2026 ECNP Congress

PS02-2053

Identification of DNA methylation-dependent functional regulatory elements in developing brain cells using massively parallel reporter assay

Justina Lugenbühl — M. Labeur, B. Wölfel, A. Fröhlich, N. Yusupov, M. Ködel, V. Sportelli, E.B. Binder · Max-Planck-Institute of Psychiatry, Genes and Environment, Munich, Germany

2026 ECNP Congress

Excessive stress causes risk for stress-related psychiatric disorders1. Epigenetic alterations, including DNA methylation, which regulates the transcriptome, contribute to this association2–4. Thus, stress-induced shifts in the DNA methylation setpoint can have detrimental effects on downstream cell biology and yield vulnerability for stress-related disorders4. Therefore, we ask which DNA methylation changes play causal roles in biological dysregulations underlying these disorders. For that purpose, we 1) detected stress-sensitive differentially methylated regions in in vitro models of brain cells exposed to stress. From these sequences, we aimed to 2) identify regulatory elements whose functions depend on the DNA methylation state using a massively parallel reporter assay (MPRA), specifically AAV-mStarrSeq (methylation-self-transcribing-active-regulatory-region-sequencing). We trust that this novel technique is a valuable contribution to the functional neuroepigenomics tool kit.

Human cohort and postmortem brain studies show the association between stress exposure and psychiatric disorders4. Yet, this relation remains to be studied causally on a methylome wide level. We exposed human neuronal organoids and human induced pluripotent stem cell (iPSC)-derived astrocytes to glucocorticoids and measured resulting DNA methylation using EPIC arrays. The exposure affected 10621 CpGs in human neuronal organoids, and 7702 CpGs in iPSC-derived astrocytes. Specifically, 6353 CpGs and 3673 CpGs were hypermethylated, while 4286 or 4029 CpGs were hypomethylated in the organoids and astrocytes, respectively.

Not all of these stress-sensitive sites have functional consequences on cell biology. To identify the functional DNA methylation-dependent regulatory elements among them, we employ the novel AAV-mStarrSeq. MStarrSeq is a DNA methylation dependent MPRA5. We combined mStarrSeq with adeno-associated virus (AAV) technology, generating AAV-mStarrSeq. AAV-mStarrSeq enables us to identify regulatory elements in any cell type, including neurons and astrocytes. Briefly, we generate a library of sequences containing stress-sensitive DNA methylation sites from the in vitro experiments. Then, we infect iPSC-derived astrocytes with AAVs containing these sequences in methylated or unmethylated state. After glucocorticoid exposure, vector DNA and RNA are sequenced, to assess DNA methylation-dependent regulatory activity. For instance, we conducted mStarrSeq on differentially methylated regions in FKBP5and ZBTB16 identified by previous in vitro studies. In HeLa cells, we observe a 45% reduction in reporter expression when an glucocorticoid responsive element in intron 7 of FKBP5 is methylated compared to unmethylated, as shown by a linear regression F(1,4) = 24.74, p = 0.008, with R2 = 0.86. Thus, this element is DNA-methylation-dependent functional regulatory element.

In sum, we can identify functional regulatory elements among stress-sensitive DNA methylation in human brain cell types. Furthermore, we describe a novel technique AAV-mStarrSeq, which can be applied to detect DNA methylation-dependent regulatory elements in any cell type. It thereby contributes to the understanding of epigenetic dysregulation in the human brain. These functional insights are crucial for the generation of treatments that target the neurobiological underpinnings of stress-related psychiatric disorders.

References

1.de Kloet, E. R. Coping with the multifaceted and multifunctional role of cortisol in the brain. Neurosci. Appl. 3, 104047 (2024). 2.Wiechmann, T. et al. Identification of dynamic glucocorticoid-induced methylation changes at the FKBP5 locus. Clin. Epigenetics 11, 83 (2019). 3.Provençal, N. et al. Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation. Proc. Natl. Acad. Sci. 117, 23280–23285 (2020). 4.Li, H. et al. Mapping DNA methylation signatures to identify epigenetic variation across subcortical regions of the human PTSD brain. Biol. Psychiatry S0006322325015811 (2025) doi:10.1016/j.biopsych.2025.10.032. 5.Lea, A. J. et al. Genome-wide quantification of the effects of DNA methylation on human gene regulation. eLife 7, e37513 (2018).