Context of the project.
The human body is constantly exposed to infectious agents. Efficient immune response is therefore fundamental to protect the cells from infection and keep individuals healthy. Yet, substantial variation is observed in the efficiency of immune response, both among individuals and populations. Although the contribution of genetic variation to the variability of host immune responses to infection is increasingly recognized, the mechanisms by which genetics alters the immune response remained poorly characterized. Indeed, genetics may alter immune response by controlling the total amount of RNA produced by a gene (transcripts), but also by altering the diversity of transcripts that are produced by a single gene. To allow the production of multiple transcripts (and proteins) from a single gene, a mechanism known as alternative splicing occurs, which consists in the cutting and reassembly of transcripts (i.e. splicing) during their maturation, each of them being known as an isoform.
The GATTACA project aimed at studying how genetic variants affect immune response at the RNA level by modulating not only the amount of transcripts produced by a gene, but also the diversity of transcripts (and ultimately proteins) that can arise from the same gene. By using an innovative approach, combining human population genetics (the genetic history of modern human populations) with functional genomics (the impact of genetic variants on human molecular phenotypes), the GATTACA project aimed to: (1) identify master regulators of isoform diversity through the study of isoform regulatory networks in response to immune stimuli, (2) decipher the genetic bases of between-individual variations in isoforms abundance and identify DNA motifs that are essential to the regulation of isoform usage in an immune context, and (3) detect the mode and intensity of natural selection on the evolution of splicing regulatory elements and splicing factors. All of these objectives have been successfully accomplished along the duration of the project.
Importance for society.
The most significant impact of the GATTACA project has been of fundamental nature in allowing to better characterize the genetic bases of variability in the immune response to pathogens. However, because the host immune response to stress is a highly complex phenotype, and problems in the immune system can result in enhanced infection, inflammation, autoimmunity, or adverse response to vaccines, these results will be crucial to understand the mechanisms that contribute to immune disorders. Indeed, the GATTACA project has allowed to identify novel regulatory variants acting in the specific context of infection, some of which might contribute to shape organismal traits, including response to vaccines, treatments and susceptibility to auto-immune disorders. Such a better understanding of the genetics bases of alternative splicing variability in response to immune challenges will open new avenues for the treatment of infectious, auto-immune and inflammatory disorders.
Work performed and overview of the results:
The first part of my work was to quantify the extent of isoform diversity in immune cells, using RNA sequencing data obtained across a cohort of 200 individuals and in response to 4 different stimuli. This work has lead to the identification and quantification of 16,173 frequent splicing events occurring in innate immune cells and has allowed to characterize how splicing of immune genes is modified during immune response. In particular, I identified 1,919 genes whose splicing is altered in response to immune stimuli, including many primary regulators of the immune response, highlighting an important role of splicing in the regulation of the human immune response. I further analysed the regulatory networks governing splicing regulation, and identified several splicing factors associated to strong variation in splicing during immune response, highlighting key regulators of the immune splicing response.
Next, I studied the genetic determinants of splicing and identified 1,271 loci that are associated to changes in isoform levels of 993 distinct genes. For 157 of these loci, we could match the splice regulatory variants that we identified to known genetic loci associated to human phenotypes, including auto-immune disorders (28 splice regulatory variants), highlighting the strong phenotypic consequences of the genetic variants we identified. I further studied how the impact of these genetic variants on isoform usage is modified upon stimulation, and showed that while the effect of most splice-altering variants is stable across conditions, some genetic variants act in a stimulation-dependent manner. In total, I identified 274 such variants, highlighting the mechanisms through which splicing is regulated during immune response. I also searched for genetic variants that would induce major shifts in isoform regulation of host cells, and found only 2 such variants, highlighting the rarity of such genetic variants. Moreover, my analyses revealed that variants that create shifts in alternative splicing act mostly by altering molecules involved in cell communication that control cellular behaviour, rather than by disrupting the splicing machinery. This is consistent with the observation that natural selection tends to remove variants that alter the splicing machinery, as such variants would prevent the normal function of the cell and have a deleterious effect on survival.
