It is known as noncoding DNA because approximately 98.5% of human DNA does not include the instructions needed to make proteins. Nonetheless, this noncoding DNA significantly influences the regulation of genes and disease.
A recent study led by Yale School of Medicine’s Steven Reilly aimed to determine how particular noncoding areas, referred to as “enhancers” and “promoters,” affect genes. Promoters are found close to genes and regulate whether or not those genes are translated into mRNA, which is subsequently utilized to synthesize proteins. Conversely, enhancers are DNA sequences that aid in activating promoters; however, they may be distant from the genes they regulate.
Researchers intend to understand better how mutations in noncoding DNA might impact gene activity and contribute to diseases by charting the relationships between enhancers, promoters, and genes.
The long-term effort, which involves more than 30 institutions, aims to identify the functional elements in the human genome. This study is part of the Encyclopedia of DNA Elements (ENCODE) Consortium.
Similar to locating light switches in a house, researchers previously mapped the human genome to determine the positions of enhancers and promoters. On the other hand, this study sought to determine the relationship between these switches and specific genes, like trying to identify which lights each switch regulates in the house.
To do this, scientists employed the gene-targeting technique CRISPR to disable short stretches of DNA individually and tracked the resulting changes in genes. Typically, CRISPR cleaves particular DNA sequences, but a modified strain silenced neighboring DNA without cleaving it in this work.
They could ascertain the effects on genes by simply turning the light switches on and off in this way. Crucially, the scientists examined a sizable section of the genome rather than merely putative enhancers or promoters.
The good news was that the things scientists previously identified as enhancers or promoters were the only ones that appeared to have any effect. Thus, there were no hidden light switches that we were unaware of. This demonstrates that their enhancer and promoter maps are the locations to look at when examining a DNA variant that may affect the disease.
The researchers revealed that a single enhancer might impact numerous genes, just like a single light switch can turn on multiple lights.
Steven Reilly, an assistant professor of genetics at Yale School of Medicine who co-led the study, said, “We originally had tended to think that one enhancer was affecting one gene, but we found it was really common for one enhancer to impact many genes. That says that if you have a mutation in an enhancer associated with a disease, you might need to look for several impacted genes, not just one.”
Over 540,000 DNA sections were used in these studies, which were carried out by the researchers collectively.
By working together methodically on this project, the team found best practices and patterns that they probably wouldn’t have found through independent experimentation.
The group collectively determined the best approach for conducting CRISPR experiments, identifying the optimal guides to direct CRISPR and the most accurate analysis methods. According to Reilly, this will assist other researchers in conducting similar experiments on their DNA regions of interest more effectively and efficiently.
Researchers will want to follow our guidelines to increase their likelihood of connecting enhancers to their target genes, especially when working with patient cell samples, where resources may be limited.
Furthermore, the researchers discovered that targeting either of the two DNA strands matters when using this type of CRISPR screening.
Reilly said, “Depending on which strand you target, you will get different results on how big of an effect CRISPR-mediated DNA repression has on genes. Knowing these differences will allow researchers to design the right analysis methods.”
“This particular finding wouldn’t have been possible without the large collaborative effort of this work.”
“We only saw this because we were analyzing hundreds of these experiments. It would help if you assembled really large datasets to see these patterns. This has been the theme of the human genome work from the beginning. The genome is huge. One person or one lab can’t tackle it all. And this work has been a cool example of how large-scale collaborations work and their necessity for this monumental task of understanding the human genome.”
Journal Reference:
- Yao, D., Tycko, J., Oh, J.W. et al. Multicenter integrated analysis of noncoding CRISPRi screens. Nat Methods (2024). DOI: 10.1038/s41592-024-02216-7