The regulatory architecture of the Hmx2-Hmx3 gene pair

The development of specialised cells and structures is dependent on transcription factor genes, whose protein products in turn regulate the expression of other genes. Transcription factor proteins interact with specific bits of DNA, often called enhancers, that lie near genes. Recently we have identified a uniquely conserved pair of genes in jawed vertebrates (mammals, birds, fish etc). These genes are in the Hmx family, which encode transcription factors, and are specific to parts of the brain, spinal cord and head ganglia that relay sensory information from the ears to the brain. This pair of genes is flanked by two enhancers that are similar to each other and more deeply conserved than any enhancers identified to date. They are highly conserved in jawless fish like lampreys, the earliest diverging group of living vertebrates, and they even work well in sea squirts, the closest invertebrate to the vertebrates which split from the vertebrate lineage at least 500 million years ago. This level of conservation is unusual and can only reflect extreme 'evolutionary constraint', that is evolution doesn't tolerate changes to the sequences because their function is so important. We don't know why this is, but hypothesise its due to their highly specific expression and essential function in the nervous system.

In this project we seek to answer two related questions that test this hypothesis: how do the conserved enhancers work to control such specific gene expression, and why is such specific expression important? We can do this through three types of experiment. One is to work out what Hmx genes actually do: we predict that they will control similar sets of target genes in widely different species and this provides part of the constraint on their evolution. A second is to uncover how the enhancers work: we can do this by breaking them apart and testing the functions of the pieces. The third is to uncover what underlies the conservation of enhancer sequences: we can do this by studying their evolution and how conserved functions map onto conserved sequences.

Together these will address the 'how' and the 'why' of the conservation and function of this gene pair. As well as revealing how an important part of the brain and associated sensory systems develop, insight gained from examining such an extreme case may shed light on some of the general unknowns about how gene regulation works and evolves. It also tells us about how a fundamental part of our own bodies, our sensory nervous system, develops, and it reveals a key set of steps in our evolutionary past.

The Hmx3-Hmx2 homeobox gene locus has peculiar, unique features. Our recently published work shows it stems from a tandem duplication that occurred in the vertebrate stem lineage, and that this also duplicated a huge conserved non-coding element (CNE) that has been maintained in duplicate alongside the genes. Both these CNEs function as enhancers and we recently found that they work well in an invertebrate, the sea squirt Ciona, labelling homologous cells in central and peripheral nervous systems.

In this project I hypothesise that the unique organisation of the locus and level of conservation stem from the role the genes occupy in the specification of ancient cell types in the central and peripheral nervous systems: in the hypothalamus, spinal cord and cranial ganglia. Testing this hypothesis means determining the downstream network under Hmx control in these different tissues in different lineages, and understanding how the conserved Hmx gene regulation operates both at the level of the inner workings of the individual enhancers and in how they interact with the two Hmx genes they are adjacent to. These are possible through:

1. Manipulating gene expression to establish the conservation (or otherwise) of the downstream target network in different tissues from species spanning the evolution of the regulatory architecture
2. Deconstructing how the unusual regulatory elements work and using knockouts to determine what they are important for in vivo.
3. Using cross-species transgenesis to determine the molecular basis of conserved regulation.

By focusing on models that span the major evolutionary change (primarily chicken as a vertebrate, the tunicate Ciona as the nearest invertebrate relative) we can understand how the system functions, can determine how it has evolved, and establish why it retains such unusual and unique regulatory architecture.