jun expression plasmids  (OriGene)

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: For each clone, 1 million cells resuspended in 100 µL of T buffer (NEON Invitrogen) were co-transfected with 500 ng of FOS and 500 ng of JUN expression plasmids (RC202597 and RC209804 respectively, Origene).

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: For each clone, 1 million cells resuspended in 100 µL of T buffer (NEON Invitrogen) were co-transfected with 500 ng of FOS and 500 ng of JUN expression plasmids (RC202597 and RC209804 respectively, Origene).

Techniques: Modification, Variant Assay, Homologous Recombination, CRISPR, Sequencing, Expressing, Clone Assay, Generated, Transfection, 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: CHIP-seq peaks from ReMap2022 confirmed the binding of transcription factors, identified by RSAT, within the ESpromoter and ATP2B4 promoters, in particular the FOS:JUN dimer corresponding to AP-1.

Article Snippet: For each clone, 1 million cells resuspended in 100 µL of T buffer (NEON Invitrogen) were co-transfected with 500 ng of FOS and 500 ng of JUN expression plasmids (RC202597 and RC209804 respectively, Origene).

Techniques: ChIP-sequencing, Binding Assay