Molecular Endocrinology of Gene Regulation By cAMP

这项研究的总体目标是了解cAMP反应元件结合蛋白CREB在基础状态下调节基因转录的机制，以及对激素和神经递质刺激细胞产生的信号的反应。这些细胞外信号激活了多种蛋白激酶，使CREB的激酶诱导结构域(KID)中的Ser133磷酸化，并增强转录激活。为了刺激靶基因的转录，有必要：1)将聚合酶复合体招募到启动子上；2)将聚合酶复合体异构化到能够在依赖于ATP的过程中熔化DNA模板的活性状态；以及3)拆解聚合酶复合体，使其在称为启动子清除的步骤中呈现稳定的伸长构象。所有这些步骤都可以由转录因子和聚合酶复合体之间的相互作用来调节。在这项资助的前一个周期中，我们证明了CREB中的构成激活结构域(CAD)是通过与启动子识别因子IID相互作用来介导聚合酶复合体招募的必要条件和充分条件，并且CREB中KID的磷酸化刺激了该复合体的活性，从而促进了转录起始的后续步骤。我们假设P-CREB通过调节IIG的招募和激活来促进异构化和启动子的清除，而重新启动主要是一种招募现象，尽管它可以通过CREB的磷酸化来增强。这里描述的研究旨在确定：1)转录反应中的哪些招募后步骤受磷酸化CREB(P-CREB)调控；2)这些步骤中的每一步都需要哪些通用转录因子来刺激；以及3)通用因子如何直接或间接地与P-CREB或其辅助激活剂相互作用。这是一个重要的问题，因为几乎所有的细胞和组织都依赖于某种形式的CREB信号，无论是调节代谢过程、内分泌组织的生长和发育，还是神经元的成熟和存活。我们阐明的P-CREB转录激活途径的至少部分特征可能会被其他受不同激素调控的转录因子所共享。

The overall goal of the research described here is to understand the mechanisms used by the cAMP response element binding protein, CREB, to regulate gene transcription in the basal state and in response to signals generate by stimulation of cells with hormones and neurotransmitters. These extracellular signals activate a variety of protein kinases that phosphorylate Ser 133 in the kinase inducible domain (KID) of CREB and enhance transcription activation. In order to stimulate transcription of a target gene, it is necessary to: 1) recruit a polymerase complex to the promoter; 2) isomerize the polymerase complex to an active state capable of melting the DNA template in an ATP-dependent process; and 3) dissemble the polymerase complex to allow the polymerase to assume its stable elongating conformation in a step called promoter clearance. All of these steps can be regulated by interactions between transcription factors and the polymerase complex. In the previous cycle of this grant, we showed that the constitutive activation domain (CAD) in CREB is necessary and sufficient to mediate recruitment of a polymerase complex through interaction with the promoter recognition factor IID, and that phosphorylation of the KID in CREB stimulates the activity of this complex to facilitate subsequent steps in transcription initiation. We hypothesize that P-CREB enhances isomerization and promoter clearance by regulating the recruitment and activation of IIg and that reinitiation is primarily a recruitment phenomenon, although it could be augmented by phosphorylation of CREB. The studies described here are designed to determine: 1) which post-recruitment steps in the transcription reaction are regulated by the phospho-CREB(P-CREB); 2) which general transcription factors are required for stimulation of each of these steps; and 3) how the general factors interact, whether directly or indirectly, with P-CREB or its co- activators. This is an important question because virtually all cells and tissues rely upon some form of signaling to CREB, whether to regulate metabolite processes, growth and development of endocrine tissues, or maturation and survival of neurons. It is probable that at least some of the features of the transcription activation pathway we elucidate for P- CREB will be shared by other transcription factors regulated by different hormones.