Systematic evaluation of the impact of promoter proximal short tandem repeats on gene expression

This study uses a massively parallel reporter assay (MPRA) to investigate how short tandem repeats (STRs) within promoter regions influence gene regulation. A custom DNA library encoding promoter variants with different STR configurations was linked to a GFP reporter and unique barcodes for expression tracking. We transfected this MPRA to HEK293T cells in triplicate which showed highly reproducible reporter expression across replicates (R=0.98). We identified 1,366 out of 19,818 loci with significant associations between repeat copy number and expression, with a strong bias toward positive effect sizes (p<1.04e-110, binomial distribution testing). We designed a second array to perform more detailed study of top regulatory STRs by modifying the repeat unit, orientation, and length, for a total of 200-300 perturbations per locus. This second array identified repeat unit sequence as the key driver of differences in expression associations across loci, whereas strand and sequence context had far weaker effects. Overall, in this work we identified optimal conditions for studying STRs using MPRAs, allowing us to perform the first in depth interrogation of the impact of STR characteristics on transcriptional regulation and study the critical roles STRs play in genome function. Overall design: This MPRA study was designed to assess the regulatory effects of short tandem repeats (STRs) located within promoter regions on gene expression. A synthetic oligonucleotide library containing promoter sequences with variable STR lengths and motifs was cloned upstream of a GFP reporter gene, with unique barcodes in the 3' UTR to track expression. The plasmid library was transfected into human cells, and barcode abundance was quantified from both RNA and input DNA to determine the transcriptional activity of each STR-containing promoter variant.