Sp proteins and Phox2b regulate the expression of the human Phox2a gene.
Phox2a is a vertebrate homeodomain transcription factor that is involved in the specification of the autonomic nervous system. We have isolated the 5' regulatory region of the human Phox2a gene and studied the transcriptional mechanisms underlying its expression. We first identified the minimal gene promoter by means of molecular and functional criteria and demonstrated that its activity relies on a degenerate TATA box and a canonical Sp1 site. We then concentrated on the region immediately upstream of the promoter and found that it stimulates transcription in a neurospecific manner because its deletion caused a substantial decline in reporter gene expression only in neuronal cells. This DNA region contains a putative binding site for homeodomain transcription factors, and its mutation severely affects the transcriptional activity of the entire 5' regulatory region, thus indicating that this site is necessary for the expression of Phox2a in this cellular context. The use of the electrophoretic mobility shift assay showed that Phox2b/PMX2b is capable of specifically interacting with this site, and cotransfection experiments demonstrated that it is capable of transactivating the human Phox2a promoter. Many data obtained from knock-out mice support the hypothesis that Phox2a acts downstream of Phox2b during the development of most of the autonomic nervous system. We have provided the first molecular evidence that Phox2b can regulate the expression of Phox2a by directly binding to its 5' regulatory region.
Flora
A
A
Department of Medical Pharmacology, University of Milan and Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology Center, 20129 Milan, Italy.
Lucchetti
H
H
Benfante
R
R
Goridis
C
C
Clementi
F
F
Fornasari
D
D
D020121:5' Untranslated Regions / Q000235:genetics; D001341:Autonomic Nervous System / Q000196:embryology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000502:physiology; D015152:Blotting, Northern; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009447:Neuroblastoma / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015533:Transcriptional Activation; D014162:Transfection
D020121:5' Untranslated Regions; D018398:Homeodomain Proteins; C109220:NBPhox protein; D009419:Nerve Tissue Proteins; C491061:PHOX2A protein, human; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA