An important DNA modification is methylation, or the addition of a methyl group to the 5th carbon of cytosine. This forms 5-methylcytosine (5mC), typically associated with repression of gene expression. Reversing this modification requires DNA demethylation, a series of enzymatic reactions which starts with the creation of 5-hydroxymethylcytosine (5hmC). But 5hmC is not just a transient step in the DNA demethylation pathway but represents an important epigenetic modification in its own right. 5hmC is a key component of the 6-base genome with an essential role in regulating gene expression, maintaining cellular identity, ensuring genomic stability, and responding to environmental changes. Here we explore the unique functions of 5hmC, how it is formed, and highlight its importance in normal development, differentiation, brain function, and its potential as a biomarker in disease pathogenesis.
What is DNA demethylation?
The DNA demethylation pathway is the biological process through which methyl groups are removed from modified cytosine bases in DNA and is facilitated through either passive or active mechanisms. Passive demethylation occurs when methylation marks are not maintained during DNA replication, leading to a gradual dilution of 5mC with each cell division. Conversely, active demethylation involves the enzymatic removal of methyl groups without the need for DNA replication. Active DNA demethylation involves a series of enzymatic reactions, primarily mediated by the TET (Ten-Eleven Translocation) family of enzymes. Active DNA demethylation is therefore a critical aspect of epigenetic regulation, allowing for dynamic changes in gene expression in response to developmental cues, environmental signals, and cellular processes.
The role of TET enzymes in DNA demethylation
Active DNA demethylation begins with the oxidation of 5mC by TET enzymes (TET1, TET2, and TET3) that convert 5mC to 5hmC. Further oxidation of 5hmC then forms the intermediaries 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), before base excision repair processes complete the demethylation process resulting in an unmodified cytosine. The activity of TET enzymes is therefore essential for maintaining epigenetic plasticity and is implicated in both normal development and pathological conditions.
Beyond demethylation: the critical role of 5hmC in biological processes
The addition of a hydroxy group to 5mC to form 5hmC is a key step in the active DNA demethylation pathway. But 5hmC is a stable epigenetic modification in its own right and can modulate gene expression independently of its role in the DNA methylation pathway. Here are some key roles 5hmC plays in biological processes.
1. Regulation of gene expression
Unlike 5mC, which generally represses gene expression, 5hmC is often associated with gene activation or a more open chromatin state, making DNA more accessible for transcription and enabling specific genes to be expressed when needed. The addition of 5hmC to DNA can therefore influence binding of specific transcription factors and other proteins, and so affect the transcriptional landscape of the cell. This allows for control over which genes are expressed and so contributes to the fine-tuning of cellular functions in response to developmental cues and environmental changes.
2. Genome stability and protection
The introduction of 5hmC affects chromatin architecture, rendering it less condensed and more accessible for transcription factors and other regulatory proteins. This alteration in chromatin structure enhances the cell’s ability to modulate gene expression rapidly in response to physiological changes, thereby playing a crucial role in cellular responsiveness and function. But 5hmC also helps protect specific regions of the genome from aberrant hypermethylation, which can lead to inappropriate gene silencing. By marking these regions, 5hmC ensures that essential genes remain active, and that genomic integrity is maintained. There is also evidence that 5hmC may also play a role in DNA repair mechanisms, contributing to the maintenance of genome stability, which is crucial for preventing mutations and ensuring proper cellular function.
3. Cellular differentiation and identity
During development, cells must undergo precise changes in gene expression to differentiate into various cell types. Both 5mC and 5hmC play a crucial combined role in modulating gene expression during differentiation, inhibiting and activating genes which results in a gene expression profile unique to each cell type. The maintenance of stable yet dynamic epigenetic patterns in the genome by 5hmC therefore facilitates the expression of genes essential for proper development. The distribution of 5hmC is tissue-specific and shown to be highly enriched in certain cell types, such as neurons and embryonic stem cells. Its presence is often crucial for the proper function and differentiation of these cells underscoring its important role in supporting the complex regulation of genes required for maintaining cell identity.
4. Environmental response and ageing
Levels of 5hmC can be influenced by environmental factors, such as stress, diet, and toxins, allowing cells to adapt their gene expression in response to changing conditions. This dynamic regulation is vital for an organism’s ability to respond to environmental challenges. However changes in 5hmC patterns affect cellular function, promote genomic instability, and influence the ability to repair DNA, all of which are key factors in the ageing process. The distribution and quantity of 5hmC in the genome often changes over time, contributing to alterations in gene expression that are associated with ageing-related decline and diseases. Understanding how 5hmC responds to environmental factors throughout life can, therefore, offer key insights into how these changes might affect epigenetic states and overall health.
5. Role in neurological function & disease
5hmC is particularly abundant in the brain, where it is involved in regulating genes essential for cognitive functions, learning, and memory. Changes in 5hmC levels have been linked to various neurological disorders, highlighting its importance in maintaining healthy brain function and its potential as a biomarker for neurodegenerative diseases. For example, aberrant patterns of 5hmC have been linked to neurodegenerative disorders such as Alzheimer’s disease, where decreased levels of 5hmC in neuronal cells may contribute to pathogenesis.
6. Cancer
A common feature in many cancers is a global reduction in 5hmC levels across the genome which often results in silencing of tumour suppressor genes. This loss is particularly evident in aggressive tumours and is often associated with a poor prognosis. The decrease in 5hmC often results from mutations or dysfunctions in TET enzymes, which contributes directly to the loss of 5hmC and subsequent epigenetic changes that drive tumorigenesis.
5hmC: beyond just demethylation
5hmC is far more than just a transient step in the DNA demethylation pathway catalysed by TET enzymes. It is a crucial epigenetic modification with broad implications for gene regulation, cellular differentiation, brain function, genome stability, and disease. Its unique roles in maintaining cellular identity and responding to environmental changes underscore its importance as a key regulator in both health and disease – as well as a potential biomarker and therapeutic target. This shows that we need to remember that genetic information is not just encoded within the 4 traditional bases of DNA – we need to consider it a 6-base genome, as both 5mC and 5hmC are also required to understand and tell the whole story of cellular and biological function.
