Publications

The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian enhancers at single-nucleotide resolution through a massively parallel reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2-3% divergence from the wild-type sequence. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing the transcribed tags. Linear regression analysis yielded highly reproducible estimates of the effect of every possible single-nucleotide change on enhancer activity. The functional consequence of most mutations was modest, with ∼22% affecting activity by >1.2-fold and ∼3% by >2-fold. Several, but not all, positions with higher effects showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.

Cell adhesion molecules govern leukocyte-endothelial cell (EC) interactions that are essential in regulating leukocyte recruitment, adhesion, and transmigration in areas of inflammation. In this paper, we synthesized hydrogel matrices modified with antibodies against vascular cell adhesion molecule-1 (VCAM1) and endothelial leukocyte adhesion molecule-1 (E-Selectin) to mimic leukocyte-EC interactions. Adhesion of human umbilical vein ECs to polyvinyl alcohol (PVA) hydrogels was examined as a function of the relative antibody ratio (anti-VCAM1:anti-E-Selectin) and substrate elasticity. Variation of PVA backbone methacrylation was used to affect hydrogel matrix stiffness, ranging from 130 to 720 kPa. Greater EC adhesion was observed on hydrogels presenting 1:1 anti-VCAM1:anti-E-Selectin than on gels presenting either arginine-glycine-asparagine (RGD) peptide, anti-VCAM1, or anti-E-Selectin alone. Engineered cell adhesion - based on complementing the EC surface presentation - may be used to increase the strength of EC-matrix interactions. Hydrogels with tunable and synergistic adhesion may be useful in vascular remodeling.