Every gene in a cell is expressed at exact levels due to complex gene regulatory networks. For example, when T cells (a type of white blood cells that fight off infections and cancer) are activated in our immune systems, thousands of proteins in these cells change. The proteins are interconnected and changes in one protein level can impact that of another.
The connections between regulatory genes and their downstream targets have been mapped to some extent — they can be thought of as a subway or metro map, with connections existing between major “hubs.” A significant disruption to these networks can alter the expression of immune genes and result in disease. Mapping networks helps us understand the impact of a disruption and its effect throughout the network, or how a drug can impact numerous proteins at once.
However, there is limited knowledge about the upstream regulators of most genes. A downstream approach has traditionally been used by scientists to map networks, wherein a gene for a protein is removed one at a time and the impact on the immune cell’s function is observed.
On July 11th, 2022, researchers at the Gladstone Institutes, UC San Francisco and Stanford School of Medicine published a study in Nature Genetics about using CRISPR technology to study the networks between thousands of genes simultaneously, as announced in a press release.
The research team has created the most detailed map to date of complex gene networks for three important immune genes involved in the role of T-cell function: IL2RA, IL-2 and CTLA4. The maps provide valuable insight into immune cell function and the development of autoimmune diseases.
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Using CRISPR Technology to Disrupt Networks of Immune Genes
An upstream approach to mapping has not been used before in primary human cells. The team behind this study developed a method involving CRISPR technology for systematic discovery of the upstream regulators of IL2RA, IL-2 and CTLA4, which are all critical immune factors in human T cells and function as the “hubs” outlining the overall network in their upstream approach.
CRISPR-Cas9 gene editing was used to simultaneously perturb thousands of genes by targeting specific points of genetic code. The focus was on genes that create transcription factors, which are proteins that switch genes on or off and therefore control multiple other genes at once, including the three immune factors of interest. Using CRISPR allowed the team to examine over a thousand transcription factors to determine which impact IL2RA, IL-2 and CTLA4.
Although it was expected that numerous connections exist between the genes regulating the three immune genes, the extent of the connectivity was more elaborate than initially thought. There were 117 regulators that controlled the levels of at least one of the three genes, 39 controlled two of the genes, and 10 regulators could alter the levels of all three genes.
The team then approached the network using the traditional downstream approach and removed 24 regulators from the T cells to decipher the full list of genes they regulate aside from IL2RA, IL-2 and CTLA4. They found that many of the regulators controlled each other through numerous interconnected feedback loops. For example, the transcription factor IRF4 influenced the activity of nine regulators, and itself is regulated by 15 other regulators. All 24 of the regulators affected the levels of IL2RA.
Impact for Patients
Mapping complex gene networks can arm researchers with a deeper understanding of autoimmune diseases and how to approach immunotherapy development.
The results of this study demonstrate how immune genes are regulated in human T cells, and that a high number of these genes are linked to diseases like lupus and rheumatoid arthritis. The map reveals how even though changes associated with these diseases can occur in different genes, the connections between them result in the same net effect on the cell.
Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology and co-author of the study, says in the press release that “when we understand the ways in which these networks and pathways are connected, it starts to help us understand key collections of genes that need to function properly to prevent diseases of the immune system.” Essentially, the maps are like instruction manuals for immune cell function and how we can engineer them for our benefit.