Lindon, Ann Catherine; (1994) Protein-protein interactions of transcription factor CREB 1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The transcription factor CREB, shown to activate the expression of target genes in response to extracellular signals mediated by cyclic AMP, is present in all cells examined for its expression. Despite the ubiquity of CREB, different cell types vary greatly in their ability to respond to cyclic AMP. Moreover, CREB has been implicated in the regulation of highly tissue-restricted patterns of gene expression. Although the mechanics of transcriptional activation by CREB have been elucidated in some detail, the modulation of its activity which can give rise to this specificity in its behaviour are poorly understood. This thesis investigates a role for the interaction of cellular factors with CREB in modifying its activity. Data are presented which show that in cell lines not responsive to cyclic AMP, CREB appears to interact through its leucine zipper with a factor inhibitory to its activity. Attempts to investigate this phenomenon by use of fusions of CREB with the DNA-binding domain of the yest GAL4 protein, and problems encountered with this approach, are discussed. ATF-1 is shown to be a good candidate for a negative regulator of CREB in the cell lines examined. Experimental procedures are developed for observing CREB-interactions with factors present in cell extracts, and the use of these techniques in cloning CREB-interacting factors from an expression library is described. Among the factors cloned are CREB, ATF-1 and a novel form of CREM. One of the clones is found to encode a novel and unusual homeodomain protein, Homeodomain protein Interacting with CREB (HIC), which is expressed in a tissue-restricted pattern, most notably in the prostate. HIC interacts with CREB in Far Western assays in a manner which appears to require the homeodomain of HIC and the leucine zipper of CREB. Additional properties of HIC are examined, including its ability to dimerize and its preferred sequence-specificity of DNA binding; and the possible consequences of its interaction with CREB are explored.

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