Mechanism of DNA replication-dependent transcriptional activation of the acetylcholinesterase gene in the Ciona intestinalis embryo.

ABSTRACT

The acetylcholinesterase-encoding gene in the ascidian Ciona intestinalis (Ci-AChE) is expressed in tail muscle cells from the gastrula stage. When the embryo was continuously treated with aphidicolin from the 32-cell stage, Ci-AChE was not expressed even when control embryos reached the tailbud stage. This result suggests that Ci-AChE acquires the competence to be transcribed after passing through a certain number of DNA replication cycles. A lacZ reporter gene containing the 5' flanking region of Ci-AChE was expressed in the tail muscle cells. Aphidicolin treatment from the 32-cell stage affected, but did not completely suppress, the expression of lacZ. A bisulfite sequencing analysis was carried out to examine the methylation status of four regions within the 5' flanking sequence and the first exon. However, all of these regions remained unmethylated from the 16-cell to 110-cell stages. The results suggested that the DNA of the Ci-AChE locus is not responsible for counting the rounds of replication. We examined the expression of the C. intestinalis MyoD (Ci-MyoD), a transcription factor that activates Ci-AChE. Aphidicolin treatment from the 32-cell stage suppressed the expression of Ci-MyoD, even when control embryos reached the gastrula stage. These results suggest that a lack of Ci-MyoD is critical to the suppression of Ci-AChE in aphidicolin-treated embryos.