1468-P: Massively Parallel Reporter Assay Reveals Position-, Promoter-, and Strand-Dependent Effects in Islet TSSs Overlapping Type 2 Diabetes GWAS Loci

Introduction & Objective: Massively parallel reporter assays (MPRAs) are an increasingly popular method of assessing the sequence-dependent activity of variants associated with complex diseases such as type 2 diabetes (T2D). Many MPRA libraries use a single plasmid configuration, a minimal promoter, and a single strand orientation, which may fail to capture the context-dependent expression of T2D-associated cis-regulatory elements. Here, we interrogate the potential activity differences introduced by variable MPRA plasmid construction.

Methods: We used an MPRA library to quantify the activity of 1,305 human pancreatic islet transcription start sites (TSSs). We cloned TSS fragments in either the upstream or downstream position of either the human insulin promoter or a synthetic housekeeping promoter. Activity was measured in the rat pancreatic islet beta cell line 832/13. We used statistical models to calculate position- and promoter-specific fragment activity and enrichment of transcription factor motifs (TFMs) and fragment strand-of-origin.

Results: The non-zero position-bias coefficients for the fragments were significantly stronger than the promoter-bias coefficients (p < 0.05) and strongly biased towards the upstream configuration. Fragments that displayed preferential activity upstream of the reporter gene were enriched for TFMs such as BHLHE22, NR2E1, and E2F, which have been implicated in insulin production and glucose metabolism. 28% of fragments with significant promoter effects overlapped a 99% T2D genome-wide annotation study credible set. Positive strand-of-origin (β = 11.91, p < 0.001) predicted significantly higher fragment activity.

Conclusion: Our MPRA library demonstrates the position-, promoter-, and strand-dependence of activity at islet TSSs that may contribute to T2D development. Future MPRA studies should seek to account for such factors when assaying T2D-associated regulatory element variants.