Cloning and functional characterization of the rat alpha2B-adrenergic receptor gene promoter region: Evidence for binding sites for erythropoiesis-related transcription factors GATA1 and NF-E2.
In the rat, the alpha2B-adrenergic receptor (alpha2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of alpha2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of alpha2B-AR expression, a genomic fragment containing 2.8 kb of the 5'-flanking region, the ORF and approximately 20 kb of the 3'-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5'-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332 bp fragment (-732/-401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0 kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for alpha2B-AR expression during fetal life.
INSERM Unit 388, Institut Louis Bugnard, CHU Rangueil, Bâtiment L3, BP 84225, 31432 Toulouse Cedex 4, France.
Stéphane
S
Schaak
Daniel
D
Cussac
Stéphane
S
Labialle
Vincent
V
Mignotte
Hervé
H
Paris
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D008969:Molecular Sequence Data; D050983:NF-E2 Transcription Factor; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D050983:NF-E2 Transcription Factor; D018343:Receptors, Adrenergic, beta-2; D014157:Transcription Factors; D004247:DNA