Investigating the biological phase separation of CBP; a balance of opposing forces

CREB binding protein (CBP) is a transcriptional coactivator protein which has histone acetyltransferase (HAT) activity and binds multiple transcription factors, and is therefore commonly found at enhancer regions. Enhancers are DNA regulatory elements which increase the transcription of their target genes by acting as transcription factor binding hubs. Recent evidence indicates phase separation, the formation of a condensate due to a high concentration of proteins and nucleic acids, as a mechanism of how enhancers donate transcriptional machinery to the promoter, to upregulate gene expression. Proteins commonly found at enhancer regions, including CBP, have been shown to phase separate; however, the importance of how phase separation affects protein activity and dynamics is only now becoming clear. I wanted to determine what drives CBP phase separation, and develop experimental systems to understand how phase separation affects CBP HAT activity.

I have shown that CBP forms phase separated condensates both at endogenous levels and when overexpressed. Utilising an overexpression model system I defined regions which influence the phase behaviour of CBP. These experiments suggested that the phase separation of CBP is not caused by a single region but it is the cumulative effect of different regions with opposing influences on phase separation. I further developed doxycycline inducible cell lines expressing GFP tagged CBP and selected phase separating mutants, to express near-endogenous levels of protein, and reduce concentration as a factor for influencing phase separation. Finally, I have started to validate a single molecule approach to monitor CBP dynamics and HAT activity within the context of the different phase separating mutants.

The toolkit developed within this doctoral work will broaden our knowledge on the diverse effects of phase separation on CBP HAT activity.