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 core 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 binding 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 study, we have analysed whether the palidromic EBS of the chicken GATA-1 promoter is a target for binding and activation by members of the cellular ets gene family products. The results herein indicate that both EBS in the palindrome are required for DNA-binding because mutations in either site reduces the activity by at least 95%. Moreover, DNA binding of ETS1 to the EBS palindrome is dramatically stabilized in the presence of a specific monoclonal antibody whose epitope maps between amino acid positions 240-260. Although each of the single sites bind, the efficiency of binding is extremely low. Furthermore, for efficient binding the two sites must be in an inverted configuration because of the fact that the oligonucleotide containing the left and right EBS in the same orientation binds 10-fold less than the oligonucleotide containing 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, alternatively-spliced ETS1, ETS2 or ERGB/Hu-FLI-1 expression vectors.
Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21702-1201.
A
A
Seth
L
L
Robinson
D M
DM
Thompson
D K
DK
Watson
T S
TS
Papas
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494041:ETS1 protein, human; C494069:ETS2 protein, human; D050981:Erythroid-Specific DNA-Binding Factors; C078433:FLI1 protein, human; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA