Long-range functional interactions between genes and regulatory elements

• Main Author: Carter, D. P. F.
• Published: University of Cambridge 2004
• Subjects: 591.35
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597329

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, <i>in vitro</i> 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 <i>in vivo.</i> 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.