaction of Ets factors with AML1 in the context of
a B cell-specific promoter might help to determine the function of Ets factors and AML1 in leukemia.
We report here the isolation ofTel-2, a novel member of the Ets transcription factor family, with high homology to Tel/ETV-6. Tel-2 is the second mammalian member of the Tel Ets family subclass whose ...prototype Tel is involved in various chromosomal translocations in human cancers. Six differentially expressed alternative splice products of Tel-2 were characterized encoding different Tel-2 isoforms which either contain or lack the amino-terminal Pointed domain and also vary at the carboxyl terminus. In contrast to Tel, which is highly expressed in several different cell types and tissues, Tel-2 is only weakly expressed in a variety of tissues and cell types, including placenta, prostate, spleen, liver, and lung. Tel-2 binds to functionally relevant Ets-binding sites of several genes and only the Tel-2 isoform containing the Pointed domain and the DNA-binding domain acts as a strong repressor of transcription. The retinoic acid receptor α and bone morphogenetic protein-6B(BMP-6) genes are specifically repressed by Tel-2 indicating a function for Tel-2 as an inhibitor of differentiation. Due to the important involvement of Tel in human cancer and the location of Tel-2 within the MHC cluster region,Tel-2 might be involved in chromosomal translocations in human cancer as well.AF116509AF116510AF218235AF218365AF218366
AML1 plays a critical role during hematopoiesis and chromosomal translocations involving AML1 are commonly associated with different forms of leukemia, including pre-B acute lymphoblastic leukemia. ...To understand the function of AML1 during B cell differentiation, we analyzed regulatory regions of B cell-specific genes for potential AML1-binding sites and have identified a putative AML1-binding site in the promoter of the B cell-specific tyrosine kinase gene, blk. Gel mobility shift assays and transient transfection assays demonstrate that AML1 binds specifically to this site in the blk promoter and this binding site is important for blk promoter activity. Furthermore, in vitro binding analysis revealed that the AML1 runt DNA-binding domain physically interacts with the paired DNA-binding domain of BSAP, a B cell-specific transcription factor. BSAP has been shown previously to be important for B cell-specific regulation of the blk gene. Physical interaction of AML1 with BSAP correlates with functional cooperativity in transfection studies where AML1 and BSAP synergistically activate blk promoter transcription by more than 50-fold. These results demonstrate physical and functional interactions between AML1 and BSAP and suggest that AML1 is an important factor for regulating a critical B cell-specific gene, blk.
Abstract
We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (
RIC
3) by comparing
RIC
...3 protein in rat
GH
4C1 and human
SH
‐
EP
1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat
RIC
3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface α7 expression in
SH
‐
EP
1 and human embryonic kidney (
HEK
) cells measured by α‐bungarotoxin binding. Contrary to expectations, endogenous
RIC
3 protein expression determined by immunoblots did not differ between untransfected
GH
4C1 or
SH
‐
EP
1 cells. si
RNA
against rat
RIC
3 exon 4 and sh
RNA
against exons 2, 5 and 6 knocked down transfected rat
RIC
3 expression in
SH
‐
EP
1 cells and simultaneously blocked toxin binding. However, no
RNA
i construct blocked binding when co‐transfected with α7 into
GH
4C1 cells. sh
RNA
against rat exons 2 and 5 knocked down rat
RIC
3 protein transfected into
GH
4C1 cells with a time course suggesting a protein half‐life of a few days. These results suggest
GH
4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known
RIC
3 splice variants.
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