Cloning and Characterization of the 5' Upstream Region of the Human Primary Response Gene EGR-3

Egr-3 is an immediate-early primary response gene encoding a zinc finger transcription factor. We cloned the human Egr-3 genomic locus including greater than 1100 bp of the 5' flanking region and analyzed this region for putative cis-acting elements. The GC-rich promoter forms part of a representative CpG island that extends into the genomic locus. The Egr-3 promoter contains a region of TATA homology located 25bp upstream from a major transcriptional start site. One serum response element and two variant Egr consensus sequences were identified. Features that distinguished Egr-3 from other human Egr gene promoters included the presence of at least five E-box motifs and a retinoblastoma response element. In addition, an overlapping tandem repeat of 16 GC-rich nonamers was identified in the flanking region that may represent a novel regulatory region for this primary response gene. Reporter constructs coupled with Egr3 flanking sequences in sense and antisense orientation were tested in transient transfection assays. The functional activity of the Egr-3 regulatory region was position-specific. Deletional analysis in serum stimuIated embryonic lung fibroblasts identified that the major elements responsible for growthinduced Egr-3 expression are located within the first 378 bp upstream of the major transcription start site. Analysis of the human Egr-3 genomic locus revealed a complex regulatory organization with significant differences from other Egr genes. These findings may provide insights into the expression of Egr-3 in normal and neoplastic tissues. Introduction The primary response genes encode a spectrum of structural and regulatory proteins including several families of nuclear transcription factors that presumably regulate a select group of target genes involved in tissueand signalspecific responses (Herschman, 1991). Among them, the Egr (early growth response) gene family is a structurally related group consisting, to date, of four zinc finger transcription factors, Egr-1, Egr-2, Egr-3, and pAT 13 (Sukhatme et al., 1988; Joseph et al., 1988; Muller et al., 1991). The Egr genes encode proteins with three tandem zinc finger motifs of the Cys2His2 subclass that are highly homologous and mediate sequence-specific DNA binding. The prototypic member of this group, Egr-1, has been most extensively studied in this regard. A GC-rich nonameric consensus sequence (GCGGGGGCG) was initially identified as an Egr-1 binding site Christy and Nathans, 1989; Cao et al., 1993), and the interaction of murine Egr1 with the GCGTGGGCG motif was characterized by Xray crystallography studies (Pavletich and Pabo, 1991). Binding to the nonameric consensus sequence has been demonstrated for all the Egr family members, which complex to this domain with different levels of affinity. Other putative Egr response elements include a homopurine/ homopyrimidine domain (TCCTCCTCCTCCTCTCC) (Wang and Deuel, 1992) and variations of the consensus sequence (Swirnoff and Milbrandt, 1995; Nakagama et al., 1995). Some of the Egr-binding sequences are present in the promoter region of various genes involved in cell proliferation, thus linking this family of regulatory proteins to transcriptional control of cellular growth processes. A potential relationship of some Egr genes and related zinc finger proteins to the development of the malignant phenotype has also been suggested. Egr-1 is located on chromosome 5q31, a region commonly deleted in therapyrelated myeloid leukemias (Nucifora et al., 1993). Dysregulated expression of Egr-1 and Egr-2 by human retrovirus-transformed cells (Wright et al., 1990) and softtissue sarcomas has been identified. Putative tumor suppressor activity of Egr-1 has been described based on studies showing inhibition of v-sis transformation in murine fibroblasts co-transfected with an expression vector containing Egr or Egr gene fragments (Huang et al., 1994). Finally, the WT1 gene, the loss of which results in the development of Wilms tumors, encodes a transcription factor with zinc finger regions that share a high level of sequence homology to the corresponding region of Egr proteins and binds the Egr consensus sequence as well (Nakagama et al., 1995). The human and murine Egr-3 genes were isolated from a serum-activated cDNA library by low-stringency hybridization with an Egr-1 probe containing the zinc finger (Patwardhan et al., 1991). 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