Androgen Receptor Regulates Transcription of the ZEB1 Transcription Factor.

The zinc finger E-box binding protein 1 (ZEB1) transcription factor belongs to a two-member family of zinc-finger homeodomain proteins involved in physiological and pathological events mostly relating to cell migration and epithelial to mesenchymal transitions (EMTs). ZEB1 (also known as δEF1, zfhx1a, TCF8, and Zfhep) plays a key role in regulating such diverse processes as T-cell development, skeletal patterning, reproduction, and cancer cell metastasis. However, the factors that regulate its expression and consequently the signaling pathways in which ZEB1 participates are poorly defined. Because it is induced by estrogen and progesterone and is high in prostate cancer, we investigated whether tcf8, which encodes ZEB1, is regulated by androgen. Data herein demonstrate that tcf8 is induced by dihydrotestosterone (DHT) in the human PC-3/AR prostate cancer cell line and that this induction is mediated by two androgen response elements (AREs). These results demonstrate that ZEB1 is an intermediary in androgen signaling pathways.

The ZEB1 transcription factor is a novel repressor of adiposity in female mice.

BACKGROUND: Four genome-wide association studies mapped an "obesity" gene to human chromosome 10p11-12. As the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is encoded by the TCF8 gene located in that region, and as it influences the differentiation of various mesodermal lineages, we hypothesized that ZEB1 might also modulate adiposity. The goal of these studies was to test that hypothesis in mice. METHODOLOGY/PRINCIPAL FINDINGS: To ascertain whether fat accumulation affects ZEB1 expression, female C57BL/6 mice were fed a regular chow diet (RCD) ad libitum or a 25% calorie-restricted diet from 2.5 to 18.3 months of age. ZEB1 mRNA levels in parametrial fat were six to ten times higher in the obese mice. To determine directly whether ZEB1 affects adiposity, wild type (WT) mice and mice heterozygous for TCF8 (TCF8+/-) were fed an RCD or a high-fat diet (HFD) (60% calories from fat). By two months of age on an HFD and three months on an RCD, TCF8+/- mice were heavier than WT controls, which was attributed by Echo MRI to increased fat mass (at three months on an HFD: 0.517+/-0.081 total fat/lean mass versus 0.313+/-0.036; at three months on an RCD: 0.175+/-0.013 versus 0.124+/-0.012). No differences were observed in food uptake or physical activity, suggesting that the genotypes differ in some aspect of their metabolic activity. ZEB1 expression also increases during adipogenesis in cell culture. CONCLUSION/SIGNIFICANCE: These results show for the first time that the ZEB1 transcription factor regulates the accumulation of adipose tissue. Furthermore, they corroborate the genome-wide association studies that mapped an "obesity" gene at chromosome 10p11-12.

Interferon regulatory factors (IRFs) repress transcription of the chicken ovalbumin gene.

Although the ovalbumin (Ov) gene has served as a model to study tissue-specific, steroid hormone-induced gene expression in vertebrates for decades, the mechanisms responsible for regulating this gene remain elusive. Ov is repressed in non-oviduct tissue and in estrogen-deprived oviduct by a strong repressor site located from -130 to -100 and designated CAR for COUP-TF adjacent repressor. The goal of this study was to identify the CAR binding protein(s). A transcription factor database search revealed that a putative interferon-stimulated response element (ISRE), which binds interferon regulatory factors (IRFs), is located in this region. Gel mobility shift assays demonstrated that the protein(s) binding to the CAR site is recognized by an IRF antibody and that mutations in the ISRE abolish that binding. In hopes of identifying the IRF(s) responsible for the tissue-specific regulation of Ov, mRNA levels for IRFs-4, -8, and -10 were measured in seven tissues from chicks treated with or without estrogen. PCR experiments showed that both IRF-8 and -10 are expressed in all chick tissues tested whereas IRF-4 has a much more limited expression pattern. Transfection experiments with OvCAT (chloramphenicol acetyltransferase) reporter constructs demonstrated that both IRF-4 and IRF-10 are capable of repressing the Ov gene even in the presence of steroid hormones and that nucleotides in the ISRE are required for repression. These experiments indicate that the repressor activity associated with the CAR site is mediated by IRF family members and suggest that IRF members also repress Ov in non-oviduct tissues.

Expression of the ZEB1 (deltaEF1) transcription factor in human: additional insights.

