Differential chromatin topology and transcription factor enhancer binding regulate spatiotemporal gene expression in limb development
Williamson, William Iain
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Many developmental genes are located in gene-poor genomic regions and are activated by long-range enhancers located up to 1Mb away. Modification and reorganisation of chromatin structure is pivotal to such long-range gene regulation. A prerequisite for enhancer activity is the binding of transcription factors and co-factors with the interplay between activating and repressive factors determining tissue, spatial and temporal specificity. Spatiotemporal control of sonic hedgehog (Shh) and the 5′ Hoxd genes (especially Hoxd13) is crucial for vertebrate limb anterior-posterior (A-P) axis and autopod patterning. Shh tissue specificity is controlled by multiple enhancers throughout an adjacent gene desert. The ~0.8Mb-distant limb enhancer (ZRS) bypasses nearby genes to activate only Shh. In contrast, limb-specific HoxD expression is regulated by multiple enhancers, with the ~200kb-distant global control region (GCR) regulatory element the most characterised. In this thesis I investigated the mechanisms of ZRS and GCR regulation of Shh and Hoxd13 respectively. The model system used was immortalised cell lines derived from the anterior and posterior distal forelimb buds of E10.5 and E11.5 mouse embryos. Cell line data were confirmed in dissected limb tissue. Increased expression of the 5′ Hoxd genes, particularly Hoxd13, correlated with the loss of the repressive, polycomb catalysed, histone modification H3K27me3 and decompaction of chromatin structure over the HoxD locus at the distal posterior forelimb bud at stage E10.5. Moreover, I show that the GCR spatially co-localises with the 5′ HoxD locus at the distal posterior region of E10.5-11 embryos. These data are consistent with the formation of a chromatin loop between Hoxd13 and the GCR at the time and place of distal limb bud development when the GCR is required to initiate 5′ Hoxd gene expression. This is the first example of A-P differences in chromatin compaction and local folding in the limb. Point mutations within the ZRS cause ectopic (anterior) Shh expression, which results in preaxial polydactyly (PPD). The ZRS contains multiple canonical ETS transcription factor binding motifs, and point mutations in two families with PPD results in the formation of additional ETS binding sites. The point mutations cause the loss or reduction of ETV4/5 transcription factor binding at a non-canonical ETS binding site and enable additional binding instead of ETS1. I show that ETV4/5, ETS1 and another ETS protein GABPα all bind to the ZRS. This work has revealed the differential effect on Shh expression of two groups of ETS factors mediated through the ZRS. The binding of ETS1/ GABPα determines the posterior Shh expression domain while ETV4/5 restricts anterior Shh expression. Two point mutations alter the ETS-binding profile, creating an additional ETS1/ GABPα site that is sufficient to drive ectopic Shh expression. DNA FISH on E11.5 forelimb and floorplate tissue sections revealed that the Shh-ZRS genomic locus is in a compact chromatin conformation in both Shhexpressing and non-expressing cells. However, I show that the ZRS co-localises with Shh to a significantly greater extent in the distal posterior limb bud and the floorplate compared with cells where Shh is not expressed. This thesis presents novel research into long-range gene regulation during limb development, elucidating the role of chromatin re-organisation and how spatial-specific enhancer activity is determined by opposing sets of binding factors.