Then, to further detect the impact of natural selection on splicing, I looked for splicing differences between human populations, and identified 515 genes showing differential splicing between individuals of African and European ancestry. These genes were enriched in immune related genes and were often under genetic control. I further assessed that between 49% and 81% of differences in splicing that are observed between Africans and Europeans, could be attributed to differences in frequency of splicing altering variants between these populations. To assess the contribution of natural selection to the differentiation of alternative splicing between populations, I next searched for signatures of positive selection (extreme differentiation allelic frequency between populations, and strong variation of haplotype length between alleles) and identified several loci showing haplotype patterns consistent with events of natural selection including loci associated to childhood resistance to tuberculosis and systemic lupus erythematous. I further identified several genes where admixture with Neanderthal in Europe introduced regulatory variants that alter splicing and are associated to increased risk of asthma or allergy. This suggests that both natural selection and admixture with Neanderthals have contributed to shape differences in isoform usage, and ultimately in immune responses, in present-day human populations. I also explored the intensity of selective constraints acting on splicing, across various gene categories corresponding to specific biological or molecular functions. My analyses showed that immune response genes and signaling receptor genes tend to accept a higher amount of splice regulatory variants compared to other genes, indicating that the intensity of natural selection is weaker in these genes categories, allowing a higher variability of splicing of immune genes.
Finally, I characterized the splice regulatory sequences involved in immune splicing regulation to identify DNA motifs that are essential to the regulation of isoform usage in an immune context. I first showed that 86% of splice regulatory variants are located within 10kb of the splicing event they control, with 38% being located directly within the boundaries of the alternatively spliced intron. To further identify RNA binding proteins involved in condition-specific regulation of splicing, I scanned the human genome for DNA motifs that, when transcribed, would be recognized by RNA binding proteins (RBP), based on known RNA binding motifs. I then assessed the impact of variants overlapping these DNA motifs on splicing regulation and identified splicing factors, the binding sites of which were over-represented around splice regulatory variants detected in our study. This work allowed me to highlight specific splicing factors involved in the regulation of splicing in response to immune challenges. Using the HOMER software to scan for novel DNA regulatory motifs enriched in the surroundings of splice altering variants, I identified a total of 8 such motifs associated to splice regulatory variants that are active across conditions and 3 motifs associated to splice regulatory variants, acting specifically in response to stimulation. Follow-up analyses are now required to validate and characterize the importance of these putative RNA motifs in the regulation of splicing, both at the basal state and in response to immune stimulation.
The following measures have been taken to ensure dissemination of the results obtained as part of the MSCA:
Participation to International conferences with peer-review committee
- American Society of Human Genetics meeting, 2016, Vancouver, Canada. Poster
- Biology of Genomes, 2017, Cold Spring Harbor laboratory, NY, USA. Poster
- Keystone symposia “Understanding the function of human genome variation” ,Uppsala, 2016. Poster.
Additional participation to International conference (invited)
- Symposium: “Computational modeling with functional and evolutionary genomics of infectious diseases”, 2017, Tel Aviv, Israel. Oral presentation
Initial analyses of natural selection performed as part of this project were published as part of a first manuscript alongside with additional analyses of expression Quantitative trait loci and response quantitative trait loci that I performed prior to the start of the Grant agreement.
- Quach H*, Rotival M*, Pothlichet J*, Loh YE*, Dannemann M, Zidane N, Laval G, Patin E, Harmant C, Lopez M, Deschamps M, Naffakh N, Duffy D, Coen A, Leroux-Roels G, Clément F, Boland A, Deleuze JF, Kelso J, Albert ML, Quintana-Murci L (2016) Genetic Adaptation and Neandertal Admixture Shaped the Immune System of Human Populations. Cell 167(3):643-656 (*equal contributors)
- Rotival M, Quach H, Quintana-Murci L. Increased plasticity of immune splicing shapes auto-immune disease susceptibility. Manuscript in preparation.