The zinc finger E-box binding transcription factor ZEB1 (deltaEF1/Nil-2-a/AREB6/zfhx1a/TCF8/zfhep/BZP) is emerging as an important regulator of the epithelial to mesenchymal transitions (EMT) required for development and cancer metastasis. ZEB1 promotes EMT by repressing genes contributing to the epithelial phenotype while activating those associated with the mesenchymal phenotype. TCF8 (zfhx1a), the gene encoding ZEB1, is induced by several potentially oncogenic ligands including TGF-beta, estrogen, and progesterone. TGF-beta appears to activate EMT, at least in part, by inducing ZEB1. However, our understanding of how ZEB1 contributes to signaling pathways elicited by estrogen and progesterone is quite limited, as is our understanding of its functional roles in normal adult tissues. To begin to address these questions, a human tissue mRNA array analysis was done. In adults, the highest ZEB1 mRNA expression is in bladder and uterus, whereas in the fetus highest expression is in lung, thymus, and heart. To further investigate the regulation of TCF8 by estrogen, ZEB1 mRNA was measured in ten estrogen-responsive cell lines, but it is only induced in the OV266 ovarian carcinoma line. Although high expression of ZEB1 mRNA is estrogen-dependent in normal human ovarian and endometrial biopsies, high expression is estrogen-independent in late stage ovarian and endometrial carcinomas, raising the possibility that deregulated expression promotes cancer progression. In contrast, TCF8 is at least partially deleted in 4 of 5 well-differentiated, grade I endometrial carcinomas, which may contribute to their non-aggressive phenotype. These data support the contention that high ZEB1 encourages gynecologic carcinoma progression.

An oviduct-specific and enhancer-like element resides at about -3000 in the chicken ovalbumin gene.

The chicken ovalbumin (Ov) gene is one of the best models to study tissue-specific gene regulation because it is only expressed in the oviduct. In this paper, a tissue-specific element was characterized by 5'-flanking region deletion in combination with in vivo gene electroporation (EP). Plasmids with varying lengths of truncated Ov 5'-flanking region fused to the Renilla luciferase reporter gene were transfected in vivo into oviduct, muscle, and pancreas. A chicken oviduct-specific and enhancer-like region (designated COSE) was implicated between -3100 and -2800. The COSE showed up-regulation of gene expression in oviduct, but not in muscle or in pancreas. The COSE region was further characterized by gel mobility shift assays using nuclear extracts from oviduct, pancreas and liver. With the region from -2900 to -2851, designated T2, there were two distinct protein-DNA complexes: one found only in oviduct extract and the other detected only in pancreas and liver. These data suggest a model where the regulation of Ov gene expression in the oviduct and non-oviduct tissues is exerted at least in part by the presence of protein modulators that bind to the COSE element.

Estrogen action: revitalization of the chick oviduct model.

Despite decades of investigation, the molecular pathways triggered by estrogen that lead to tissue-specific cell proliferation, differentiation and survival are only superficially understood. If we are to modulate the actions of estrogen selectively in these processes, continued investigation using biologically relevant models is essential. The chick oviduct emerged as an early model for investigating the mechanism of action of steroid hormones because of its exquisite responsiveness to them. Unfortunately, because of experimental limitations, this model has been neglected in the past decade. Reviving this model has become intellectually attractive and technically feasible.

Comparison of the responsiveness of the pGL3 and pGL4 luciferase reporter vectors to steroid hormones.

The ovalbumin gene (Ov) gene is responsive to estrogen, glucocorticoid, androgen, and progesterone. In our efforts to characterize the regulation of the Ov gene by steroid hormones, we have repeatedly observed that many vector backbones and promoters are responsive to steroids. In order to determine which vectors are most suitable for these types of analyses, vectors from Promega's pGL3 and newly engineered pGL4 Dual-Luciferase Reporter Assay System were tested with both estrogen and/or corticosterone. The results confirmed that both series are induced by glucocorticoids in transient transfections of primary oviduct tubular gland cells, which contain glucocorticoid receptors, but not in MCF-7 cells, which do not. Modest effects that were dependent upon backbone and promoter context were observed with both series of vectors with estrogen. Thus, use of these vectors for experiments analyzing the effects of steroid hormones, especially glucocorticoids, should be done with caution. However the new pGL4 series does have some advantages over the older series, and a comparison of transcription factor binding sites is reported.

Transcriptional activation by the zinc-finger homeodomain protein delta EF1 in estrogen signaling cascades.

The transcription factor delta EF1 is a key player in estrogen-signaling cascades in vertebrates. In this pathway, estrogen induces the expression of the gene encoding delta EF1, and then delta EF1 activates transcription of downstream targets. Yet, the molecular mechanisms of transcriptional activation by delta EF1 have remained obscure. Furthermore, most work has concentrated on the capacity of delta EF1 to repress gene expression, rather than its ability to activate transcription. To investigate this activation potential in an endogenous signaling pathway, we characterized ovalbumin (Ov) gene induction by delta EF1. Gel mobility shift assays demonstrate that delta EF1 binds to the 5' flanking region of the Ov gene at two sites, one at -810 to -806 and one at -152 to -148 with respect to the start point of transcription. Correspondingly, these sites are required for induction by estrogen or by delta EF1 in transfection experiments. Furthermore, the activation potential of delta EF1 is not restricted to the chick homolog, as the human ZEB and the mouse delta EF1 homologs also induce Ov gene expression. To characterize the molecular mechanisms whereby delta EF1 activates gene expression, its C-terminal acidic domain was deleted and shown to be necessary for activation of transcription. Furthermore, the acidic domain has intrinsic activation potential, as it can induce the heterologous thymidine kinase promoter. These data establish delta EF1 as an activator of transcription whose action may be DNA-context and cell-type specific, but not species specific.

Upstream stimulatory factor (USF) is recruited into a steroid hormone-triggered regulatory circuit by the estrogen-inducible transcription factor delta EF1.

In the past decade, investigation into steroid hormone signaling has focused on the mechanisms of steroid hormone receptors as they act as signaling molecules and transcription factors in cells. However, the majority of hormone-responsive genes are not directly regulated by hormone receptors. These genes are termed secondary response genes. To explore the molecular mechanisms by which the steroid hormone estrogen regulates secondary response genes, the ovalbumin (Ov) gene was analyzed. Three protein-protein complexes (Chirp-I, -II, -III), which do not contain the estrogen receptor, are induced by estrogen to bind to the 5'-flanking region of the Ov gene. The Chirp-III DNA binding site, which is required for estrogen induction, binds a complex of proteins that contains the estrogen-inducible transcription factor deltaEF1. Experiments undertaken to identify proteins complexed with deltaEF1 led to the elucidation of a novel mechanism of action of upstream stimulatory factor-1 (USF-1), which involves its tethering to the Ov gene 5'-flanking region by deltaEF1. Gel mobility shift assays and co-immunoprecipitation experiments identify USF-1 as a component of Chirp-III. However, USF-1 is not able to bind to the Chirp-III site independently. In addition, USF-1 overexpression is able to induce Ov gene promoter activity in transfection experiments. USF-1 can also potentiate the induction of the Ov gene by the transcription factor deltaEF1. Moreover, mutating the deltaEF1 binding sites in the 5'-flanking region of the Ov gene abrogates induction of the gene by USF-1. These data begin to establish a molecular mechanism by which hormone-inducible transcription factors and ubiquitous transcription factors cooperate to regulate estrogen-induced secondary response gene expression.

The zinc finger/homeodomain protein deltaEF1 mediates estrogen-specific induction of the ovalbumin gene.

Regulation of the ovalbumin (Ov) gene is strictly controlled by precise developmental, tissue-specific, and hormonal cues. The Ov gene is transcriptionally activated by four classes of steroid hormones: estrogens, androgens, glucocorticoids, and progestins. Although it has served as a model to study multi-hormone gene regulation for the past 30 years, the pathways that relay each hormone signal to the Ov gene are largely unclear. Extensive linker-scanner and point mutation analysis has revealed elements necessary for its induction by estrogen, androgen, progesterone, or glucocorticoid but has failed to identify any elements that are specific to the action of any one steroid hormone. These observations in conjunction with the observation that the Ov gene is indirectly regulated by steroid hormones suggest that these signals may all induce the same transcription factor. However, here we have identified two cis-acting DNA elements in the 5' flanking region of the Ov gene that are required for induction by estrogen, but not by androgen or progesterone. These elements span -152 to -146 and -810 to -806 with respect to the start point of transcription. This implies that estrogen induces the Ov gene by a separate pathway than do androgens or progestins. Gel mobility shift assays demonstrate that the estrogen-specific sequences bind the estrogen inducible transcription factor deltaEF1, suggesting that deltaEF1 plays a distinct role in mediating the estrogen signal to the Ov gene.

Tissue-protective effects of estrogen involve regulation of caspase gene expression.

Estrogen plays a critical role in the protection from apoptosis in several cell types because the withdrawal of estrogen leads to increased apoptosis in tissues such as the brain, endothelium, testes, and uterus. Our recent report demonstrated that the chick oviduct also regresses through apoptotic mechanisms during estrogen deficiency. Despite these observations, very little is known concerning the intracellular mechanisms by which estrogen opposes apoptosis. To better understand how estrogen exerts its antiapoptotic effects, several key apoptotic genes were examined for their regulation by estrogen. Our results show that mRNA expression levels of Bcl-2, hsp-70, c-myc, Bcl-X(l), caspase-3, and caspase-6 remain essentially constant when apoptosis is stimulated by estrogen withdrawal. However, the genes for caspase-1 and caspase-2 are rapidly stimulated, at least for the most part, at the transcriptional level after the withdrawal of estrogen. This increase in caspase-2 mRNA is followed by an increase in enzyme activity. Furthermore, although mRNA expression levels are unaffected, both caspase-3 and caspase-6 proenzymes are activated in the estrogen-withdrawn cells. Taken together, these results demonstrate that estrogen has the potential to oppose apoptosis by regulating caspase activity through both transcriptional and posttranscriptional mechanisms in reproductive tissues.