Regulation of Temporal and Spatial Gene Expression in Th

我们的单位感兴趣的是在昼夜节律的时间框架基因表达的调控模式的分子机制。昼夜节律场提供了假设驱动研究最适合填补的空白类型的绝佳例子。在描述生物钟的内部运作方面取得巨大成功之后，分子时间生物学领域正在迅速将注意力转移到全球范围内转录的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答案。例如，我们现在知道BMAL/CLOCK蛋白复合物与称为E-Box的顺式作用元件之间的相互作用位于主时钟机制与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核心性质，目前没有模型可以解释这样一个混杂的DNA调控位点如何能够引导转录机制特异性地进入昼夜节律基因的启动子。 时间基因表达单元的长期目标是确定控制昼夜节律系统中基因表达特异性的机制。我们的具体目标是确定DNA位点和蛋白质因子，其功能是实现a）昼夜节律靶向选择（激活哪个基因，在哪里？）和B）精确的幅度调制（多少？）。我们假设存在尚未鉴定或未识别的调节因素，赋予昼夜节律值到一个电子盒。我们进行这项研究的基本原理是，这些因素的鉴定必将揭示新的和关键的途径，调节CCG表达的位置，相位和幅度。 为了验证这一假设，UTGE的研究集中在两个平行系统上，在这两个系统中，基因表达的精心编排是显而易见的。第一个系统是BMAL/CLOCK转录因子和昼夜节律E-Box之间的相互作用。我们的方法涉及系统地解剖DNA上下文周围的一个子集的完美的电子盒已知是响应或耐火的输入从时钟。我们锁定到新的序列，有可能专门修改BMAL/时钟的行动和调查的同源结合因子的生物学。第二个系统涉及Fos相关抗原（Fra）-2应答，特别是在松果体中。在这个从属振荡器中，Fra-2基因的强大昼夜节律诱导发生在基因表达的大规模重编程期间。我们研究这种反应，通过一代转基因大鼠，表达松果体特异性显性阴性形式的Fra-2蛋白。然后，我们评估了一个Fra-2包含AP-1因子在时间的目标选择转录组分析的作用。 由于我们在这两个领域的研究，我们变得特别感兴趣的可能性之间的串扰的AP-1家族的转录因子和调制的时钟基因表达的BMAL/时钟复合物在转录振荡系统。 我们最近的成就包括： 1)在视网膜与松果体中昼夜E-Box的差异使用的体内验证。 2)大鼠松果体中Fra-2调节靶点的鉴定。 3)发现一种新的顺式作用元件（CTRR），影响BMAL/CLOCK反式激活，并在松果体中显示节律性结合。 4)发现Fra-2/JunD复合物对BMAL/CLOCK依赖性通路的调节作用。 5)发现强昼夜冷诱导糖蛋白30基因也是一个性二型基因， 5)在啮齿动物肝脏中固醇调节元件结合蛋白（SREBP）-1的蛋白水解激活中发现了急性可重置的昼夜节律。 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。通过系统地剖析空间（AA-NAT，Fra-2，SREBP）和时间（AVP，Fra-2，SREBP）的严格控制和非常强大的调控系统，我们希望找到新的机制来控制基因表达。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.