By Katy Pallister

Up to 80 percent of the white blood cell population in humans are neutrophils. Although neutrophils are classically thought to be the servants to other reigning cell types in regulating the immune system, a recent study has provided evidence for their more prominent function in autoimmune diseases, such as rheumatoid arthritis, which may eventually lead to novel therapies.

Researchers from the Functional Gene Control group at the MRC London Institute of Medical Sciences (LMS), in collaboration with the Wellcome Sanger Institute and the Josep Carreras Leukaemia Research Institute in Spain, have used an innovative approach in neutrophils to interpret the function of genetic variants associated with autoimmune diseases. This study builds on a previous large collaborative project, called BLUEPRINT, which revealed how variation in blood cells’ characteristics and numbers can affect a person’s risk of developing complex diseases such as heart disease, and autoimmune diseases including rheumatoid arthritis, asthma, coeliac disease and type 1 diabetes.

“Often, to understand the function of specific parts of DNA, scientists either delete these regions or introduce mutations to them artificially. Instead, here we are leveraging the natural differences in the DNA sequence between multiple individuals,” senior author, Dr Mikhail Spivakov of LMS, explained. “Mainly, we are looking at the effects of differences in the DNA sequence (‘mutations’) on the binding of an important protein, PU.1, that regulates the function and development of neutrophils and other blood cell types.”

Particular regions in DNA, called enhancers and promoters, control gene expression, switching a gene on or off. These regions are bound by proteins called transcription factors, including PU.1. In this paper, the researchers found that mutations which affect the binding of PU.1, affect the activity of the enhancer and promoter regions, as well as the connections between these regions in the 3D space of the cell’s nucleus that are necessary for their correct function. Thousands of DNA variants were found to affect the binding of PU.1 in neutrophils. Interestingly, in many cases, these mutations were not located at the place where the binding was affected.

“We then looked at the publicly available data on mutations that are associated with immune function, such as autoimmune disease susceptibility, and found that a number of these mutations also affect PU.1 binding in neutrophils,” Spivakov said. “This implicates both PU.1 and neutrophils in these diseases because a lot of the affected binding sites are specific to this cell type.”

“The classic molecular genetics way of proving that point would be to mutate those sequences – just remove them to prevent PU.1 from binding or repress it artificially without modifying the DNA sequence – but it’s quite a harsh perturbation,” Spivakov continued. “Whereas we are not touching the cells before we measure what’s going on, so it could be argued that we’re getting a cleaner picture. We’ve also been able to observe thousands of mutations, as opposed to introducing one or two artificially and then hoping that the cells have still retained that kind of natural behaviour.”

The interdisciplinary team involved in the study isolated neutrophils from 100 donors to detect the effects of natural genetic variation on PU.1 binding, as well as the same cells from a smaller number of donors to study variation in the physical contacts between DNA regions that control gene activity.

Ultimately, this collaborative study has the potential to lead to treatments for autoimmune diseases, when integrated with other findings. As transcription factors emerge as novel therapeutic targets, leveraging nature’s natural mutagenesis to draw the associations between transcription factor binding and autoimmunity, as this study has done, could be a powerful path forward.

“If you were to design a drug, then you would still need to do perturbation studies to really study the effects of an individual mutation. I think that’s really a challenge, particularly for autoimmune diseases, which are difficult to model adequately both in human cells in vitro, and in laboratory animals. This is why studies such as ours, that use high-throughput molecular readouts in unperturbed cells, are particularly valuable for these diseases,” Spivakov concluded.

‘Genetic perturbation of PU.1 binding and chromatin looping at neutrophil enhancers associates with autoimmune disease’ was published on 16 April 2021 in the journal Nature Communications. Read the full article here.