Light directs zebrafish period2 expression via conserved D and E boxes.
Light directs zebrafish period2 expression via conserved D and E boxes.
复制标题
DOI：
10.1371/journal.pbio.1000223
复制
发表时间：
2009-10
期刊：
影响因子：
9.8
通讯作者：
Foulkes NS
中科院分区：
文献类型：
--
作者：
Vatine G;Vallone D;Appelbaum L;Mracek P;Ben-Moshe Z;Lahiri K;Gothilf Y;Foulkes NS
脊椎动物时钟基因中高度保守的启动子模块赋予光调节基因表达。对于大多数物种来说，光代表了引导内源生物钟的主要环境信号。斑马鱼是研究这一过程的一个令人着迷的脊椎动物模型，因为与哺乳动物不同，斑马鱼的大部分组织直接暴露在光线下会导致局部时钟夹带。重要的是，光在体内和体外的大多数斑马鱼细胞类型中诱导一组基因的表达，包括某些时钟基因。然而，将光与基因表达联系起来的机制仍然知之甚少。为了阐明这一关键机制，我们在这里重点关注光如何调节斑马鱼 period2 (per2) 基因的转录。使用转基因鱼和基于稳定转染细胞系的检测，我们在 per2 启动子内定义了光响应模块 (LRM)。 LRM 位于转录起始位点附近，对于光驱动的基因表达以及光依赖性生物钟调节来说是必要且充分的。奇怪的是，LRM 序列在其他脊椎动物的 per2 基因中高度保守，甚至在缺乏直接光敏感外周时钟的物种中也是如此。此外，我们发现人类 LRM 可以替代斑马鱼 LRM 在斑马鱼细胞中赋予光调节转录。 LRM 包含对其功能至关重要的 E 盒和 D 盒元件。 E-box 通过介导 BMAL/CLOCK 活动来指导生物钟调节，而 D-box 则赋予光驱动表达。促甲状腺激素胚胎因子的斑马鱼同源物可有效结合 LRM D-box 并反式激活表达。我们证明，tef mRNA 水平是光诱导的，并且 tef 表达的敲低会减弱体内 per2 启动子的光驱动转录。总之，我们的结果支持了一个模型，其中 E 盒结合因子和 D 盒结合因子之间的光依赖性串扰是 per2 表达的核心决定因素。这些发现扩展了对光驱动时钟的机制以及脊椎动物中光对时钟基因表达的调节如何进化的一般理解。光是动物用来使生物钟与昼/夜环境同步的主要信号。这一重要特性的核心是光触发基因表达变化的能力。然而，我们仍然缺乏对这是如何发生的完整了解。斑马鱼在这方面特别有趣，因为直接光照会诱导其大部分组织中时钟基因的表达，进而调整内在时钟的相位。在这里，通过研究一个关键的光调节斑马鱼时钟基因 per2 的启动子，我们确定了光响应模块 (LRM)，它对于光控表达是必要且充分的。有趣的是，LRM 在缺乏广泛的光传感组织的其他脊椎动物的 per2 基因中也高度保守。此外，人类LRM可以替代斑马鱼对应物，对斑马鱼细胞中的基因表达进行直接光调节。 LRM 包含对其功能至关重要的 E 盒和 D 盒增强剂。 E 盒是时钟调节的目标，而 D 盒则指导光驱动表达。我们发现 D-box 结合转录因子 tef 的表达本身是由光诱导的，并且对于正常光诱导的 per2 表达至关重要。这些结果增进了我们对光夹带机制以及脊椎动物中光调控时钟基因表达如何进化的理解。
A highly conserved promoter module in a vertebrate clock gene confers light-regulated gene expression. For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line–based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates. Light is the principal signal used by animals to synchronize their circadian clocks with the day/night environment. Central to this vital property is the ability of light to trigger changes in gene expression. However, we still lack a complete understanding of how this occurs. The zebrafish is particularly interesting in this regard since direct light exposure induces the expression of clock genes in most of its tissues and in turn adjusts the phase of the intrinsic clocks. Here, by studying the promoter of one key light-regulated zebrafish clock gene, per2, we have identified a Light Responsive Module (LRM) that is necessary and sufficient for light controlled expression. Interestingly, the LRM is also highly conserved in the per2 genes of other vertebrates that lack widespread light-sensing tissues. In addition, the human LRM can substitute for its zebrafish counterpart to confer direct light regulation of gene expression in zebrafish cells. The LRM contains E- and D-box enhancers critical for its function. While the E-box is a target of clock regulation, the D-box directs light driven expression. We show that the expression of the D-box binding transcription factor, tef, is itself induced by light and is essential for normal light-induced per2 expression. These results advance our understanding of the mechanisms underlying entrainment by light and how light-regulated clock gene expression has evolved in vertebrates.
登录
查看更多内容
DOI：
10.1073/pnas.141090998
发表时间：
2001-07-03
影响因子：
11.1
作者：
Doi, M;Nakajima, Y;Fukada, Y
通讯作者：
Fukada, Y
影响因子：
2.5
作者：
Gothilf, Y;Toyama, R;Klein, DC
通讯作者：
Klein, DC
影响因子：
--
作者：
Appelbaum, L;Toyama, R;Gothilf, Y
通讯作者：
Gothilf, Y
影响因子：
5.3
作者：
Abe, M;Herzog, ED;Block, GD
通讯作者：
Block, GD
影响因子：
64.5
作者：
Balsalobre, A;Damiola, F;Schibler, U
通讯作者：
Schibler, U