Abstract
The structure and function of the 5'-flanking region of the mouse and human serotonin 1a receptor gene have been analyzed by RNA 5' end mapping, DNA-protein interaction, and transient expression assays. A large number of mRNA 5' termini, detected by mapping 5' ends from mouse brain RNA, were found dispersed over a region of about 700 base pairs flanking the receptor coding sequence. Consistent with the apparently heterogeneous pattern of transcription initiation, the flanking DNA sequence lacked typical TATA box elements and was rich in guanine and cytosine. The mouse and human 5'-flanking sequences were 63% homologus and similarly organized. A guanine-cytosine-rich DNA sequence motif related to the sequence 5'-GGGG(C/A)GGGG-3' was repeated within the 5'-flanking region and located at or near several mRNA 5' ends. This DNA sequence motif bound to proteins in a crude HeLa cell nuclear extract. A cDNA encoding a protein that interacts with this sequence was cloned and found to be the MAZ (Pur-1, Zif87) protein. The interaction between MAZ and the receptor gene 5'-flanking region proximal to the protein coding sequence was examined by DNase I footprinting, and four sites of MAZ interaction were identified. Three of the four MAZ binding sites also were shown to interact with transcription factor Sp1. Overproduction of MAZ or Sp1 in transient transfection assays increased expression directed by the human 5'-flanking sequence, although MAZ was substantially more effective. This result suggests that MAZ and Sp1 both participate in regulating expression from the serotonin 1a receptor gene promoter, and it raises the possibility that MAZ may act at a variety of promoters through the guanosine-cytosine-rich sequences generally thought to serve as binding sites for the Sp1 family of transcription factors. Analysis of one of the guanosine-cytosine-rich DNA sequences also revealed that it can serve as a transcription initiator sequence in vitro. This initiator sequence differs from previously characterized initiators and may represent a new class of this transcriptional control sequence.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U33820, mouse serotonin 1a receptor gene promoter; Z11168, human serotonin 1a receptor gene promoter; U33819, MAZ DNA-binding protein
The serotonin 1a (5-HT1a) receptor has several features that make it an interesting candidate for study of gene regulation. It is expressed in a restricted pattern in the brain. 5-HT1a receptor mRNA has been detected by in situ hybridzation in the hippocampus, midbrain raphe nuclei, and cerebral cortex [7,8,9]. 5-HT1a receptor expression is regulated during development; it is present transiently in the rat cerebellum (10 –13)
Analysis of G/C-rich transcriptional control elements is complicated by the relatively large number of different DNA-binding proteins that are capable of interaction with GC-rich DNA sequences [30]. Consistent with this fact, we have found that the MAZ binding sites located in the 5-HT1a receptor 5Ј-flanking region interact with transcription factor Sp1 [31]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U33820, mouse serotonin 1a receptor gene promoter; Z11168, human serotonin 1a receptor gene promoter; U33819, MAZ DNA-binding protein. The serotonin 1a (5-HT1a) receptor has several features that make it an interesting candidate for study of gene regulation It is expressed in a restricted pattern in the brain. The levels of 5-HT1a receptor and its mRNA apparently are regulated by hormones [14], and the 5-HT1a receptor itself may be an important regulator of gene expression through its coupling to a G-protein that negatively regulates adenylate cyclase [15, 16] All of these characteristics probably contribute to the roles thought to be played in the brain by the 5-HT1a receptor, influencing mood, behavior, and regulation of neuroendocrine function [17,18,19,20,21,22,23,24,25,26]
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