Graduate School of Systems Life Sciences, Kyushu University, Hakozaki, Fukuoka, Japan.
PLoS One. 2013 Apr 30;8(4):e63113. doi: 10.1371/journal.pone.0063113. Print 2013.
In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC) is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes"). Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP), a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per), vrille (vri), and PAR domain protein 1ε (Pdp1ε). Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.
在果蝇中，时钟/周期异二聚体（CLK/CYC）是包含 E 盒序列的生物钟基因的主要激活剂（以下简称“E 盒生物钟基因”）。尽管已经进行了广泛的研究来探讨生物钟基因的反馈调节，但对于与 CLK/CYC 一起作用的其他因素知之甚少。在这里，我们表明果蝇 C 端结合蛋白（dCtBP），一种转录共因子，参与 E 盒生物钟基因的调节。在生物钟细胞中过表达 dCtBP 会延长或消除生物钟的运动节律，同时上调一部分 E 盒生物钟基因，如 period（per）、vrille（vri）和 PAR 结构域蛋白 1ε（Pdp1ε）。体外共表达 dCtBP 和 CLK 也会增加 per、vri、Pdp1ε 和 cwo 的启动子活性，这取决于 dCtBP 的表达量，而没有 CLK 时则没有观察到这种效果。当我们使用携带 NAD+结构域氨基酸取代的突变型 dCtBP 时，这些时钟基因在体外的激活并未观察到。这些结果表明，dCtBP 通常通过 E 盒序列作为 CLK/CYC 的假定共激活因子发挥作用。
