Site-specific regulation of DNA methylation by the STOX1 transcription factor: Identification of a STOX1A-DNMT3A-FOXRED2 axis for the management of cellular stresses

Background DNA methylation regulates gene expression, yet how transcription factors give rise to locus-specific DNA methylation remains poorly understood. Here, we explore the epigenetic effect of the transcription factor STOX1, implicated in preeclampsia, Alzheimer’s disease and various malignancies. Methods We used genome-wide DNA methylation profiling (850K Illumina microarray), expression profiling following demethylating aza-cyitidin treatment, Pyrosequencing locus-specific methylation analysis, as well as direct sequencing of bisulphite treated samples. Focusing on the epigenetic regulation of the gene FOXRED2 by STOX1, we performed CRISPR-Cas9 genome editing, coimmunoprecipitation experiments and various treatments to implement oxidative and endoplasmic reticulum stresses in cell culture, including siRNA Knock-down of specific genes, followed in particular by live-cell fluorescent imaging. Statistical tests were ANOVAs followed by Dunnett’s post-hoc t-tests. Results Significant differential methylation greater than 20% (and thus possibly connected to transcription alterations) was observed for 1359 CpG sites out of 17,494 CpGs (2.14% of the CPG analyzed) which had a nominal p-value < 0.05. The vast majority were hypomethylated (1158), whereas a smaller subset showed hypermethylation (201). Among the most differentially methylated, five CpGs within the promoter region of FOXRED2 (FAD Dependent Oxidoreductase Domain Containing 2) showed strong hypermethylation, accompanied by marked transcriptional repression of the gene. We identified an evolutionary conserved STOX1 binding motif (STRE1) 322 bp upstream of the associated CpG island. Using genome editing, we demonstrated that this motif is necessary for STOX1A-dependent promoter methylation, providing direct evidence that transcription factor binding prompts DNA methylation at a specific genomic locus. Gene knockdown experiments suggest that the methylation is primarily deposited by DNMT3A. Epigenetic repression of FOXRED2 leads to survival of stressed cells instead of their apoptosis. Conclusions These results establish a mechanistic framework, in which transcription factor binding triggers locus-specific DNA methylation, with implications for epigenetic regulation. Our work posits an epigenetically regulated STOX1-DNMT3A-FOXRED2 axis as a cascade involved in important human diseases.