TRANSACTIVATION OF GATA-1 PROMOTER WITH ETS1, ETS2 AND ERGB HU-FLI-1 PROTEINS - STABILIZATION OF THE ETS1 PROTEIN-BINDING ON GATA-1 PROMOTER SEQUENCES BY MONOCLONAL-ANTIBODY/

Ets family proteins activate transcription via binding to the GGAA cor e sequence located in the promoter/ enhancer elements of many cellular and viral genes. GATA-1 is an erythroid-specific transcription factor . The promoter of the chicken GATA-1 gene contains multiple ets bindin g sites (EBS), two of them are present in palindromic form. The GATA-1 promoter has been shown to be activated by the E26 virus. In this stu dy, we have analysed whether the palidromic EBS of the chicken GATA-1 promoter is a target for binding and activation by members of the cell ular ets gene family products. The results herein indicate that both E BS in the palindrome are required for DNA-binding because mutations in either site reduces the activity by at least 95%. Moreover, DNA bindi ng of ETS1 to the EBS palindrome is dramatically stabilized in the pre sence of a specific monoclonal antibody whose epitope maps between ami no acid positions 240-260. Although each of the single sites bind, the efficiency of binding is extremely low. Furthermore, for efficient bi nding the two sites must be in an inverted configuration because of th e fact that the oligonucleotide containing the left and right EBS in t he same orientation binds 10-fold less than the oligonucleotide contai ning the EBS palindrome. Additionally, we show that the transcription of a reporter gene (CAT) either linked to the GATA-1 EBS palindrome or GATA-1 promoter can be activated by cotransfection with ETS1, alterna tively-spliced ETS1, ETS2 or ERGB/Hu-FLI-1 expression vectors.