Journal: bioRxiv

Article Title: Unraveling a novel dual-function regulatory element showing epistatic interaction with a variant that escapes genome-wide association studies

doi: 10.1101/2024.03.18.585566

Figure Lengend Snippet: (A) WashU epigenome browser view of LAX1 region with the position of all eQTLs for LAX1 in the ELIXIR database, those having a PIP score > 0.7 are shown in red. Localization of Encode cCRES was shown with promoter-like elements in red and enhancer-like elements in orange. The density of CHIP-seq peaks available in ReMap2022 is also displayed. Overall, these results revealed a potentially functional SNP in the LAX1 promoter (rs11240391). (B) Luciferase assays to assess the impact of the rs11240391 variant on LAX1 promoter activity without stimulation (NS: no stimulation in grey) or with PMA/ionomycin stimulation for 6 hours (S: stimulated in black). Relative luciferase activity was lower with the “G” allele than with the “T” allele. The graph shows the mean values ± SEM. P-values were calculated using a two-sided Student’s t-test. (C) Prediction of transcription factor binding sites disruption at rs11240391 using RSAT. The genomic region containing the variant is displayed. The P-value ratio was calculated by dividing the best probability of transcription factor binding by the worst probability. All transcription factors exhibit a higher binding affinity to the T allele of SNP rs11240391. (D) CHIP-seq peaks from ReMap2022 confirmed the binding of transcription factors, identified by RSAT, in the region containing rs11240391. (E) Luciferase assays to evaluate the impact of rs11240391 on the fixation of transcription factors by adding expression plasmid of JUN or FOS or both. The major allele T was represented in grey, and the minor allele G was represented in black. A higher relative luciferase activity was observed in the presence of the "T" allele, which only increased with the simultaneous addition of JUN and FOS. Values were generated in triplicate from 3 independent experiments. The plot shows the mean values ± SEM. P-values were calculated using a two-sided Student’s t-test.

Article Snippet: The rs11240391 single nucleotide polymorphism (SNP) was genotyped using the TaqMan allelic discrimination technique (4351379, C_319219_10, Thermofisher, Waltham, MA, USA) on the QuantStudio 6 Flex instrument.

Techniques: ChIP-sequencing, Functional Assay, Luciferase, Variant Assay, Activity Assay, Binding Assay, Disruption, Expressing, Plasmid Preparation, Generated

Journal: bioRxiv

Article Title: Unraveling a novel dual-function regulatory element showing epistatic interaction with a variant that escapes genome-wide association studies

doi: 10.1101/2024.03.18.585566

Figure Lengend Snippet: (A) Generation of cell lines with a modified rs11240391 variant allele by homologous recombination using a 101 pb ultramer, a single guide RNA (gRNA3), and CRISPR-Cas9 technology. Sanger sequencing chromatograms show the genomic sequence of the wild-type Jurkat clone (WT Jurkat) and of the clone in which a “T” allele has been replaced by a “G” allele (T/G Jurkat). (B) qPCR analysis of LAX1 gene expression on wild-type clone after homologous recombination experiment (WT HR ) and two heterozygous clones (T/G 1 and T/G 2) for the rs11240391 SNP. Values were generated in triplicate from 3 independent experiments. The plot shows the mean values ± SEM. P-values were calculated using a two-sided Student’s t-test. (C) qPCR analysis of LAX1 gene expression in wild-type Jurkat cells (WT HR ) and T/G clone heterozygotes (T/G 1 and T/G 2) for the SNPs rs11240391, untransfected (grey) or transfected (black) with both expression plasmids for FOS and JUN. Values were generated in triplicate from 3 independent experiments. The plot shows the mean values ± SEM. P-values were calculated using a two-sided Student’s t-test. (D) Monitoring of Jurkat cell activation by anti-CD69 staining through flow cytometry after PMA/ionomycin stimulation. The values represent the average ± SEM of two independent experiments performed in duplicate, indicating the percentage of cells positive for anti-CD69 staining (acquisition of 2000 cells per clone). The comparison was carried out between a wild-type clone after a homologous recombination experiment (WT HR ) and two heterozygous clones (T/G 1 and T/G 2) for the rs11240391 SNP. (D) T cell activation was assessed based on CD69 staining and flow cytometric gating strategy. Plots showing the results at different time points after PMA/Ionomycin stimulation of wild-type clone after CRISPR-Cas9 editing (WT HR ) and heterozygous clones (T/G 1 and T/G 2) for the rs11240391 SNP. Representative experiments on 2000 cells of each type in function to Forward Scatter-Horizontal (FSC-H) and anti-CD69 staining with Fluorescein Isothiocyanate (FITC). A higher percentage of CD69-positive cells was observed for T/G 1 and T/G 2 clones.

Article Snippet: The rs11240391 single nucleotide polymorphism (SNP) was genotyped using the TaqMan allelic discrimination technique (4351379, C_319219_10, Thermofisher, Waltham, MA, USA) on the QuantStudio 6 Flex instrument.

Techniques: Modification, Variant Assay, Homologous Recombination, CRISPR, Sequencing, Gene Expression, Clone Assay, Generated, Transfection, Expressing, Activation Assay, Staining, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Unraveling a novel dual-function regulatory element showing epistatic interaction with a variant that escapes genome-wide association studies

doi: 10.1101/2024.03.18.585566

Figure Lengend Snippet: (A) Design for recruitment of cohort, amplification of DNA, and genotyped of rs11240391. (B) Association of rs11240391 with severe malaria. The graph shows the percentage of the GG risk genotype (black) versus heterozygous TG and major homozygous TT (grey) in the control and severe malaria groups. P-values were calculated using logistic regression analyses and the graph displays the Odd Ratio (OR) with its 95% confidence interval. (C) Normalized LAX1 expression obtained by microarray in children with uncomplicated malaria (UM) and cerebral malaria (CM). Lower expression of LAX1 is observed in CM. (D) WashU epigenome browser view of recombination rate of YRI (Yoruba in Ibadan, Nigeria). Black lines correspond to the 5 SNPs previously identified (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) and the SNP in the LAX1 promoter (rs11240391). The green line corresponds to the tagSNP (rs10900585) previously associated with severe malaria in GWAS analyses. (E) Linkage disequilibrium (LD) between different SNPs of the ATP2B4 and LAX1 genes in our Senegalese cohort. LD is expressed as r 2 multiplied by 100. SNPs with an r 2 > 0.6 are considered in linkage disequilibrium and are colored red. (F) Epistatic interaction between the haplotype of 5 SNPs and rs11240391 computed by logistic regression. The plot shows the percentage of individuals carrying either both protective haplotype/genotype (mm+Mm/TT+TG) (m: minor haplotype, M: major haplotype) or both risk haplotype/genotype (MM/GG). Individuals carrying the GG genotype and the MM haplotype are at increased risk of severe malaria. P-values were calculated using logistic regression analyses and the graph displays the Odd Ratio (OR) with a 95% confidence interval. (G) Luciferase assays to functionally assess the epistatic effect between the haplotype including the 5 SNPs in the ESpromoter and the LAX1 promoter SNP rs11240391. Graphs showing the relative luciferase activity under the control of LAX1 promoter with “T” or “G” allele at rs11210391 alone or in combination with the ESpromoter containing either the major haplotype (maj: TCCGA) or minor haplotype (min: CTTGG) for the 5 SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255). Values were generated in 84triplicate from 3 independent experiments. The plot shows the mean values ± SEM. P-values were calculated using a two-sided Student’s t-test.

Article Snippet: The rs11240391 single nucleotide polymorphism (SNP) was genotyped using the TaqMan allelic discrimination technique (4351379, C_319219_10, Thermofisher, Waltham, MA, USA) on the QuantStudio 6 Flex instrument.

Techniques: Amplification, Control, Expressing, Microarray, Luciferase, Activity Assay, Generated