Structural and functional characterization of the 5′ upstream promoter of the humanPhox2agene: possible direct transactivation by transcription factor Phox2b

Publisher Website
Google Scholar
Abstract
The specification of neurotransmitter identity is a critical step in neural development. Recent progresses have indicated that the closely related homeodomain factors Phox2a and 2b are essential for development of noradrenergic (NA) neuron differentiation, and may directly determine the neurotransmitter identity. With a long-term goal of understanding the regulatory cascade of NA phenotype determination, we isolated and characterized a hPhox2a genomic clone encompassing approximately 7.5 kb of the 5′ upstream promoter region, the entire exon–intron structure, and approximately 4 kb of the 3′ flanking region. Using mRNAs isolated from the Phox2a-expressing human cell line, both primer extension and 5′-rapid amplification of cDNA ends analyses identified a single transcription start site that resides 172 nucleotides upstream of the start codon. The transcription start site was preceded by a TATA-like sequence motif and transcripts from this site contained an additional G residue at the 5′ position, supporting the authenticity of this site as the transcriptional start site of hPhox2a. We assembled hPhox2a–luciferase reporter constructs containing different lengths of the 5′ upstream sequences. Transient transfection assays of these reporter constructs in both hPhox2a-positive and -negative cell lines show that 1.3-kb or longer upstream sequences of the hPhox2a gene may confer NA cell-specific reporter gene expression. Furthermore, cotransfection assays in the Phox2a-negative HeLa cell line show that forced expression of Phox2b, but not that of Phox2a or MASH1, significantly transactivates the transcriptional activity of hPhox2a. This study will provide a frame to further delineate the regulatory cascade of NA neuron differentiation.