How do genetics and epigenetics interact to influence the activity of a context-dependent enhancer?

Behaviours such as excess ethanol intake and anxiety have a major impact on people's health across the life-course. Thus, understanding the genetic and environmental processes that control ethanol intake and anxiety, across the generations, will provide opportunities to develop future preventative interventions and treatments.

Context-dependant enhancers act as gene switches and are responsible for turning specific genes on and off in specific cells of the body and at specific times. The importance of these enhancers to health, and their possible role in disease, has been highlighted by numerous genetic studies (GWAS) which demonstrate that >95% of complex diseases, that negatively affect our ability to age in a healthy manner, can be attributed to DNA changes within regulatory sequences such as enhancers. Critically, we previously discovered that environmental factors, that can include dietary fat intake, can also impact the health span of the next generation by affecting the activity of enhancer sequences.

Unfortunately, we know very little about the biology of these gene switches, how they are affected by genetics and sex in different species or how diet alters their activity, and their effects of ethanol intake and anxiety, through the generations.

We hypothesised that context-dependant enhancers which control genes encoding neuropeptides (short proteins produced in the brain and known to influence behavior) play a role in the interaction of genetics, sex and diet to influence behaviours that negatively impact the health-span such as ethanol intake and anxiety.

To address this hypothesis, we identified an enhancer (GAL5.1) that has remained almost unchanged in both mice and humans. GAL5.1 turns on a neuropeptide gene called GAL in specific cells of the brain where GAL controls ethanol intake and anxiety. Importantly we found that this enhancer contained DNA sequence changes (polymorphisms) that had been associated with anxiety and alcohol abuse in human males. Moreover, we deleted this enhancer from mice and found that, not only was the GAL gene (called Gal in mice) nearly turned off, but that these mice didn't drink as much ethanol and males did not suffer as much anxiety. This is the first time that a highly context-dependent enhancer has been identified in both mice and humans that controls ethanol intake and anxiety; behaviours with a strong impact on health through the life course.

Identification of this enhancer provides our internationally recognised multidiscipline team (Aberdeen; McEwan, MacKenzie, Manchester; Murgatroyd) with a unique opportunity to identify

i) The molecular mechanisms regulating a context-dependent enhancer which regulates behaviours (ethanol intake and anxiety) with a serious impact on human health,

ii) How diet interacts with DNA sequence changes (polymorphisms) and sex to affect the activity of a context-dependant enhancer and compare the effects in different species(Mouse/human).

iii) How a context-dependant enhancer is affected by maternal diet in the womb to impact the behaviour of subsequent generations.

Identification of GAL5.1, and its characterisation as a context-dependent enhancer, provides an unparalleled opportunity to determine the molecular, genetic and dietary dependent mechanisms influencing the activity of an enhancer whose activity may have a direct impact on ethanol intake and anxiety and our ability to age healthily

Context-dependant enhancers support the expression of genes essential to health in specific groups of cells and in specific amounts. Because of their importance to life-long health, it is critical to know the molecular, genetic and environmental mechanisms influencing their behaviour. To this end, we identified a conserved enhancer (GAL5.1) that controls Gal gene expression in hypothalamus and amygdala. CRISPR deletion of this enhancer in mice reduced Gal expression, alcohol intake and anxiety in males. In parallel, UK biobank analysis revealed a significant association between ethanol abuse and anxiety in men. Thus, identification of GAL5.1 provides a unique opportunity to investigate the biology of a health relevant context-specific enhancer by addressing the following questions;
i) Where are the specific sequences within GAL5.1 that regulate its response to signal transduction cues such as PKC? This will be determined using a combination of cell culture-based experiments, mouse CRISPR manipulation and ethanol intake and anxiety analyses.
ii) How are these mechanisms affected by polymorphic variation and different types of DNA-methylation (5mC/5hmC) and how does this vary between species? This will be answered using SssI/TET1 treated plasmid reporter assay-based studies in transformed and primary cell culture.
iii) How does maternal diet affect the differential methylation (5mC/5hmC) of GAL5.1 and does GAL5.1 play a role in regulating the consequences of maternal high-fat diet on the behaviour of subsequent generations? This will be established by comparing the effects of maternal high fat diet on offspring behaviour, qPCR and DNA-methylation analysis.
Exploiting this opportunity will give us a unique insight into how context-specific enhancers control behaviours with an impact on life long health and how their influence is governed by genetic variation and diet in maintaining health through the life course.