Transcriptional-Regulatory Convergence Across Functional MDD Risk Variants Identified by Massively Parallel Reporter Assays

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 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 1 000 SNPs prioritized from 39 neuropsychiatric trait/disease GWAS loci, with SNPs selected 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. Further, 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 subset of which are unmasked by retinoids.

Sex significantly impacts the function of major depression-linked variantsin vivo

“…Likewise, female glutamatergic TFs were enriched for PPIs with SMAD3 (3/8), along with RXRA (3/8), replicating in vivo our recent in vitro findings of retinoid-interacting rSNPs in MDD-associated loci (26). Female total hippocampal TFs, on the other hand, were enriched for upstream regulation by several TFs, including ATF2 (9/19), BRCA1 (8/19), and TCF3 (5/19).…”

“…MDDassociated SNP rs1467013 was previously demonstrated to be functional in a classical luciferase reporter assay in three different cell lines (75). A prior in vitro MPRA identified rs301807, but not rs301806, as an rSNP (26), while both were identified as functional here. This highlights the importance of in vivo context for obtaining relevant insights about functional variation within GWAS loci-in this case, revealing two rSNPs in close (~2kb) proximity that likely influence expression of the same target (RERE).…”

“…S2-S4). We calculated empirical p (pemp) values using 50,000 simulated 'allelic' comparisons between subsets of random barcodes coupled to the minimal promoter alone (26,51) to account for technical and barcode-mediated noise. Significance was called at FDR-corrected pemp<0.2, a stringency comparable to a recent study of sex-interacting eQTLs (21).…”

“…We next asked whether there were any shared transcriptional-regulatory mechanisms underlying MDD rSNP effects in the hippocampus. We tested whether rSNPs perturbed specific transcription factor (TF) binding motifs more frequently than expected by chance (defined by their rates in rSNPs vs. non-effect SNPs in the assay) (26). We assessed motif disruptions using motifbreakR (64) and RSAT var-tools (65) (see Online Methods) defining rSNPs at a nominal LMM pemp of 0.05.…”

“…We overcome these prior limitations in functional regulatory SNP (rSNP) identification to interrogate the biological contexts under which MDD rSNPs act by delivering an MPRA library of MDD-associated variants (26) into the adult mouse hippocampus in vivo. Building on prior brain MPRAs of enhancers (27) and an RE variant (28), our approach greatly extends in vivo MPRA methods to identify rSNPs and their sex interactions, including those which are cell type-specific.…”