Transcriptional-regulatory convergence across functional MDD risk variants identified by massively parallel reporter assays

Bernard Mulvey,Joseph D Dougherty

doi:10.1038/s41398-021-01493-6

Bernard Mulvey, Joseph D Dougherty

Open Access

https://doi.org/10.1038/s41398-021-01493-6

Copy DOI

Journal: Translational Psychiatry	Publication Date: Jul 22, 2021
Citations: 12	License type: open-access

Affiliation: Washington University in St. Louis

Abstract

Family and population studies indicate clear heritability of major depressive disorder (MDD), though its underlying biology remains unclear. The majority of single-nucleotide polymorphism (SNP) linkage blocks associated with MDD by genome-wide association studies (GWASes) are believed to alter transcriptional regulators (e.g., enhancers, promoters) based on enrichment of marks correlated with these functions. A key to understanding MDD pathophysiology will be elucidation of which SNPs are functional and how such functional variants biologically converge to elicit the disease. Furthermore, retinoids can elicit MDD in patients and promote depressive-like behaviors in rodent models, acting via a regulatory system of retinoid receptor transcription factors (TFs). We therefore sought to simultaneously identify functional genetic variants and assess retinoid pathway regulation of MDD risk loci. Using Massively Parallel Reporter Assays (MPRAs), we functionally screened over 1000 SNPs prioritized from 39 neuropsychiatric trait/disease GWAS loci, selecting SNPs based on overlap with predicted regulatory features—including expression quantitative trait loci (eQTL) and histone marks—from human brains and cell cultures. We identified >100 SNPs with allelic effects on expression in a retinoid-responsive model system. Functional SNPs were enriched for binding sequences of retinoic acid-receptive transcription factors (TFs), with additional allelic differences unmasked by treatment with all-trans retinoic acid (ATRA). Finally, motifs overrepresented across functional SNPs corresponded to TFs highly specific to serotonergic neurons, suggesting an in vivo site of action. Our application of MPRAs to screen MDD-associated SNPs suggests a shared transcriptional-regulatory program across loci, a component of which is unmasked by retinoids.

Highlights

Major depressive disorder (MDD) is a common but debilitating psychiatric disorder affecting hundreds of millions worldwide [1], exacting substantial tolls on both individuals [2] and societies [3]
Many MDD loci contain more than one functional single-nucleotide polymorphism (SNP) We identified >1000 SNPs from MDD-associated GWAS loci, prioritizing SNPs overlapping with epigenetic data from neural samples, and cloned them into an Massively Parallel Reporter Assays (MPRAs) library (Fig. 1)
We leveraged MPRA to screen over 1000 SNPs from loci associated with MDD, related phenotypes, and broader psychiatric disease, demonstrating the utility of this technique for dissecting the functional regulatory architecture of psychiatric GWAS loci, and defining shared upstream regulatory features across loci

Summary

INTRODUCTION

Major depressive disorder (MDD) is a common but debilitating psychiatric disorder affecting hundreds of millions worldwide [1], exacting substantial tolls on both individuals [2] and societies [3]. Delivery of a library of DNA elements to cells, followed by RNA collection and sequencing, enables quantitative estimation of the expression driven by each element as a ratio of expressed RNA barcode to delivered DNA barcode These assays have recently been adapted to systematically identify SNPs with functional allelic TR differences from GWAS loci for several diseases [51,52,53,54,55,56,57,58,59]. For the first MPRA and single-condition analysis of vehicle samples from the second assay, empirical p values (Pemp) were calculated via simulated allelic comparisons between random subsets of “basal” barcodes (see Supplemental Methods) following an analogous procedure from a multiplex CRISPR study [69], with significance defined as Pemp < 0.05 unless specified otherwise This ensures that a representative cross-section of expression variability driven by barcode sequences is accounted for when assessing TR differences. We examined TFs for enrichment among highly-expressed genes in adult and developing human brain using the ABAEnrichment package’s Wilcoxon approach [83], effectively weighting TFs by the number of functional SNPs implicated by motifbreakR (see Supplemental Methods)

RESULTS

DISCUSSION

CODE AVAILABILITY