Tiit Örd, Daima Örd, Priit Adler, Tõnis Örd
Activating Transcription Factor 4 (ATF4) is an important regulator of gene expression in stress responses and developmental processes in many cell types. Here, we catalogued ATF4 binding sites in the human genome and identified overlaps with trait-associated genetic variants. We probed these genetic variants for allelic regulatory activity using a massively parallel reporter assay (MPRA) in HepG2 hepatoma cells exposed to tunicamycin to induce endoplasmic reticulum stress and ATF4 upregulation. The results revealed that in the majority of cases, the MPRA allelic activity of these SNPs was in agreement with the nucleotide preference seen in the ATF4 binding motif from ChIP-Seq. Luciferase and electrophoretic mobility shift assays in additional cellular models further confirmed ATF4-dependent regulatory effects for the SNPs rs532446 (GADD45A intronic; linked to hematological parameters), rs7011846 (LPL upstream; myocardial infarction), rs2718215 (diastolic blood pressure), rs281758 (psychiatric disorders) and rs6491544 (educational attainment). CRISPR-Cas9 disruption and/or deletion of the regulatory elements harboring rs532446 and rs7011846 led to the downregulation of GADD45A and LPL, respectively. Thus, these SNPs could represent examples of GWAS genetic variants that affect gene expression by altering ATF4-mediated transcriptional activation.
Tiit Örd, Daima Örd, Priit Adler, Tõnis Örd. Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs. 2023-10-31. https://doi.org/10.1371/journal.pgen.1011014
An episomal barcode MPRA tested reference and alternative alleles of 581 trait-associated SNP-containing 175-bp sequences in HepG2 cells without stress treatment. Five independent transfections performed on separate days were analyzed by poly(A)+ RNA barcode sequencing and MPRAnalyze.
An episomal barcode MPRA tested reference and alternative alleles of 581 trait-associated SNP-containing 175-bp sequences in HepG2 cells after tunicamycin-induced endoplasmic-reticulum stress. Five independent transfections performed on separate days were analyzed by poly(A)+ RNA barcode sequencing and MPRAnalyze.
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