Molecular Biology Today (2001) 2(2): 13-20. Human Primary Response Gene EGR-3 13 Cloning and Characterization of the 5' Upstream Region of the Human Primary Response Gene EGR-3 Jun Liu1*, Marion Nau2, Liam Grogan2, Powell Brown2, Introduction Carmen Allegra2, Edward Chu1, and John Wright2 The primary response genes encode a spectrum of 1Department of Medicine and Pharmacology, Yale Cancer structural and regulatory proteins including several families Center, Yale University School of Medicine and VA of nuclear transcription factors that presumably regulate a Connecticut Healthcare System, New Haven, CT 06520, select group of target genes involved in tissue- and signal- USA specific responses (Herschman, 1991). Among them, the 2Medicine Branch, Division of Clinical Science, National Egr (early growth response) gene family is a structurally Cancer Institute, National Institutes of Health, Bethesda, related group consisting, to date, of four zinc finger MD 20889, USA transcription factors, Egr-1, Egr-2, Egr-3, and pAT 13 (Sukhatme et al., 1988; Joseph et al., 1988; Muller et al., *Address correspondence to: 1991). The Egr genes encode proteins with three tandem Jun Liu, M.D., VA CT Healthcare System, Cancer Center- zinc finger motifs of the Cys2- His2 subclass that are highly 111D, 950 Campbell Avenue, West Haven, CT 06516, USA 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 Abstract consensus sequence (GCGGGGGCG) was initially identified as an Egr-1 binding site Christy and Nathans, Egr-3 is an immediate-early primary response gene 1989; Cao et al., 1993), and the interaction of murine Egr- encoding a zinc finger transcription factor. We cloned 1 with the GCGTGGGCG motif was characterized by X- the human Egr-3 genomic locus including greater than ray crystallography studies (Pavletich and Pabo, 1991). 1100 bp of the 5' flanking region and analyzed this Binding to the nonameric consensus sequence has been region for putative cis-acting elements. The GC-rich demonstrated for all the Egr family members, which promoter forms part of a representative CpG island complex to this domain with different levels of affinity. Other that extends into the genomic locus. The Egr-3 putative Egr response elements include a homopurine/ promoter contains a region of TATA homology located homopyrimidine domain (TCCTCCTCCTCCTCTCC) 25bp upstream from a major transcriptional start site. (Wang and Deuel, 1992) and variations of the consensus One serum response element and two variant Egr sequence (Swirnoff and Milbrandt, 1995; Nakagama et al., consensus sequences were identified. Features that 1995). Some of the Egr-binding sequences are present in distinguished Egr-3 from other human Egr gene the promoter region of various genes involved in cell promoters included the presence of at least five E-box proliferation, thus linking this family of regulatory proteins motifs and a retinoblastoma response element. In to transcriptional control of cellular growth processes. addition, an overlapping tandem repeat of 16 GC-rich A potential relationship of some Egr genes and related nonamers was identified in the flanking region that may zinc finger proteins to the development of the malignant represent a novel regulatory region for this primary phenotype has also been suggested. Egr-1 is located on response gene. Reporter constructs coupled with Egr- chromosome 5q31, a region commonly deleted in therapy- 3 flanking sequences in sense and antisense related myeloid leukemias (Nucifora et al., 1993). Dys- orientation were tested in transient transfection regulated expression of Egr-1 and Egr-2 by human assays. The functional activity of the Egr-3 regulatory retrovirus-transformed cells (Wright et al., 1990) and soft- region was position-specific. Deletional analysis in tissue sarcomas has been identified. Putative tumor serum stimuIated embryonic lung fibroblasts identified suppressor activity of Egr-1 has been described based on that the major elements responsible for growth- studies showing inhibition of v-sis transformation in murine induced Egr-3 expression are located within the first fibroblasts co-transfected with an expression vector 378 bp upstream of the major transcription start site. containing Egr or Egr gene fragments (Huang et al., 1994). Analysis of the human Egr-3 genomic locus revealed Finally, the WT1 gene, the loss of which results in the a complex regulatory organization with significant development of Wilms tumors, encodes a transcription differences from other Egr genes. These findings may factor with zinc finger regions that share a high level of provide insights into the expression of Egr-3 in normal sequence homology to the corresponding region of Egr and neoplastic tissues. 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). The putative Egr-3 protein *For correspondence. Email [email protected]; Tel. (203) 932-5711 Ext. 4033; Fax. (203) 937-4869. featured three tandem zinc finger motifs that were 90% © 2001 Caister Academic Press Further Reading Caister Academic Press is a leading academic publisher of advanced texts in microbiology, molecular biology and medical research. 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Northern blot analysis of discordant patterns of mRNA expression by related Egr genes. A. Kinetics of Egr-1 and Egr-3 expression following serum stimulation. Left panel: Egr-3 expression and right panel: Egr-1 expression. MRC-9 cells were synchronized in G0 by serum deprivation, and total RNAs were extracted at 0, 0.5, 1, 2, 3, and 4 hr following 20% serum stimulation. Ten µg of total RNA was electrophoresed and hybridized with 32P-labeled Egr-3 cDNA probe. B. Comparison of Egr-1, Egr-2 and Egr-3 expression by human HTLV-1 transformed. T-lymphocyte cell line 702 and fetal lung cell line MRC-9. Cells were harvested at 60 min following serum stimulation of quiescent cells. Ten µg of total RNA was electrophoresed per lane and hybridized with the respective 32P-labeled cDNA probes. homologous to the Egr-1 zinc finger domains as well as a adult rat tissues despite high levels of Egr-1 expression in significant degree (~35%) of similarity in the N-terminus to rat brain, lung, and heart. In addition, although Egr-1 is the corresponding region of Egr-1 and Egr-2. Analysis of highly expressed in rat PCJ 2 cells following nerve growth an Egr-3 genomic clone identified a gene structure similar factor stimulation, Egr-3 is not activated under similar to Egr-1 and Egr-2 with one intron approximately 1.3 kb in conditions.