Preventing dedifferentiation of neurons: a role for H3K9me- and HP1 associated heterochromatin?

As an organism develops, and when it repairs its tissues, stem cells divide to produce daughter cells that differentiate into specific cell types. These cells are the building blocks of the body and can have very diverse functions, such as hair follicle cells in the skin, or nerve cells (neurons) in the brain. As a cell differentiates, it shuts down expression of stem cell genes and turns on genes required for its specific function.

It will also make sure that other genes (related to other functions or properties) are kept switched off. These 'epigenetic' mechanisms that prevent these genes from being expressed are very important. If silent genes are re-expressed, it can prevent the cell from functioning properly, cause cells to proliferate uncontrollably, or lead to cell death.

It is becoming increasingly evident that erosion of epigenetic silencing, and the expression of unwanted genes, is a key driver of aging. Furthermore, metabolism, which is influenced by the genetic background, the environment and diet, can affect the epigenetic control of gene expression.

Here, we will investigate the mechanisms that keeps neural stem cell (NSC) genes off in neurons. We have previously shown that the mutation of a specific transcription factor (a protein that binds to DNA to regulate gene expression) can cause developing neurons to have an identity crisis and revert back to a NSC-like state. This only happens in immature neurons, suggesting that there are epigenetic alterations that 'lock' NSC genes into a highly repressed state.

A strong candidate is HP1 heterochromatin, which is characterised by the presence of the HP1 protein and a modification on histones (the proteins that DNA is wrapped around) called 'H3K9me'. Evidence from studies in Drosophila (fruit fly) and mouse cells show that HP1 heterochromatin is present at NSC genes in neurons. Our key hypothesis is that HP1 heterochromatin prevents neurons from 'dedifferentiating' back into NSCs.

We will test this hypothesis and investigate how one-carbon metabolism, a central metabolic pathway involved in aging and histone modification, affects HP1 heterochromatin in neurons.

We will first comprehensively profile HP1 heterochromatin in developing neurons, young adult neurons and old neurons. This will be done in Drosophila and mice using a technique called Targeted DamID that we developed. These data will determine whether mechanisms required for maintaining cell fate identity deteriorate with age.

Then, to test our main hypothesis, we will use a cutting-edge technique called 'epigenome editing'. This utilises CRISPR-Cas9 technology (more widely known for its use in altering the sequence of DNA in the genome) that has been adapted to recruit effector proteins to specific sites in the genome. A histone demethylase will be recruited to NSC genes in neurons to remove the H3K9 methylation and prevent the formation of HP1 heterochromatin.

We can then check whether NSC genes are de-repressed and whether it can cause or enhance dedifferentiation of neurons into NSCs. Finally, we will characterise and genetically manipulate one-carbon metabolism enzymes to modulate levels of SAM and SAH. These are the metabolites involved in H3K9me modification of histones and are known to change during ageing.

We will assess the impact on H3K9me abundance and cell fate maintenance when the levels of these metabolites change in developing and ageing neurons. Together, these studies will provide important insights into neuronal cell fate plasticity and mechanisms that prevent ageing in the brain.