A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor Egr-1.

Abstract

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

The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A

“…Such inhibitor activity has been described for a number of transcription factors (Gashler et al, 1993;Brown et al, 1995;Nerlov and Zi , 1994;Dubendor et al, 1992). However, certain features exhibited by HBP1 (its masked activation domain and RB binding characteristics) are very reminiscent of the viral transcription factor E1A (Figure 8).…”

Stimulation of p53-mediated Transcriptional Activation by the p53-binding Proteins, 53BP1 and 53BP2

“…Cells were lysed, and both nuclear and cytoplasmic fractions were recovered following the protocol of Gashler et al (38). The nuclear fraction was lysed in 100 l of lysis buffer (100 mM Tris-HCl, pH 9.0, 150 mM NaCl, 1% Nonidet P-40, 0.2 M phenylmethylsulfonyl fluoride, 5 g/ml pepstatin, 2 g/ml leupeptin).…”

The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2.

“…A serine-threonine-rich region is found near the N terminus of ANT (amino acids 13 to 53) ( Figures 8A and 8B). Sequences rich in serine and threonine have been implicated in transcriptional activation (Seipel et al, 1992;Gashler et al, 1993). A potential nuclear localization site is present within the ANT protein from amino acids 252 to 255, suggesting that ANT may be targeted to the nucleus ( Figure 8A; Chelsky et al, 1989 To identify the temporal and spatial expression patterns of the ANT gene, an ANT cDNA was hybridized at high stringency to an RNA gel blot containing total RNA from various plant organs (Figure 78).…”