Hill, Robert

Lettice, Laura

Graham, Katy Alexandra

Medical Research Council

Cancer Research UK

Chief Scientist Office of Scotland

the Stroke Association

2023-07-11T11:47:28Z

2023-07-11T11:47:28Z

2023-07-11

Enhancers are capable of driving gene expression over linearly vast distances, allowing precise patterns of spatiotemporal gene expression. They are able to do this independent of orientation to the promoter, and a single gene often has multiple enhancers. There is still limited understanding of how developmental enhancers drive transcription. It must be a highly regulated process, previous evidence has shown that alterations in expression levels can result in developmental malformations. Furthermore, there is debate surrounding the mechanisms of how enhancers interact with their distal promoters. The models currently most popular in the field are looping and transcriptional hubs. Sonic hedgehog (Shh) gene expression is a good model to further our understanding of both developmental transcriptional regulation and distal enhancer-promoter interactions. Shh expression is regulated by many tissue and spatial specific enhancers with, in some instances, single enhancers driving expression in single embryonic domains. The enhancers are all located within a single TAD, at a range of distances from the Shh promoter. Over the years, my lab has taken a special interest in the limb enhancer, ZRS. The ZRS drives transcription in the distal posterior mesenchyme of the developing limb bud in a domain called the ZPA. We have been able to identify a network of activator and repressor binding sites within the ZRS that restricts transcription in the absence of histological boundaries providing an interesting model for me to explore mechanisms for how a developmental enhancer drives transcription. Throughout this thesis I will address three main aims. Firstly, I will establish transcriptional characteristics at the wild-type Shh locus. Before I start exploring how an enhancer drives transcription using different mouse models, I first need a strong understanding of what transcription looks like in wild-type animals. I addressed this using nascent RNA-FISH. Using nascent RNA-FISH I have been able to determine the bursting frequency of Shh in the ZPA. Furthermore, this technique has allowed me to ascertain if an active enhancer can be transcribed through. Meanwhile, the use of RNAscope has allowed me to establish the overall pattern of expression across the ZPA. Determining whether there is a clear-cut boundary or a gradient type pattern. Secondly, I will decipher the role of discrete functional elements of the ZRS in transcription. Previous work from members of my lab has identified different binding sites located throughout the ZRS. For example, there are four known Hox binding sites. Mutations in these sites are known to cause down-regulation of Shh. I used mutants for these sites to determine the action of HOXD proteins on the ZRS and how this impacts transcription. Furthermore, I have investigated how pioneer factor binding of the ZRS influences transcriptional characteristics. This was done by investigating how the mutation of a Lim homeodomain binding site impacted transcriptional characteristics of Shh. To contrast, I then explored how up-regulatory mutations of the ZRS effect transcription by using mice where a ZRS repressor site, the WMS, was disrupted. This work revealed that HOXD proteins, Limb homeodomain proteins and proteins binding the WMS all have different influences on Shh transcription, revealing a range of different roles. Finally, I will explore long-range regulation of distal enhancer-promoter interactions. There have been multiple models proposed to explain how enhancers interact with their target promoters. Two of these models for explaining long range regulation are the looping model and the transcriptional hub model. These two models can be differentiated by the action of a single enhancer on multiple promoters, the looping model predicts promoter choice while the transcriptional hub predicts simultaneous promoter activation. To test these models, I looked at the ability of Shh enhancers to drive transcription of multiple promoters in different contexts. Firstly, I used a mouse line carrying a LacZ reporter integrated within the Shh TAD. This provided a second internal promoter where expression is driven by Shh enhancers in their cognate tissues. Secondly, I performed experiments examining activation of two endogenous genes in adjacent TADs, Shh and Mnx1.

https://hdl.handle.net/1842/40767

http://dx.doi.org/10.7488/era/3524

en

The University of Edinburgh

Crane-Smith Z, Schoenebeck J, Graham KA, Devenney PS, Rose L, Ditzell M, Anderson E, Thomson JI, Klenin N, Kurrasch DM, Lettice LA and Hill RE (2021) A Highly Conserved Shh Enhancer Coordinates Hypothalamic and Craniofacial Development. Front. Cell Dev. Biol. 9:595744. doi: 10.3389/fcell.2021.595744

Sonic hedgehog gene

Shh gene

enhancers

defined periodicity

ZRS influences

Transcriptional dynamics of the Sonic hedgehog gene

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy