Regulation of Temporal and Spatial Gene Expression in Th

我们单位对昼夜节律时间框架内基因表达调控模式背后的分子机制感兴趣。昼夜节律领域提供了假设驱动的研究最适合填补的空白类型的极好例子。在描述生物钟的内部工作机制方面取得巨大成功后，分子时间生物学领域正迅速将其注意力转移到全球范围内转录昼夜变化的可视化上。但是，在快速增长的数据库中，在我们对分子钟如何工作的理解取得显著进展的背后，有几个重要的问题仍然没有得到回答。例如，我们现在知道BMAL/Clock蛋白复合体和一个被称为E-Box的顺式作用元件之间的相互作用位于主时钟机械和一组特定的时钟和时钟控制基因之间的间期。然而，尽管这种相互作用的中心性质，目前还没有任何模型可以解释这样一个混杂的DNA调控位点如何将转录机制专门引导到昼夜节律基因的启动子上。 时间基因表达单位的长期目标是确定在昼夜节律系统中控制基因表达特异性的机制。我们的具体目标是确定起作用的DNA位点和蛋白质因子，以实现昼夜节律目标选择(在哪里激活哪个基因？)以及b)精确的幅度调制(幅度多少？)我们假设存在尚未识别或未被识别的调控因素，这些因素赋予E-Box昼夜节律价值。我们进行这项研究的基本原理是，识别这些因素必然会揭示调控CCG表达的位置、相位和幅度的新的关键途径。 为了验证这一假设，UTGE的研究重点放在两个平行的系统上，在这些系统中，基因表达的精心编排是显而易见的。第一个系统是BMAL/Clock转录因子与昼夜节律的E-Box之间的相互作用。我们的方法包括围绕已知对来自时钟的输入响应或难以响应的完美E盒的子集的DNA上下文的系统剖析。我们抓住了新的序列，有可能特异性地修改BMAL/时钟作用，并研究同源结合因子的生物学。第二个系统与Fos相关抗原(Fra)-2反应有关，特别是在松果体。在这个奴隶振荡器中，Fra-2基因的强劲昼夜诱导发生在基因表达的大规模重新编程期间。我们通过产生表达松果体特异的显性阴性形式的Fra-2蛋白的转基因大鼠来研究这种反应。然后，我们通过转录组分析评估了含有AP-1因子的FrA-2在时间靶选择中的作用。 由于我们在这两个领域的研究，我们对AP-1转录因子家族与BMAL/CLOCK复合体在转录振荡系统中调控CLOCK基因表达之间存在相互作用的可能性特别感兴趣。 我们的最新成就包括： 1)昼夜节律E-Box在视网膜和松果体中的区别使用的体内验证。 2)大鼠松果体中FrA-2调控靶点的鉴定。 3)发现了一个新的顺式作用元件(CTRR)，它影响BMAL/Clock反式激活，并在松果体中呈现节律性结合。 4)发现了Fra-2/Jund复合体在BMAL/时钟依赖途径中的调节作用。 5)发现强昼夜节律冷诱导糖蛋白30基因也是性二型基因 5)在啮齿动物肝脏中斯特罗调节元件结合蛋白(SREBP)-1的蛋白水解性激活过程中，发现了一种可迅速重置的昼夜节律。 意义 上述研究工作已经对几个领域产生了新的见解，有助于更深入地了解控制昼夜节律转录的微妙机制，并为未来的项目提供了令人兴奋的新线索。通过系统地剖析严格控制和非常强大的空间调控系统(AA-NAT、Fra-2、SREBP)和时间调控系统(AVP、Fra-2、SREBP)，我们期望找到控制基因表达的新机制。它们的相关性可能会蔓延到与时间生物学无关的领域。

Our Unit is interested in the molecular mechanisms underlying regulated patterns of gene expression in circadian time frames. The circadian field provides excellent examples of the types of gaps that hypothesis-driven research is best suited to fill. After having enormous success in describing the inner workings of the biological clock, the field of molecular chronobiology is rapidly shifting its attention to the visualization of circadian changes in transcription on a global scale. But in the midst of rapidly growing databases and behind the remarkable progress in our understanding of how molecular clocks work, several important questions remain unanswered. For example, we now know that the interaction between the BMAL/CLOCK protein complex and a cis-acting element known as the E-Box lies at the interphase between the master clock machinery and a specific set of clock and clock-controlled genes. In spite of the central nature of this interaction however, no current models can explain how such a promiscuous DNA regulatory site can steer the transcriptional machinery specifically onto the promoters of circadian genes. The long-term goal of the Unit on Temporal Gene Expression is to identify the mechanisms controlling the specificity of gene expression in the circadian system. Our specific aim is to identify DNA sites and protein factors that function to achieve a) circadian target selection (which gene to activate and where?) and b) precise amplitude modulation (by how much?). We hypothesize the existence of yet unidentified or unrecognized regulatory factors that confer circadian value onto an E-Box. Our rationale for conducting this research is that identification of such factors is bound to reveal novel and critical pathways that modulate the location, phase and amplitude of CCG expression. To test this hypothesis the UTGE research focuses on two parallel systems where careful orchestration of gene expression is evident. The first system is the interaction between the BMAL/CLOCK transcription factor and the circadian E-Box. Our approach involves the systematic dissection of the DNA context around a subset of perfect E-Boxes known to be responsive or refractory to the input from the clock. We latch onto novel sequences with the potential to specifically modify BMAL/CLOCK action and investigate the biology of the cognate binding factors. The second system pertains to the Fos Related Antigen (Fra)-2 response, particularly in the pineal gland. The robust circadian induction of the Fra-2 gene in this slave oscillator takes place during a massive reprogramming of gene expression. We study this response through the generation of transgenic rats that express a pineal-specific dominant negative form of the Fra-2 protein. We then evaluate the role of a Fra-2 containing AP-1 factor in temporal target selection by transcriptome analysis. As a result of our research in these two areas we became particularly interested in the possibility of a cross-talk between the AP-1 family of transcription factors and the modulation of clock gene expression by the BMAL/CLOCK complex in transcriptionally oscillatory systems. Our most recent accomplishments include: 1) the in vivo validation of the differential use of a circadian E-Box in retina vs. pineal. 2) identification of Fra-2-modulated targets in the rat pineal gland. 3) the discovery of a novel cis-acting element (CTRR) that affects BMAL/CLOCK transactivation and that displays rhythmic binding in the pineal gland. 4) the discovery of a modulatory role of the Fra-2/JunD complex on the BMAL/CLOCK-dependent pathway. 5) discovering that the strongly circadian Cold inducible glycoprotein 30 gene is also a sexually dimorphic gene 5) the discovery of an acutely resettable circadian rhythm in the proteolytic activation of Sterol regulatory element binding protein (SREBP)-1 in the rodent liver. Significance The research efforts outlined above have already generated new insight into several areas, contributed to a deeper understanding of the subtle mechanisms that control circadian transcription, and provided exciting new leads for future projects. By systematically dissecting tightly controlled and very robust regulatory systems in space (AA-NAT, Fra-2, SREBP) and time (AVP, Fra-2, SREBP) we expect to find novel mechanisms for the control of gene expression. Their relevance would likely spill over into fields unrelated to chronobiology.