Massively parallel characterization reveals context-dependent and non-additive regulatory effects of closely spaced variant pairs
- 1Institute of Genomics, University of Tartu
Abstract
Precise control of gene expression relies in part on cis-regulatory elements (CREs), including enhancers. Genetic variation within enhancers can alter their regulatory activity and contribute to variation in gene expression, yet the effects of multiple nearby variants within the same enhancer remain poorly understood. To address this, we designed a massively parallel reporter assay (MPRA) to test the regulatory effect of 7,285 pairs of close-proximity single-nucleotide variants (SNVs) selected from blood-specific and broadly active enhancers. To enrich for functionality, we required at least one variant in each pair to be annotated as an eQTL in eQTLGen dataset. We measured the regulatory activity of all four haplotypes in K562 leukemia cells, where 57% of tested variant pairs had at least one derived haplotype that differed significantly in activity from the ancestral haplotype. The effects of individual variants frequently depended on the allelic background provided by the neighboring variant, with some variants showing opposite effects in different allelic backgrounds. Among a smaller, high-confidence subset selected for analysis of additivity, 59% (105/178) of variant pairs showed non-additive effects, and non-additive pairs were located closer together than additive pairs. Among pairs in which both single-derived haplotypes increased activity, the double-derived haplotype generally showed a smaller effect than expected under additivity. Together, our results demonstrate that nearby variants within the same enhancer can jointly shape regulatory activity and highlight the importance of considering local allelic context when interpreting the functional effects of regulatory variation.
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BibTeX
@misc{kreevan2026massively,
title = {{Massively parallel characterization reveals context-dependent and non-additive regulatory effects of closely spaced variant pairs}},
author = {R. Kreevan and V. Pankratov and D. Yermakovich and N. Shukla and M. Metspalu and A. Eriksson and M. Dannemann and I. Gallego Romero and T. Org},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.10.750584},
url = {https://www.biorxiv.org/content/10.64898/2026.09.10.750584v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Massively parallel characterization reveals context-dependent and non-additive regulatory effects of closely spaced variant pairs
AU - R. Kreevan
AU - V. Pankratov
AU - D. Yermakovich
AU - N. Shukla
AU - M. Metspalu
AU - A. Eriksson
AU - M. Dannemann
AU - I. Gallego Romero
AU - T. Org
PY - 2026
DA - 2026/09/10
PB - bioRxiv
DO - 10.64898/2026.09.10.750584
UR - https://www.biorxiv.org/content/10.64898/2026.09.10.750584v1
AB - Precise control of gene expression relies in part on cis-regulatory elements (CREs), including enhancers. Genetic variation within enhancers can alter their regulatory activity and contribute to variation in gene expression, yet the effects of multiple nearby variants within the same enhancer remain poorly understood. To address this, we designed a massively parallel reporter assay (MPRA) to test the regulatory effect of 7,285 pairs of close-proximity single-nucleotide variants (SNVs) selected from blood-specific and broadly active enhancers. To enrich for functionality, we required at least one variant in each pair to be annotated as an eQTL in eQTLGen dataset. We measured the regulatory activity of all four haplotypes in K562 leukemia cells, where 57% of tested variant pairs had at least one derived haplotype that differed significantly in activity from the ancestral haplotype. The effects of individual variants frequently depended on the allelic background provided by the neighboring variant, with some variants showing opposite effects in different allelic backgrounds. Among a smaller, high-confidence subset selected for analysis of additivity, 59% (105/178) of variant pairs showed non-additive effects, and non-additive pairs were located closer together than additive pairs. Among pairs in which both single-derived haplotypes increased activity, the double-derived haplotype generally showed a smaller effect than expected under additivity. Together, our results demonstrate that nearby variants within the same enhancer can jointly shape regulatory activity and highlight the importance of considering local allelic context when interpreting the functional effects of regulatory variation.
ER -