Epigenetic Inheritance - but short of short RNAs!
It is not for nothing that DNA is described as “the molecule of life”. The DNA sequences that make up our genes contain the essential code for making a living being. However, there is an additional layer of information without which DNA alone could not give rise to the complexity we observe in nature. This “epigenetic” information serves to regulate the expression of genes, turning them on or off in the right context.
The most well-known types of epigenetic information are methylation of DNA, chemical modification of the histone proteins associated with DNA (which can include methylation, acetylation and many more), and small non-coding RNAs, short RNA molecules that do not code for proteins. These signals affect gene expression in a variety of ways and often work together to reinforce each other. In humans, for instance, the silencing histone modification H3K9me3 is almost always found together with DNA methylation. However, this means it is hard to separate their relative effects on gene expression.
This is even more true when we consider epigenetic effects that last multiple generations. Things have become even more interesting in recent years with the discovery that some epigenetic information is inherited. This means that the genetic information found in DNA sequence is not the only thing that can be passed from parent to offspring. However, whilst the fact that some epigenetic information is inherited is not in doubt, what’s much less clear is exactly which of the many different types of epigenetic signals are the important ones that are transmitted between the generations.
One signal that has been demonstrated to pass between generations is small non-coding RNAs. These have been shown to be important in epigenetic inheritance in nematode worms and plants. Small RNAs are very appealing candidates for transgenerational epigenetic inheritance because they can recognise specific sequences in genes and target them for epigenetic silencing, allowing them to reinitiate silencing every generation. But do they underpin all transgenerational epigenetic inheritance? This is still up for debate. In our recently published study, our team from the Sarkies lab uncovered evidence that some epigenetic inheritance is not due to small RNAs.
Figure 1: epigenetic silencing of a gene in Drosophila causes the eyes to become white rather than the usual red. The eye colour can be maintained for many generations without changing the sequence of the gene
We studied a particular case of epigenetic inheritance in the fruit fly Drosophila melanogaster, where a change in eye colour from red to white can be caused by epigenetic silencing of a pigment gene, which can be inherited for multiple generations without any change in DNA sequence. We set out to investigate whether small RNAs might be responsible. To do this, we first sequenced small RNAs in a variety of genetically identical lines that displayed epigenetic differences. This includes white-eyed, red-eyed and “mosaic” flies, in which eye colour is a patchwork of white and red. We found no small RNAs mapping to the gene responsible for eye colour, strongly suggesting that they are not involved in its regulation or inheritance. To confirm this, we also blocked the function of a number of genes involved in different small RNA pathways to see if this had any effect on eye colour or its inheritance. Again, we found that this did not change the silencing process, arguing against the involvement of small RNAs in this case of epigenetic inheritance.
Science is nothing if not unpredictable though, and something very unexpected jumped out at us. As controls for our experiment, we also measured eye colour in two unrelated mutants frequently used as “markers” because they have visible effects on the adult fly that are easy to track. These were mutations in the Serrate gene (which give the flies serrated wings) and Stubble gene (which make the bristles on the back short and “stubbly”). The Serrate mutation behaved as expected, showing no effect on eye colour just like the small RNA mutants. Unusually, the Stubble mutation had a clear effect on eye colour, making it more red!
In following up on this unexpected result, we discovered a previously unknown effect of Stubble mutation on a gene called Pho, which helps direct an epigenetic histone modification known as H3K27me3 to its target genes. We suspect this effect has gone unreported because of its relatively mild effect. However, we hope that others will bear this in mind when thinking about using Stubble mutation as a control for other studies investigating H3K27me3, and urge them to opt for the safer Serrate or other marker mutations where possible. At the same time, this finding suggests that the H3K27me3 mark, rather than small RNAs, might be the most important factor in transgenerational epigenetic inheritance in this particular case.
Overall, whilst our study did not definitively solve how this case of transgenerational epigenetic inheritance works, it is very important in taking us forward by ruling out some possible explanations. In the future, we hope to be able to zone in on what is really underpinning the phenomenon. But it already tells us that there might be many mechanisms capable of transmitting information between the generations, with different ones operating in different organisms. This makes it even more interesting to continue researching transgenerational epigenetic inheritance in many different organisms.
Read the full article at: https://royalsocietypublishing.org/doi/10.1098/rsob.240298
Max Fitz-James and Peter Sarkies
11th March 2025
