Dissecting the Roles of the Transcriptional Coactivator p300 Regulating the Oncogenic Self-Renewal Gene NANOG.

Abstract

Eukaryotic gene expression is controlled by the concerted actions and interactions of DNA-regulatory elements, transcriptional activators and associated coactivators. Successful assembly of the proper activator and coactivators at a target gene promoter leads to the stimulation of RNA polymerase activity and the transcription of the gene into messenger RNA. Studying the interactions leading up to transcriptional initiation is challenging due to the relatively weak and promiscuous nature of activator-coactivator interactions. This dissertation documents the identification of coactivator functions that control the expression of the medically relevant target gene NANOG.

NANOG is an embryonic transcription factor that confers tumorigenic and self-renewing potential when expressed in human cancer cells, yet its regulation is poorly understood and few methods are currently available to block NANOG function. We reasoned that modulating the coactivator(s) that regulate NANOG would allow for control over NANOG expression. To achieve this control, we identified the histone acetyltransferase (HAT) p300 as a necessary and direct coactivator of NANOG expression in a variety of cancer cells.

In order to better manipulate p300, we identified critical p300 domains involved in NANOG expression by systematically deleting, mutating, and inhibiting all potential p300 interaction surfaces and catalytic functions. The activator-binding domain CH1 was found to be essential for p300-driven NANOG expression, suggesting the CH1 domain may act in localizing p300 to the NANOG promoter prior to transcription. Additionally, p300 HAT domain activity was found to be necessary for maintaining high levels of histone acetylation at the NANOG promoter and for maintaining NANOG expression in cancer cells. These results allow us to propose a model for the activator-coactivator interactions that drive NANOG transcription. This model will assist in guiding future drug discovery and chemical probe design by providing the first validated targets capable of downregulating NANOG expression in cancer cells.