Étude du polymorphisme du gène majeur d’histocompatibilité de classe IIb (MHIIb) chez l’omble de fontaine (Salvelinus fontinalis)

Classical major histocompatibility complex class II (MHCII) molecules are cell-surface glycoproteins specialized in the presentation of peptides, mainly derived from extracellular pathogens, to the antigen receptors of CD4+ T cells in the adaptive immune system.  They are encoded, with those of the MHC class I, by the most polymorphic genes known to date, with multiple loci and high allelic diversity at each one.  Moreover, the polymorphism within MHCII genes is not restricted to coding sequences.  It has also been observed in promoters where it was shown to affect the expression level of the genes.  Variation in gene regulation is believed to be an important factor from which modification in morphology, physiology or behaviour can be observed in all organisms. Repeated DNA sequences with functional roles in this regulation have been identified within the non-coding parts of the genomes.  On the other hand, pathogen-driven selection is also believed to be important in the evolution and maintenance of the polymorphism of the MHC genes in vertebrates.  Studies have shown the implication of different MHC alleles in disease resistance or susceptibility.  In this study, our aims were to characterize the polymorphism of the MHIIb gene in brook charr (Salvelinus fontinalis), to document its effects on the survival conferred by specific alleles and/or genotypes following an infection and on the variation of the expression level of the gene in different environmental conditions.      In a first part, we identified a total of 6 MHIIb alleles, designated Safo-DAB*0101 to Safo-DAB*0601, showing a high similarity to coding sequences from teleost fish and human.  Analysis of the b1 domain sequences indicates the effect of a positive selection pressure to select polymorphic mutations in that region of the molecule.  Four of these alleles were tested in a challenge experiment against the pathogen Aeromonas salmonicida to evaluate their effect on fish survival.  We found that one allele, DAB*0101, was significantly associated with resistance to furonculosis.  In addition to homozygotes for this allele, its resistance effect was also detected in the heterozygote individuals of the DAB*0101/*0201 genotype.  In contrast, other allelic combinations, namely heterozygous genotypes DAB*0201/*0301 and DAB*0301/*0401 were significantly associated with increased susceptibility.  Given that its frequency was relatively low in the population, the negative frequency dependant selection hypothesis could explain the advantage associated with the allele DAB*0101 over the other alleles and highlight the importance of this mechanism to sustain variation at the MHC in brook charr.     In a second part, we reported the identification of a polymorphic minisatellite formed of a 32 nucleotides motif in the second intron of MHIIb gene, and for which distinctive repeat numbers of the motif were associated to each alleles (69, 27, 20, 40, 19 and 25 repeats for the DAB*0101 to DAB*0601 alleles respectively). Relative expression levels of four alleles were determined in heterozygous fish at temperature of 18 ºC and 6 ºC.  Results indicate that alleles carrying the longest minisatellite showed a 1.67 to 2.56-fold reduction in the transcript expression relatively to the shortest one.  In contrast, no significant differences were seen in the expression levels between alleles with comparable minisatellite length at both temperatures.  Furthermore, the repressive activity associated to the longest minisatellite was more effective at temperature of 18 ºC in fish from three different genotypes.  We finally observed a significant 2.08-fold up-regulation of the total MHII transcript amount at 6 ºC.  The results support the implication of repeated DNA sequences in the regulation of the gene transcriptional activity and suggest that a temperature-sensitive minisatellite could potentially be submitted to selective forces and therefore play an important role in gene expression and evolution in ectothermic organisms.

Keyphrases: Major histocompatibility complex, Histocompatibility antigens class II, Polymorphism, Repetitive DNA sequences, Gene expression regulation, Disease resistance, Bacterial infection, Selection, Evolution, Salmonids