Institute of Genomics, University of Tartu, Tartu, Estonia.
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.
PLoS Genet. 2023 Oct 31;19(10):e1011014. doi: 10.1371/journal.pgen.1011014. eCollection 2023 Oct.
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.
激活转录因子 4（ATF4）是许多细胞类型中应激反应和发育过程中基因表达的重要调节剂。在这里，我们对人类基因组中的 ATF4 结合位点进行了编目，并鉴定了与特征相关的遗传变异与 ATF4 结合位点的重叠。我们使用在暴露于衣霉素的 HepG2 肝癌细胞中进行的大规模平行报告基因分析（MPRA）来探测这些遗传变异的等位基因调节活性，以诱导内质网应激和 ATF4 上调。结果表明，在大多数情况下，这些 SNP 的 MPRA 等位基因活性与 ChIP-Seq 中 ATF4 结合基序的核苷酸偏好一致。在其他细胞模型中的荧光素酶和电泳迁移率变动分析进一步证实了 SNP rs532446（GADD45A 内含子；与血液学参数相关）、rs7011846（LPL 上游；心肌梗死）、rs2718215（舒张压）、rs281758（精神疾病）和 rs6491544（受教育程度）的 ATF4 依赖性调节作用。CRISPR-Cas9 敲除和/或删除含有 rs532446 和 rs7011846 的调节元件分别导致 GADD45A 和 LPL 的下调。因此，这些 SNP 可能代表 GWAS 遗传变异的例子，通过改变 ATF4 介导的转录激活来影响基因表达。
