CHARACTERIZATION OF THE HUMAN 5-HT2A RECEPTOR GENE PROMOTER

The regulation of 5-HT2A receptor (5-HT(2A)R) expression has been impl icated in a variety of pathological processes and has been shown to be extremely complicated and controversial. In order to understand the m echanisms of regulation of this receptor, it is important to character ize its promoter. In this report, the 5' end of the human 5-HT(2A)R ge ne was cloned and characterized. Anchored PCR mapped multiple transcri ption initiation sites at nucleotides -1157, -1137, -1127, and -496. T ransfection of chimeric growth hormone plasmids containing various DNA fragments into 5-HT(2A)R-positive human cell lines (SHSY-5Y, neurobla stoma; HeLa, cervix carcinoma) showed that the 0.74 kb HaeIII/PvuII fr agment, which encompasses the initiation sites between -1157 and -1127 and 5' of the downstream initiation site (at -496), exhibited signifi cant promoter activity. This promoter activity was not affected by the sequence upstream of the 0.74 kb fragment. The sequence downstream (t he 0.45 kb PvuII/SmaI fragment) strongly repressed this promoter activ ity, suggesting the presence of a silencer. Sequence analysis combined with gel retardation and Dnase 1 footprinting assay identified multip le cis and trans elements for this fragment, including Sp1, PEA3, cycl ic AMP response element (CRE)-like sequence, and E-boxes, Two novel tr anscription factors have been detected by gel retardation and DNase 1 footprinting assay; one of them may be specific for human. The transcr iption factors and promoter activities were low in the negative cell l ine NCI-H460 (human lung large cell carcinoma). Interestingly, the 0.3 9 kb fragment, isolated from the 3' end of the 0.74 kb fragment, exhib ited the highest promoter activity. The possibility that this 0.39 kb fragment may be an alternative promoter is discussed. These new data a re essential for further study of the regulation of 5-HT(2A)R gene exp ression.