Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A(2A) adenosine receptor.
By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5'-flanking region and a 0.5-kb 5'-untranslated region of the rat A(2A) adenosine receptor (A(2A)-R) gene. Various lengths of the 5'-flanking region of the A(2A)-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A(2A)-R/NF1), located between bases -2846 and -2827 of the A(2A)-R gene, functions as a repressor for A(2A)-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A(2A)-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A(2A)-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A(2A)-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A(2A)-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A(2A)-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A(2A)-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A(2A)-R. Moreover, addition of the A(2A)-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A(2A)-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A(2A)-R/NF1) that plays a functional role in modulating the expression of A(2A)-R.
Lee
Yi Chao
YC
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
Lai
Hsing Lin
HL
Sun
Chung Nan
CN
Chien
Chen Li
CL
Chern
Yijuang
Y
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D000906:Antibodies; D001253:Astrocytes / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002490:Central Nervous System / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation / Q000235:genetics*; D005809:Genes, Regulator / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D051839:NFI Transcription Factors; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D043705:Receptor, Adenosine A2A; D018047:Receptors, Purinergic P1 / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1
D000906:Antibodies; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D043705:Receptor, Adenosine A2A; D018047:Receptors, Purinergic P1; D012097:Repressor Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human