Scientists have developed a powerful new gene-editing platform that allows them, for the first time, to systematically test the function of key proteins in mammalian cells.
Using the technology, researchers have created the first comprehensive functional map of key sites on proteins called histones, which act as spools around which DNA is wound. Histones help package the genome and play a crucial role in controlling which genes are switched on or off.
By revealing which of these key sites are essential for healthy cells, this research – led by scientists at The Institute of Cancer Research, London – could help scientists better understand cancer.
Funding for the research was provided by UK Research and Innovation, the Royal Society and The Institute of Cancer Research (ICR), which is both a research institute and a charity. The findings were published in Nature Genetics.
Overcoming a long-standing obstacle
Small chemical tags attached to histones can alter gene activity, influence cell identity and help maintain genome integrity. Although scientists have identified more than 100 different histone modifications, understanding exactly what many of them do has proven remarkably difficult.
One major obstacle has been the complexity of mammalian genomes. Histone genes exist in many near-identical copies scattered throughout the genome, making it extremely challenging to alter every copy of a specific histone site and observe the consequences. Previous approaches often introduced unintended changes and lacked the precision required for systematic studies.
The new study overcame these limitations. The researchers developed a high-throughput CRISPR prime-editing platform capable of precisely editing all copies of mammalian histone H3 genes in their natural genomic locations. The system allows scientists to introduce targeted mutations, reverse them if required and even combine multiple mutations to investigate how different histone modifications interact.
Discovering which histone sites matter most
To demonstrate the power of the platform, the team systematically altered every lysine residue within histone H3. Lysines are amino acids that frequently carry regulatory chemical modifications, making them key control points for gene activity.
The researchers replaced individual lysines with another amino acid, arginine, preventing the usual chemical modifications from occurring. By comparing these mutant cells with carefully matched control cells, they were able to measure the contribution of each lysine to cell fitness and function.
The experiments revealed a small group of particularly important residues. Mutations affecting H3K4, H3K9, H3K14, H3K18 and H3K79 reduced cellular fitness, suggesting that the normal modifications occurring at these sites are essential for healthy cell growth and survival. Some mutations were so poorly tolerated that researchers struggled to create fully edited cell lines because cells carrying too many mutations were outcompeted by healthier cells.
In contrast, mutations at several other lysine sites had little effect on cellular fitness under the conditions tested. This suggests that histone H3 lysines make different contributions to cell function and that some may play roles in specific biological contexts that were not captured in this study. Together, the findings provide one of the clearest pictures so far of how individual histone H3 residues contribute to mammalian cell function.
The platform also allowed the researchers to study combinations of mutations. They found that some pairs of mutations had much stronger effects than either mutation alone. In particular, simultaneous mutations at positions H3K27 and H3K36 impaired stem-cell self-renewal and altered patterns of gene expression, revealing previously hidden interactions between chromatin-regulating pathways.
One of the study's notable findings involved H3K56, a histone site previously linked to DNA damage responses in yeast and fruit flies. The researchers showed that mammalian cells carrying H3K56 mutations were more sensitive to DNA damage, providing evidence that this genome-protective function has been conserved through evolution.
Laying the foundations for future cancer research
First author Daniel Price, Higher Scientific Officer at the ICR, said: “Our study establishes a scalable framework for functional interrogation of histone alterations in mammalian cells.
“The work is fundamentally a basic-science advance rather than a medical breakthrough, but the implications could be significant in the long term. Abnormal chromatin regulation is a hallmark of many cancers, and large-scale sequencing studies have identified recurrent mutations in histones and chromatin-regulating proteins across numerous tumour types.
“Although the clinical impact is not immediate, the technology provides a powerful new way to investigate how chromatin regulation contributes to cancer and other diseases, which may eventually inform future treatments.”
Senior author Dr Alex Radzisheuskaya, Group Leader of the Chromatin Biology Group at the ICR, said: “Our findings provide an unprecedented view of the histone residues that regulate mammalian genomes, and they offer researchers a powerful new toolkit for decoding the biology of chromatin.
“By finally making it possible to systematically test the function of individual histone residues, we have opened the door to a deeper understanding of how cells maintain their identity and protect their DNA, as well as how these processes go wrong in diseases such as cancer.
“We hope that both the methodology and the collection of precisely engineered cell lines generated during this project will become a valuable resource for scientists studying gene regulation, genome stability, development, cancer and epigenetic therapies.”
Image credit: Anja from Pixabay (modified)