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Genetics of Alternative Transcript Abundance upon immune Cellular Activation

Periodic Reporting for period 1 - GATTACA (Genetics of Alternative Transcript Abundance upon immune Cellular Activation)

Reporting period: 2015-10-01 to 2017-09-30

Efficient immune response is fundamental to protect the cells from infection and keep individuals healthy. Yet, substantial variation in immune response is observed, both among individuals and populations. Genetics may contribute to these variations by controlling the total amount of RNA produced by a gene (transcripts), but also by altering the diversity of transcripts (or isoforms) that are produced by a single gene. This diversity is allowed by a mechanism known as alternative splicing, through which transcripts are cut and reassembled (i.e. spliced) during their maturation.

To understand how genetic variants that alter splicing contribute to the variability of human immune responses, 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 acting on splicing regulatory elements and splicing factors. All of these objectives have been successfully accomplished along the duration of the project.

The most significant impact of the GATTACA project has been fundamental in nature by allowing us to better characterize the genetic bases of variability in the immune response to pathogens. However, these results will also 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 are associated to organismal traits, including response to vaccines, treatments and susceptibility to immune disorders, opening new avenues for the treatment of infectious, auto-immune and inflammatory disorders.
The first part of my work was to measure 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 quantified 16,173 frequent splicing events occurring in innate immune cells and identified 1,919 genes with altered splicing in response to immune stimuli, including essential regulators of the 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 associated to changes in isoform levels, 157 of which were previously associated to human phenotypes, including several auto-immune disorders. I further showed that while the effect of most splicing-altering variants is independent of the stimulation state, some genetic variants act in a stimulation-dependent manner. In total, I identified 274 such variants and analysed their neighbouring DNA sequences to identify specific motifs enriched in their surroundings. I thus highlighted both motifs of known splicing factors and novel regulatory motifs, contributing to regulate splicing of immune cells at basal state and in response to immune stimulation.

Finally, to understand the impact of natural selection on splicing, I searched for splicing differences between human populations, and identified 515 genes showing differential splicing between individuals of African and European ancestry. I found that population differences in frequency of splicing altering variants accounted for up to 81% of these differences in splicing. I further identified several splicing altering variants overlapping strong signatures of positive selection (extreme differentiation of allelic frequency between populations, and strong variation of haplotype length between alleles), including loci associated to childhood resistance to tuberculosis and systemic lupus erythematous, suggesting that natural selection has contributed to shape differences in isoform usage, and ultimately in immune responses, of present-day human populations.

--- Exploitation/Dissemination ---
The following measures have been taken to ensure dissemination of the results obtained as part of the MSCA:
* Participation to International conferences
- 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.
- Symposium: “Computational modeling with functional and evolutionary genomics of infectious diseases”, 2017, Tel Aviv, Israel. Oral presentation

* Publications:
- Quach H*, Rotival M*, et al (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. Submission planned to Nature Communications.
All manuscripts include reference to EU funding.

* Outreach activities:
- UPA Conferences 2015 at the “Museum National d’Histoire Naturelle” organised by the “Union des Professeurs des classes préparatoires aux grandes écoles Agronomiques, biologique géologiques et vétérinaire)”.
- Teaching to master level students at the Institut Pasteur : “Human Population Genetics and Genetic Epidemiology course”.
- Press release and radio interventions were organized following the publication in Cell (Quach et al, 2016) of the work done during the MSC Fellowship.
All communications included reference to EU funding.
The GATTACA project has allowed us to go beyond the state of the art in characterizing the genetic and evolutionary bases of the regulation of splicing in response to immune stimuli. For instance, it has allowed the definition of new immune targets of evolutionary importance, through the identification of genetic variants that affect our response to immune challenges at the isoform level and have been targeted by natural selection, some of which are associated to susceptibility to auto-immune disorders, vaccine efficiency or response to therapeutic treatments.

In addition, the splicing factors and regulatory motifs that were identified by this project will contribute to increase our ability to identify rare genetic variants that lead to disease by altering splicing in response to immune stimuli. Finally, the computational pipelines that I developed over the course of the project, to process RNA-sequencing data, evaluate the impact of immune stimulation on both mRNA levels and splicing, and to detect natural selection (purifying selection and positive selection) acting on regulatory variants, will benefit to future studies from both our lab, and the community.

From a more personal perspective, I have acquired new skills in population genetics, cellular genomics and immunology that have allowed me to obtain a permanent position in the host lab of the Marie-Curie Fellowship. I have also actively transferred my skills in functional genomics to my host lab and institution, and to other researchers in the field through (i) training of students within the lab, (ii) teaching at the Institut Pasteur and (iii) presentation in international conferences (see dissemination).
Graphical overview of The GATTACA project