Zheng Xiangzhong, Koh Kyunghee, Sowcik Mallory, Smith Corinne J, Chen Dechun, Wu Mark N, Sehgal Amita
Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Neurosci. 2009 Sep 2;29(35):10920-7. doi: 10.1523/JNEUROSCI.2133-09.2009.
The Drosophila PAR domain protein 1 (Pdp1) gene encodes a transcription factor with multiple functions. One isoform, PDP1epsilon, was proposed to be an essential activator of the core clock gene, Clock (Clk). However, a central clock function for PDP1epsilon was recently disputed, and genetic analysis has been difficult due to developmental lethality of Pdp1-null mutants. Here we report the discovery of a mutation that specifically disrupts the Pdp1epsilon isoform. Homozygous Pdp1epsilon mutants are viable and exhibit arrhythmic circadian behavior in constant darkness and also in the presence of light:dark cycles. Importantly, the mutants show diminished expression of CLK and PERIOD (PER) in the central clock cells. In addition, expression of PDF (pigment-dispersing factor) is reduced in a subset of the central clock cells. Loss of Pdp1epsilon also alters the phosphorylation status of the CLK protein and disrupts cyclic expression of a per-luciferase reporter in peripheral clocks under free-running conditions. Transgenic expression of PDP1epsilon in clock neurons of Pdp1epsilon mutants can restore rhythmic circadian behavior. However, transgenic expression of CLK in these mutants rescues the expression of PER in the central clock, but fails to restore behavioral rhythms, suggesting that PDP1epsilon has effects outside the core molecular clock. Together, these data support a model in which PDP1epsilon functions in the central circadian oscillator as well as in the output pathway.
果蝇PAR结构域蛋白1（Pdp1）基因编码一种具有多种功能的转录因子。一种异构体，即PDP1epsilon，被认为是核心生物钟基因Clock（Clk）的必需激活因子。然而，PDP1epsilon的核心生物钟功能最近受到质疑，并且由于Pdp1基因敲除突变体的发育致死性，遗传分析一直很困难。在这里，我们报告了一个特异性破坏Pdp1epsilon异构体的突变的发现。纯合Pdp1epsilon突变体是可行的，并且在持续黑暗以及存在光暗循环的情况下表现出无节律的昼夜行为。重要的是，突变体在核心生物钟细胞中CLK和周期蛋白（PER）的表达减少。此外，色素分散因子（PDF）在一部分核心生物钟细胞中的表达降低。Pdp1epsilon的缺失还会改变CLK蛋白的磷酸化状态，并在自由运行条件下破坏外周生物钟中per-荧光素酶报告基因的周期性表达。在Pdp1epsilon突变体的生物钟神经元中PDP1epsilon的转基因表达可以恢复有节律的昼夜行为。然而，CLK在这些突变体中的转基因表达挽救了核心生物钟中PER的表达，但未能恢复行为节律，这表明PDP1epsilon在核心分子生物钟之外还有作用。总之，这些数据支持了一个模型，其中PDP1epsilon在核心昼夜振荡器以及输出途径中发挥作用。
