Chromatin dynamics at the Sonic Hedgehog locus: a study using limb derived Sonic Hedgehog inducible cell lines to investigate chromatin architecture.

摘要

Enhancers are cis-regulatory sequences which promote the expression of target genes in audspatial and temporal fashion. They can be located within genes or between them and can actudat distances of over 1 Mb. There are several different mechanisms by which enhancersudregulate gene expression. Some, such as those regulating the Hox genes, are located close toudeach other in the genome in a structure referred to as a regulatory archipelago. These comeudtogether and act in combination to regulate gene expression, with different enhancerudcombinations resulting in different patterns of expression. On the other hand, enhancers canudact individually, with designated enhancers responsible for regulating the expression of theudsame gene in different tissues or at different stages of development. Indeed, this is the caseudfor the Sonic Hedgehog gene (Shh) where several different enhancers located within a geneudsparse region referred to as a gene desert, act separately leading to Shh expression in areasudsuch as the brain, the lungs, the notochord and neural tube and the limbs.udWithin the developing mouse embryo, Shh is expressed over roughly a two day period fromudE10 to E12 in a posterior distal region referred to as the Zone of Polarising Activity (ZPA).udEctopic expression in anterior regions has been observed in some common congenital diseasesudwhich affect the limbs, sometimes resulting in the formation of extra digits. The reason forudthis mis-expression is largely due to defects in the Shh limb enhancer commonly referred to asudthe Zone of Polarising Activity Regulatory Sequence (ZRS). Mutations within this highlyudconserved sequence create additional protein binding sites thus activating the enhancer in theudwrong locations. The associated diseases are known collectively as the ZRS associatedudsyndromes and can range from the less severe phenotype of preaxial polydactyly type IIud(characterised by an extra digit near the thumb) to the more severe Werner MesomelicudSyndrome (WMS), where patients present with a clear displacement of their tibia.udThe mechanism by which the ZRS functions is yet to be fully elucidated, with current studiesudproducing conflicting data. What is known, is that the region encapsulating the Shh gene isudhighly compact, with both the gene and its enhancers located in a highly conserved ToplogicaludAssociated Domain (TAD) as proven by Hi-C experiments. The boundaries of this domainudare likely created by the binding of the protein CTCF to specified binding sites located at theudeither end of the locus. This restricts the ability of the enhancers to regulate the expression ofudgenes outside the TAD. To study the exact mechanism by which the ZRS is activated and regulates Shh expression,udthe Hill laboratory has used cultured cell lines derived from the posterior regions of an E11.5udlimb bud. Gene expression in these cells is highly reflective of the posterior limb bud, withudthe key exception being Shh, which is not expressed. However, using different drug treatmentsudor biological manipulations Shh can be activated thereby making this the perfect system toudanalyse the mechanisms leading to Shh activation.udIn this investigation the cell lines were used to determine how the position of the ZRS changesudupon activation. Using techniques such as Fluorescent in situ hybridisation (FISH) with eitherudfosmid probes or directly labelled probes called MYtags, it was confirmed that the Shh locusudis indeed highly compact in both Shh expressing and non-expressing cells. However, nouddifferences were observed in terms of the distance between the ZRS and Shh between theseudtwo conditions in our cell lines. Next, both carbon copy chromosome conformation captureud(5C) and circular chromosome conformation capture (4C) were used to look at changes to theudShh locus in different conditions. This confirmed Hi-C experiments and other recentudpublications suggesting that Shh is located within a TAD, the position of which is highlyudconserved between different conditions and cell lines. Furthermore, treatments activating theudShh gene resulted in significant deviations to the chromatin interactions within the locusudsuggesting a repositioning of structures when the gene is active.udIt is believed that the use of Shh inducible limb derived cell lines will prove extremely usefuludin future scientific endeavours to study the mechanisms of mammalian limb development.udThese provide a quick and easy means of accessing large numbers of Shh expressing cells, audfeature which is increasingly important in an era where large cell numbers are needed forudconducting chromosome conformation capture experiments such as Hi-C, 5C and 4C.