Functional dissection of a single enhancer sequence at the mouse a-globin locus (360G-Wellcome-109097_Z_15_A)

Enhancers are cis-regulatory DNA elements that bind transcription factors and chromatin remodelers and drive expression of target genes in a spatiotemporal context. Despite their importance in cell fate specification in development and differentiation, up to now enhancers have largely been studied in assays that are poor predictors of their in vivo requirement and function. In this project, I will probe enhancer sequence requirements for gene regulation in situ, using the mouse a-globin locus as a model. In order to directly dissect the critical sequences, I will first produce a mouse where a-globin expression is driven by a single element (R2) by deleting the other four endogenous enhancers (R1, R3, R4 and Rm). After validating this mouse for its chromatin environment and transcriptional output, I will perform a high-throughput screen of modified R2 enhancer sequences for transcriptional output using an ES cell in vitro haematopoietic differentiation system. From these results I will select three mutant sequences for molecular characterisation in vivo. Resulting data from primary mouse tissues will provide insight into transcription factor binding interdependence, recognition site ‘grammar’ and how binding affects the enhancer-promoter interactions critical for transcription.