Fig. 2

Risk-SNP (rs304152) in the MEF2C gene significantly reduces the transcription of its own gene by inhibiting ATF4 binding to the enhancer region. (A) Schematic representation of constructs used in the luciferase reporter assays. (B) The construct containing the rs304152-G allele (MT) showed approximately 1.7-fold less luciferase activity than the construct containing the rs304152-T allele (WT). The experiment was repeated three times. Statistics were calculated using Two-way ANOVA (n = 4 wells/group: **, P = 0.006; ***, P < 0.001). Error bars represent means ± SEM. (C) A scheme illustrating ChIP-qPCR assay for determining allele-specific DNA–protein interactions of WT and MT form of MEF2C enhancer region with anti-ATF4 antibody in NSC-34 cells. (D) ChIP-qPCR results showed less ATF4 occupancy at the MT form of MEF2C enhancer. Statistics were calculated using Student’s t-test: *, P = 0.01. (E) Immunofluorescence staining for ATF4 (red) and MEF2C (green) in NSC-34 cells transfected with ATF4 for 12 h. Right: quantification of ATF4 and MEF2C immunoreactivity levels. A total of 21 cells/group were counted (7 cells/well) from n = 3 wells/group (NSC-34 and NSC-34 + ATF4). Statistics were calculated using linear mixed model (LMM) (***, P < 0.001). (F) The scatter plot showed a correlation between ATF4 and MEF2C immunoreactivity levels in the cells. (G) A scheme summarizing that the mutant form of MEF2C enhancer resulted in MEF2C transcriptional impairment by inhibiting ATF4 transcription factor binding to the enhancer region. Scheme created with BioRender.com