Long-range functional interactions between genes and regulatory elements

The mouse b-globin locus contains four genes that are expressed throughout development in the same order they are arranged along chromosome 7. Upstream of the genes is a powerful regulatory element called the locus control region (LCR). The LCR is absolutely required for high-level expression of the b-globin genes. The models to explain how LCRs (and enhancers in general) are able to activate expression of the genes fall into two categories: contact and non-contact. The contact models propose that the LCR and the gene form a physical interaction that leads to active transcription. The non-contact models propose that the LCR alters the chromatin topology or nuclear organisation of the locus, or acts as a nucleation point for factors that polymerise along the chromatin fibre to activate the gene. A contact model has been inferred from indirect observations, in vitro experiments, and oversimplified assumptions. In the absence of a physical assay that is able to determine the three-dimensional arrangements of the b-globin locus the mechanisms of LCR activation remains open to speculation. In this thesis I describe the development of a novel technique called RNA tagging and recovery of associated proteins (RNA TRAP). RNA TRAP uses the power of RNA FISH to target an enzyme to the site of a specific actively transcribing gene. The enzyme catalayses the deposition of a biotin tag onto the chromatin in the vicinity of transcription, which acts as a handle to isolate and identify chromatin elements in proximity to the actively transcribed gene. Using RNA TRAP I have shown that elements of the LCR are in significantly physical proximity to the genes they activate in vivo. This is the first physical demonstration of the interaction of the LCR with a gene, and gives insight into the regulation of genes by enhancers.