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Cis,regulatory modules (CRMs) are the functional DNA elements that encode the spatial and temporal expression patterns of a gene. Each gene is regulated by multiple CRMs, which may be hundreds of kilobases distant from the promoter. Many vertebrate CRMs have been characterised in isolation, but how CRMs act together to regulate complex patterns of expression is relatively unknown. Two CRMs that regulate expression of the muscle, specifying master gene myoD1 have previously been identified. Three additional potential CRMs were identified using a comparative genomics approach.The regulatory roles of these CRMs were investigated alone and in combination. Reporter plasmids containing all thirty,two possible combinations of these CRMs were made,and their expression was assayed in an immunologically,defined subpopulation of transfected mouse myoblast cells by flow cytometry. A statistical mechanics,based model used this exhaustive expression dataset to parameterise the interactions between the CRMs. This identified the ability of particular regulatory modules to have both enhancing and repressive effects upon transcription, dependent upon their surrounding CRM ‘context’. The physical proximity of the regulatory modules and myoD1 promoter in native chromatin during gene expression was confirmed by chromosome conformation capture analysis. To characterise the molecular basis of these properties further, a phylogenetic sequence comparison method was used to identify conserved transcription factor binding sites (TFBSs)within each CRM. A combination of experimental, bioinformatic and literature,derived evidence was used to prioritise binding sites fora large scale mutagenesis study to disentangle the molecular interactions between a prominent CRM pair. This allowed the mechanistic underpinnings of context sensitivity in this particular system to be identified. |
