The long term objective is to identify genes regulated by EGR2, a human protein with three zinc-finger domains. EGR2 gene expression is induced in fibroblasts and lymphocytes by mitogens such as phorbol 12-myristate 13-acetate. It is a member of a family of at least four genes which encode Proteins with nearly identical zinc-finger domains but little similarity elsewhere and with different induction patterns. The first goal is to identify target DNA sequences with a method shown to work for several zinc finger proteins in other organisms, then use this data to seek target genes. 1.Produce recombinant EGR2 protein in a bacterial expression vector. 2.Use recombinant EGR2 protein in vitro to bind DNA of selected targets, including restriction fragments of lambda DNA, restriction fragments of DNA from the 5' regulatory region of EGR2, and cloned genomic DNA for which the fragments have endlinkers suitable for amplification by the polymerase chain reaction. 3.Determine the protected sequence of the bound fragment by DNase I footprinting. 4.Search the GenEmbl databank for gene regulatory regions containing the target motif. If such a gene, X, is identified, obtain the genomic and cDNA clones. 5.Use the recombinant protein to generate polyclonal sera and monoclonal antibodies. 6.Create transfectants expressing EGR2 from inducible and from constitutive promoters. Use anti-EGR2 antibodies to verify EGR2 expression and determine time course. 7.If clones are available, test gene X regulation by EGR2 in transfectants: a.Northern blot analysis of gene X expression following EGR2 induction. b.Assay nuclear factor preps from transfectants for protection of the genomic sequence. Show inhibition with anti-EGR2 antibodies. C.Use the genomic DNA of gene X as a promoter for a reporter plasmid. Examine expression of reporter in EGR2 transfectants. 8.If above methods are unsuccessful, use next alternative: prepare cDNA from transfectants with and without EGR2 expression induced, and use to generate a subtractive cDNA library. Test the resulting cDNA clones for expression in the EGR2 transfectants, with and without induction of EGR2. 9.As an additional alternative, use 2D high resolution electrophoresis of transfectant to identify protein spots which correlate with EGR2 expression. This could eventually be used for protein microsequencing.
| Gottschalk, A R; Joseph, L J; Quintans, J (1994) Fc gamma RII cross-linking inhibits anti-Ig-induced erg-1 and erg-2 expression in BCL1. J Immunol 152:2115-22 |
| Gottschalk, A R; Joseph, L J; Quintans, J (1993) Differential induction of Egr-1 expression in WEHI-231 sublines does not correlate with apoptosis. Eur J Immunol 23:2011-5 |
