human tagged orf clone c-jun (jun)  (OriGene)

Journal: Cell Genomics

Article Title: Unraveling an enhancer-silencer regulatory element showing epistatic interaction with a variant that escaped genome-wide association studies

doi: 10.1016/j.xgen.2025.100889

Figure Lengend Snippet: Identification of the functional variant rs11240391 corresponding to the FOS and JUN binding site (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. 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 gray]) or with PMA/ionomycin stimulation for 6 h (S, stimulation [in black]). Values were generated from three independent experiments performed in triplicate. 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, and asterisks indicate significance (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (C) Prediction of transcription factor (TF) binding-site disruption at rs11240391 using RSAT. The genomic sequence containing the variant is displayed. The p -value ratio was calculated by dividing the best and worst probability of TF binding. All TFs exhibit a higher binding affinity to the T allele of SNP rs11240391. The FOS::JUN motif is the most affected by the allele change. (D) ChIP-seq peaks from ReMap2022 confirmed the binding of TFs, identified by RSAT, in the region containing rs11240391. (E) Luciferase assays to evaluate the impact of rs11240391 on the fixation of TFs by adding expression plasmid of JUN or FOS or both. The major allele T was represented in gray, 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 three independent experiments. The plot shows the mean values ±SEM. p values were calculated using a two-sided Student’s t test, and asterisks indicate significance (∗∗∗∗ p < 0.0001). (F) EMSA for SNP rs11240391. Nuclear extracts (NE:10 μg) from Jurkat cells stimulated for 2 h with PMA/ionomycin were incubated with biotinylated doubled-stranded oligonucleotides containing either the major T allele (rs11240391 - T) or the alternative G allele (rs11240391 - G). Two different competitions were performed either with an excess of non-biotinylated duplex identical to the biotinylated duplex or with an excess of the non-biotinylated duplex containing a specific AP-1 site. Complexes were separated on 6% nondenaturing polyacrylamide gels. Positions of specific (filled arrow) and non-specific (open arrow) AP-1 retardation are indicated. The intensity of the specific AP-1 complex band decreased in the presence of the biotinylated duplex containing the G allele.

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

Journal: Cell Genomics

Article Title: Unraveling an enhancer-silencer regulatory element showing epistatic interaction with a variant that escaped genome-wide association studies

doi: 10.1016/j.xgen.2025.100889

Figure Lengend Snippet: Lower LAX1 expression in heterozygous T/G Jurkat clones is associated with higher T cell activation (A) Generation of cell lines with a modified rs11240391 variant allele by HR using a 101-bp ultramer, a single guide RNA (gRNA3), and CRISPR-Cas9 technology. Sanger sequencing chromatograms show the genomic sequence of the wild-type (WT) Jurkat clone and of the clone in which a T allele has been replaced by a G allele (T/G Jurkat). (B) RT-qPCR analysis of LAX1 gene expression on WT clone after HR experiment (WT HR ) and two heterozygous clones (T/G 1 and T/G 2) for the rs11240391 SNP. Values were generated in triplicate from three independent experiments. The plot shows the mean values ±SEM. p values were calculated using a two-sided Student’s t test, and asterisks indicate significance (∗∗∗∗ p < 0.0001). (C) RT-qPCR analysis of LAX1 gene expression in WT Jurkat cells (WT HR ) and T/G clone heterozygotes (T/G 1 and T/G 2) for the SNPs rs11240391, untransfected (gray) or transfected (black) with both expression plasmids for FOS and JUN. Values were generated in triplicate from three independent experiments. The plot shows the mean values ±SEM. p values were calculated using a two-sided Student’s t test, and asterisks indicate significance (ns, not significant; ∗ p < 0.05, ∗∗ p < 0.01). (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 2,000 cells per clone). The comparison was carried out between a WT clone after an HR experiment (WT HR ) and two heterozygous clones (T/G 1 and T/G 2) for the rs11240391 SNP. The percentage of CD69 + cells was higher in heterozygous clones than in WT HR . (E) T cell activation in Jurkat clones was assessed based on CD69 staining and flow cytometric gating strategy. Plots showing the results at different time points after PMA/Ionomycin stimulation of WT clone after CRISPR-Cas9 editing (WT HR ) and heterozygous clones (T/G 1 and T/G 2) for the rs11240391 SNP. Representative experiments were conducted on 2,000 cells of each type based on forward scatter-horizontal (FSC-H) and anti-CD69 staining using fluorescein isothiocyanate (FITC). The number represents the percentage of cells. The orange window corresponds to CD69-positive cells. 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: Expressing, Clone Assay, Activation Assay, Modification, Variant Assay, CRISPR, Sequencing, Quantitative RT-PCR, Gene Expression, Generated, Transfection, Staining, Flow Cytometry, Comparison

Journal: Cell Genomics

Article Title: Unraveling an enhancer-silencer regulatory element showing epistatic interaction with a variant that escaped genome-wide association studies

doi: 10.1016/j.xgen.2025.100889

Figure Lengend Snippet: Model of the dual enhancer-silencer function of ESpromoter, epistatic interaction, and gene regulation (A) Chromatin interactions place promoters in close physical proximity, facilitating the recruitment of TFs needed to activate or repress transcription of their associated genes. The presence of an enhancer-silencer promoter (ESpromoter) within a regulated gene cluster could facilitate the assembly or maintenance of the TFs and cofactors by tightening the promoter-promoter interactions or by providing specific transcriptional regulators required for neighboring gene regulation. (B) Expression of the long transcripts ATP2B4 and LAX1 is regulated by a dual enhancer-silencer regulatory element (ESpromoter) in the same cell line through genetic variants associated with SM. ESpromoter functions as an enhancer for the long ATP2B4 transcripts independently of the haplotype of the five SNPs it contains (min, minor haplotype or Maj, major haplotype) while it functions as a silencer for LAX1 gene with an allele-specific intensity. The five SNPs within the ESpromoter act synergistically with rs11240391 to inhibit LAX1 gene expression. This epistatic interaction results in a stronger silencing effect in the presence of the G allele of rs11240391, for which no FOS::JUN binding is possible. Unidentified co-activators and co-repressors are also thought to modulate the activating or repressing effect on ATP2B4 and LAX1 , respectively.

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: Expressing, Gene Expression, Binding Assay