IDENTIFICATION OF STRESS-SENSITIVE DNA METHYLATION-DEPENDENT FUNCTIONAL REGULATORY ELEMENTS IN DEVELOPING BRAIN CELLS USING MASSIVELY PARALLEL REPORTER ASSAY

Excessive stress elevates risk for psychiatric disorders. Epigenetic alterations, including DNA methylation (DNAm), contribute to this association by altering transcription and downstream cell biology. We ask which stress-sensitive DNAm changes have causal regulatory effects.
We profiled human induced pluripotent stem cell (iPSC)‑derived astrocytes exposed to glucocorticoids (GCs) on EPIC arrays and identified 7,702 GC‑responsive CpGs (3,673 hypermethylated; 4,029 hypomethylated).
Yet, not all stress-sensitive sites affect downstream biology. We test which sites functionally regulate target gene expression in a DNAm‑dependent manner. For that purpose, we developed AAV‑mSTARRSeq (adeno-associated virus – methylation-self-transcribing-active-regulatory-region-sequencing). MStarrSeq is a DNAm dependent massively parallel reporter assay. We combined mStarrSeq with AAV technology, generating AAV-mStarrSeq, which enables identification of regulatory elements in any cell type, including brain cells. We build a sequence library enriched for stress‑sensitive CpGs, deliver methylated or unmethylated inserts to iPSC‑derived astrocytes via AAV, and quantify DNAm‑dependent regulatory activity after GC exposure. For instance, in HeLa cells, DNAm of a GC-responsive element in intron 7 of FKBP5 reduced the expression by 45% (linear regression F(1,4) = 24.74, p = 0.008, R² = 0.86). Thus, this element is a DNAm-dependent functional regulatory element.
In sum, we can identify functional regulatory elements among stress-sensitive DNAm sites in iPSC-derived astrocytes. We describe the novel AAV-mStarrSeq, which detects DNAm-dependent regulatory elements in any cell type. It thereby contributes to the understanding of epigenetic dysregulation in the brain. These functional insights are crucial for developing treatments targeting the neurobiological underpinnings of stress-related psychiatric disorders.