Chromatin dynamics at the Sonic Hedgehog locus: a study using limb derived Sonic Hedgehog inducible cell lines to investigate chromatin architecture.
Douglas, Adam Thomas
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Enhancers are cis-regulatory sequences which promote the expression of target genes in a spatial and temporal fashion. They can be located within genes or between them and can act at distances of over 1 Mb. There are several different mechanisms by which enhancers regulate gene expression. Some, such as those regulating the Hox genes, are located close to each other in the genome in a structure referred to as a regulatory archipelago. These come together and act in combination to regulate gene expression, with different enhancer combinations resulting in different patterns of expression. On the other hand, enhancers can act individually, with designated enhancers responsible for regulating the expression of the same gene in different tissues or at different stages of development. Indeed, this is the case for the Sonic Hedgehog gene (Shh) where several different enhancers located within a gene sparse region referred to as a gene desert, act separately leading to Shh expression in areas such as the brain, the lungs, the notochord and neural tube and the limbs. Within the developing mouse embryo, Shh is expressed over roughly a two day period from E10 to E12 in a posterior distal region referred to as the Zone of Polarising Activity (ZPA). Ectopic expression in anterior regions has been observed in some common congenital diseases which affect the limbs, sometimes resulting in the formation of extra digits. The reason for this mis-expression is largely due to defects in the Shh limb enhancer commonly referred to as the Zone of Polarising Activity Regulatory Sequence (ZRS). Mutations within this highly conserved sequence create additional protein binding sites thus activating the enhancer in the wrong locations. The associated diseases are known collectively as the ZRS associated syndromes and can range from the less severe phenotype of preaxial polydactyly type II (characterised by an extra digit near the thumb) to the more severe Werner Mesomelic Syndrome (WMS), where patients present with a clear displacement of their tibia. The mechanism by which the ZRS functions is yet to be fully elucidated, with current studies producing conflicting data. What is known, is that the region encapsulating the Shh gene is highly compact, with both the gene and its enhancers located in a highly conserved Toplogical Associated Domain (TAD) as proven by Hi-C experiments. The boundaries of this domain are likely created by the binding of the protein CTCF to specified binding sites located at the either end of the locus. This restricts the ability of the enhancers to regulate the expression of genes outside the TAD. To study the exact mechanism by which the ZRS is activated and regulates Shh expression, the Hill laboratory has used cultured cell lines derived from the posterior regions of an E11.5 limb bud. Gene expression in these cells is highly reflective of the posterior limb bud, with the key exception being Shh, which is not expressed. However, using different drug treatments or biological manipulations Shh can be activated thereby making this the perfect system to analyse the mechanisms leading to Shh activation. In this investigation the cell lines were used to determine how the position of the ZRS changes upon activation. Using techniques such as Fluorescent in situ hybridisation (FISH) with either fosmid probes or directly labelled probes called MYtags, it was confirmed that the Shh locus is indeed highly compact in both Shh expressing and non-expressing cells. However, no differences were observed in terms of the distance between the ZRS and Shh between these two conditions in our cell lines. Next, both carbon copy chromosome conformation capture (5C) and circular chromosome conformation capture (4C) were used to look at changes to the Shh locus in different conditions. This confirmed Hi-C experiments and other recent publications suggesting that Shh is located within a TAD, the position of which is highly conserved between different conditions and cell lines. Furthermore, treatments activating the Shh gene resulted in significant deviations to the chromatin interactions within the locus suggesting a repositioning of structures when the gene is active. It is believed that the use of Shh inducible limb derived cell lines will prove extremely useful in future scientific endeavours to study the mechanisms of mammalian limb development. These provide a quick and easy means of accessing large numbers of Shh expressing cells, a feature which is increasingly important in an era where large cell numbers are needed for conducting chromosome conformation capture experiments such as Hi-C, 5C and 4C.