Molecular Endocrinology of Gene Regulation By cAMP

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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.