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
