Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos.
The Brachyury genes of two divergent ascidians, As-T of Halocynthia roretzi and Ci-Bra of Ciona intestinalis, are expressed exclusively in notochord precursor cells. A previous study showed that the notochord-specific expression of Ci-Bra is controlled by a minimal promoter that is composed of three distinct regions: a region responsible for repression of expression in non-notochord mesoderm cells, a region for activation of expression in notochord cells, and a region for activation of expression in non-notochord mesoderm cells, distal to proximal to the transcription initiation site, respectively. We examined various deletion constructs of the As-T/lacZ fusion gene and demonstrate that a module between -289 and -250 bp of the 5'-flanking region is responsible for notochord-specific expression of the reporter gene. Gel-shift assays suggested the binding of nuclear protein(s) to this module. The 5'-flanking region of As-T contains a potential T-binding motif (-ACCTAGGT-) around -160 bp. Deletion of this motif from the p(-289)As-T/lacZ diminished the reporter gene expression. In addition, coinjection of p(-289)As-T/lacZ and synthetic As-T mRNA resulted in ectopic expression of lacZ in non-notochord cells, suggesting that the T-binding motif is responsible for autoactivation of the gene. These findings revealed striking differences between the minimal promoters of As-T and Ci-Bra so far revealed, with respect to their notochord-specific expression. Furthermore, reciprocal injections of reporter gene constructs, namely As-T/lacZ into Ciona eggs and Ci-Bra/lacZ into Halocynthia eggs, suggest alterations in the cis-regulatory elements and trans-activation factors that have occurred during evolution of the two ascidian species.
Takahashi
H
H
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. taka@ascidian.zool.kyoto-u.ac.jp
Mitani
Y
Y
Satoh
G
G
Satoh
N
N
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D005075:Biological Evolution; D002938:Ciona intestinalis / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005326:Fetal Proteins; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D007763:Lac Operon; D008969:Molecular Sequence Data; D009154:Mutation; D009672:Notochord / Q000196:embryology; D010063:Ovum / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020825:T-Box Domain Proteins / Q000235:genetics; D014561:Urochordata / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D000098908:Brachyury Protein
D004247:DNA; D017931:DNA Primers; D005326:Fetal Proteins; D020825:T-Box Domain Proteins; D000098908:Brachyury Protein