Identification of the Novel Player δEF1 in Estrogen Transcriptional Cascades

Estrogen acting via the estrogen receptor complex is responsible for the transcriptional regulation of many target genes in both reproductive and nonreproductive tissues. In fact, estrogen is being implicated in a growing number of human disease states, including reproductive cancers (17), heart disease (2), osteoporosis (20), and Alzheimer’s disease (3). The mechanisms underlying these physiological manifestations have been the focus of a considerable amount of research in recent years. Mechanisms such as cross talk with other signaling pathways and the interaction of the estrogen receptor complex with both activating and inhibitory transcription complexes have emerged from this recent research. However, a large number of genes still exist that are known to be regulated by estrogen in an indirect manner but whose mechanism of regulation is not fully understood (6). A long-standing hypothesis for the mechanism of action of estrogen, as well as other steroid hormones, is the existence of a regulatory transcriptional hierarchy (1). The best evidence for the existence of this type of hierarchy is the control of molting in Drosophila melanogaster (33). This process is under the control of the steroid hormone ecdysone and proceeds through a transcriptional cascade. While this hypothesis has existed for many years and has been shown to exist in the Drosophila system, there has been little progress in elucidating this type of mechanism in vertebrate systems. The ability to identify genes that are differentially regulated under various conditions was greatly aided by the description of differential display in 1992 by Liang et al. (13). This technique is ideally suited to the study of hormonal regulation, as it allows the comparison of gene expression in a given tissue plus and minus the hormone. Differential display was employed to identify genes that are induced in the chick oviduct following treatment with 17-β-estradiol. This line of investigation identified previously cloned chicken transcription factor δEF1 (δ-crystallin/E2-box factor) as being regulated by estrogen in the chick oviduct. This is the first report of the regulation of δEF1 by estrogen. δEF1 was originally cloned in a Southwestern screen designed to identify proteins that bind to the intronic enhancer region of the lens-specific δ-crystallin gene (8). Since its cloning in 1993, homologs have also been identified in mice (9), hamsters (7), rats (9), and humans (34). Analysis of the amino acid sequence reveals intriguing structural features of this protein. δEF1 contains multiple DNA binding motifs with two clusters of zinc fingers, one at the N terminus and one at the C terminus, as well as a homeodomain in the middle of the protein (8). While DNA binding activity has been determined for the zinc finger clusters, the homeodomain failed to show an interaction with DNA (12). In fact, the δEF1 homeodomain has a striking amino acid sequence similarity to the POU homeodomains that have been suggested to play a role in protein-protein interaction rather than in DNA binding (8). Although δEF1 was originally identified in the chick lens, its expression is not limited to the lens. Expression of the δEF1 gene has been detected in mesodermal tissues, as well as the central nervous system (8), and many target genes have been identified (7, 10, 22–34). Some functions for this gene have been determined by investigation of genes that are regulated by δEF1, as well as by creation of transgenic animals. δEF1 is an essential gene, as demonstrated by knockout mice. Mice completely lacking δEF1 die in utero (11). Mutations that remove the N-terminal zinc finger cluster result in mice that have thymic abnormalities, including a deficiency in normal T cells (11). δEF1 mutations that lack the C-terminal zinc finger cluster result in bone abnormalities (32). The data presented herein add a role for δEF1 in the regulation of pathways triggered by estrogen in the chick oviduct and perhaps in other tissues. Estrogen is responsible for both the proliferation and differentiation of tubular gland cells in the chick oviduct. Tubular gland cells are responsible for the production of egg white proteins during egg development (26). Ovalbumin is the major egg white protein found in chicken eggs. Induction of the ovalbumin gene requires administration of estrogen, and maximal induction requires estrogen and a second steroid hormone, which is likely to be glucocorticoid in vivo (24). The proximal 900 bp of the ovalbumin 5′-flanking region are essential for induction by estrogen (27), but this region contains no canonical estrogen response elements (EREs). Furthermore, induction of the ovalbumin gene is sensitive to cycloheximide, a protein synthesis inhibitor (18). The lack of EREs and the cycloheximide-sensitive nature of the induction by estrogen place the ovalbumin gene in the class of secondary response genes (6). Therefore, the ovalbumin gene provides a good model system in which to study regulatory hierarchies that are triggered by estrogen. To this end, the role of δEF1 in a hierarchy of estrogen signaling was tested. Indeed, there exists a transcriptional cascade that is triggered by estrogen and culminates in induction of the ovalbumin gene. The role of δEF1 in the induction or repression of other genes following treatment with estrogen needs to be examined. The potential for understanding the regulatory mechanisms of many other secondary response genes is therefore greatly aided by the identification of the regulation of transcription factor δEF1 by estrogen.