Regulation of Temporal and Spatial Gene Expression in the Circadian System

我们单位对基因表达调控模式背后的分子机制感兴趣，无论是在时间上还是在空间上。在99财年，我们已经在大鼠芳香胺N-乙酰基转移酶(NAT)启动子区域发现并鉴定了一个功能性的E-box顺式作用元件，它可以介导昼夜节律复合体Clock/BMAL的反式激活，显然只在视网膜(主振荡器)而在松果体(从振荡器)中不起作用。我们还在继续努力通过比较Cyclin、NAT和加压素(AVP)E盒来确定构成昼夜节律E盒的关键元件，这些E盒支持昼夜节律转录复合体Clock/BMAL的不同水平的反式激活(分别增加0、8和40倍)。此外，我们已经成功地使用NAT启动子通过协作转基因计划将主要的负形式的Fra-2转录因子驱动到大鼠松果体。这一成果使我们能够在体内鉴定出第一个Fra-2靶基因：FRA-2、II型脱碘酶、Nectadrin(或CD24)和ID-1(一种碱性HLH类转录因子)。第二个项目来自我们在99财年发现的一个新基因，通过酵母双杂交系统，它与周期(PER)1特异地相互作用，周期(PER)1是细胞机械的核心组件之一，在转录输出中产生自我维持的24小时周期。我们目前正在研究这个新基因及其与PER-1的相互作用，以及它的组织分布和功能(S)。击倒对手的方法也在进行中。

Our Unit is interested in the molecular mechanisms underlying regulated patterns of gene expression, both in time and space. During FY 99 we have identified and characterized a functional E-BOX cis-acting element in the promoter region of the rat Arylalkylamine N-acetyltransferase (NAT) promoter that can mediate transactivation by the circadian complex CLOCK/bMAL apparently only in the retina (a master oscillator) but not in the pineal (slave oscillator). We are also continuing our efforts to identify the critical elements that constitute a circadian E-BOX by comparing the cyclin, NAT and vasopressin (AVP) E-BOXES which support different levels of transactivation by the circadian transcription complex CLOCK/bMAL (0, 8 and 40- fold increase respectively).In addition, we have successfully used the NAT promoter to drive a dominant negative form of the Fra-2 transcription factor to the rat pineal gland through a collaborative transgenesis program. This achievement has enabled us to identify the first Fra-2 target genes in vivo: Fra-2, type II deiodinase, Nectadrin (or CD24) and Id-1 (a basic- HLH class transcription factor). A second project is derived from our discovery during FY 99 of a novel gene, by a yeast two hybrid system, that interacts specifically with Period (Per) 1, one of the core components of the cellular machinery that generates the self-sustained 24 hr cycle in transcriptional output. We are currently characterizing this novel gene and its interaction with Per 1, as well as its tissue distribution and function(s). A knockout approach is also underway.