Characterization of the human 5-HT2A receptor gene promoter.
The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes and has been shown to be extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, it is important to characterize its promoter. In this report, the 5' end of the human 5-HT2AR gene was cloned and characterized. Anchored PCR mapped multiple transcription initiation sites at nucleotides -1157, -1137, -1127, and -496. Transfection of chimeric growth hormone plasmids containing various DNA fragments into 5-HT2AR-positive human cell lines (SHSY-5Y, neuroblastoma; HeLa, cervix carcinoma) showed that the 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites between -1157 and -1127 and 5' of the downstream initiation site (at -496), exhibited significant promoter activity. This promoter activity was not affected by the sequence upstream of the 0.74 kb fragment. The sequence downstream (the 0.45 kb PvuII/SmaI fragment) strongly repressed this promoter activity, suggesting the presence of a silencer. Sequence analysis combined with gel retardation and Dnase 1 footprinting assay identified multiple cis and trans elements for this fragment, including Sp1, PEA3, cyclic AMP response element (CRE)-like sequence, and E-boxes. Two novel transcription factors have been detected by gel retardation and DNase 1 footprinting assay; one of them may be specific for human. The transcription factors and promoter activities were low in the negative cell line NCI-H460 (human lung large cell carcinoma). Interestingly, the 0.39 kb fragment, isolated from the 3' end of the 0.74 kb fragment, exhibited the highest promoter activity. The possibility that this 0.39 kb fragment may be an alternative promoter is discussed. These new data are essential for further study of the regulation of 5-HT2AR gene expression.
Zhu
Q S
QS
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033, USA.
Chen
K
K
Shih
J C
JC
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D003850:Deoxyribonuclease I; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011985:Receptors, Serotonin / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D011985:Receptors, Serotonin; D014157:Transcription Factors; D004247:DNA; D003850:Deoxyribonuclease I