Modeling Human Genetic Variation in Non-Coding DNA in Humanized Mice

Single nucleotide polymorphyms;Non-coding DNA;Transgenic;Bacteria artificial chromosome;Gene regulation;Disease susceptibility;Cytokine.;Immunology

Dabitao, Djeneba kSen, Ranjan ;

Johns Hopkins University

关键词: Single nucleotide polymorphyms;    Non-coding DNA;    Transgenic;    Bacteria artificial chromosome;    Gene regulation;    Disease susceptibility;    Cytokine.;    Immunology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60531/DABITAO-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The increasing availability of sequencing data from genome-wide association studies and whole genome sequencing of the human genome has enabled rapid identification of genetic variations—mainly single nucleotide polymorphisms (SNPs)— in non-coding DNA of the human genome. However, it has been difficult to find the biological functions of the numerous SNPs in the genome. This gap in knowledge can be explained in part by our poor understanding of the function of non-coding DNA, and by the challenge of experimentally assigning function to SNPs that map to these non-coding regions. To clearly define the function of non-coding SNPs, we created genetically humanized mice to model human genetic variation in non-coding DNA in vivo. To generate the mice, we used a bacterial artificial chromosome (BAC) system harboring two genetically different human IL10 SNP haplotypes. The IL10 SNP haplotypes are ;;ATA” and ;;GCC,” which have been associated with differential IL-10 levels and disease susceptibility in humans. We found a robust allele-specific human IL-10 expression in both macrophages and CD4+ T cells. Specifically, GCC-hIL10BAC encodes for a high human IL-10 level relative to ATA-hIL10BAC in CD4+ T cells both in vitro and in vivo. The reverse was observed in macrophages. Accordingly, by complementing Il10 null mice with the GCC-hIL10BAC, namely Il10-/-/GCC-hIL10BAC mice, we were able to completely reverse disease outcome. The Il10-/-/GCC-hIL10BAC mice were susceptible to persistent leishmania infection as evidenced by a high parasite burden in the liver and spleen. In contrast, like Il10 null mice, the Il10-/-/ATA-hIL10BAC mice were refractory to disease.Therefore, our data demonstrate that human IL10 promoter SNP haplotypes alone can modulate IL-10 levels and disease risk. In the second part of this dissertation, we examined the regulation of IL-10 and its homolog, IL-24, as a means to indirectly demonstrate that we are not missing important regulatory elements within the hIL10BAC. We chose IL-24 from the remaining cytokines within the Il10 gene cluster because the gene encoding for IL-24 is localized at the extreme end of the Il10 locus in both mouse and man and also human IL24 gene is not included in the hIL10BAC. Thus, finding co-regulation of IL-10 and IL-24 expression would suggest that the two homologs share common regulatory elements. Interestingly, we found that IL-10 and IL-24 are regulated by distinct cell-type-specific regulatory pathways. Optimal IL-24 expression requires Stat6 and Stat4 in macrophages and NK cells; meanwhile, IL-10 expression is independent of Stat6 and dependent on Stat4 only in IL-12-treated NK cells. We also discovered an unexpected role for Type-I Interferons in mediating differential regulation of IL-10 and IL-24 expression in macrophages and NK cells. Thus, our results suggest that IL-24 and IL-10 are unlikely to share common regulatory elements within the Il10 locus. Altogether, our results undoubtedly demonstrate that we can model human genetic variation in non-coding DNA in vivo using genetically humanized hIL10BAC mice. In the future, the hIL10BAC approach can be extended to other human genes to accelerate rational development of safe and efficient personalized therapies, including vaccines.