No abstract

The wt1 gene, a putative tumor suppressor gene located at the Wilms tumor (WT) locus on chromosome 11p13, encodes a zinc finger-containing protein that binds to the same DNA sequence as EGR-1, a mitogen-inducible immediate-early gene product that activates transcription. The transcriptional regulatory potential of WT1 has not been demonstrated. In transient transfection assays, the WT1 protein functioned as a repressor of transcription when bound to the EGR-1 site. The repression function was mapped to the glutamine- and proline-rich NH2-terminus of WT1; fusion of this domain to the zinc finger region of EGR-1 converted EGR-1 into a transcriptional repressor.

Egr-l is an immediate-early response gene induced by diverse signals that initiate growth and differentiation. Its cDNA sequence predicts a protein with zinc fingers. We have generated an antiserum to the Egr-1 gene product and identified it as an 80-kilodalton short-lived protein in serum-stimulated mouse fibroblasts. The rat Egr-l product has also been identified in nerve growth factor-induced PC12 cells. In addition, we show by cell fractionation and immunocytochemistry that the Egr-l protein is located in the nucleus. We also demonstrate that it is phosphorylated. In vitro-generated Egr-l protein binds with high affinity to the sequence CGCCCCCGC in a zinc-dependent manner.

Egr-1 is an immediate-early response gene induced transiently and ubiquitously by mitogenic stimuli and also regulated in response to signals that initiate differentiation. The Egr-1 gene product, a nuclear phosphoprotein with three zinc fingers of the Cys2His2 class, binds to the sequence CGCCCCCGC and transactivates a synthetic promoter construct 10-fold in transient-transfection assays. We have analyzed the structure and function of the Egr-1 protein in detail, delineating independent and modular activation, repression, these results and points to a role for sustained Egr-1 expression in these differentiated cell types. In the developing mouse, the predominant expression of Egr-1 in cartilage and bone, coordinate with c-fos induction, suggests a role for these coregulated genes in skeletal development (68). Recently, Nguyen et al. have shown that Egr-1 is essential for macrophage differentiation of the myeloid leukemia cell line HL60 and further that Egr-1 functions to restrict differentiation, since constitutive Egr-1 expression is incompatible with the differentiation of HL60 cells along the granulocyte lineage (50). Characterization of the Egr-1 gene product has shown that this serine/threonine/proline-rich nuclear phosphoprotein (6,12,73) activates transcription through the sequence CGC CCCCGC in transient transfection assays (6,8,36,53). Recently, Pavletich and Pabo have cocrystallized the three zinc fingers of Egr-1 with its cognate sequence, defining a DNA-binding domain (54). However, to date the regions responsible for activation and nuclear localization have not been mapped. A body of work suggests the modular nature of transcription factors, in which functional domains are structurally independent and able to confer activity on heterologous proteins (reviewed in reference 55). We have used deletion analysis and gene fusions to dissect the functional domains of Egr-1. We report here that the serine/ threonine-rich N terminus of Egr-1 encodes its transactivation function. Remarkably, a compact 34-amino-acid region, residues 281 to 314, represses transcription in gene fusion experiments. The zinc finger domain encodes the DNAbinding activity, and this region in conjunction with a basic sequence 5' of the fingers is responsible for nuclear localization of Egr-1. 4556 on May 11, 2018 by guest