Fig. 4.

Testing HARs and HAR sequence variants for enhancer activity with reporter assays. a Example of a transient transgenic reporter assay to test a HAR (HACNS426) for enhancer activity in mouse embryos. The experiment compares enhancer activity of the major allele (G) to that of the autism-associated minor allele (A) that is never homozygous in healthy controls and is predicted to change transcription factor binding. Top: constructs carrying each of the two HACNS426 alleles fused to the human CUX1 promoter and the GFP reporter gene are separately injected into single-cell mouse embryos. Bottom: to assay enhancer activity, brain slices from embryonic day E16.5 are stained for GFP. The authors observed differences in GFP expression with the G allele (above) versus A allele (below). The major strength of the approach is the spatial and cellular resolution of in vivo measurements, while weaknesses include not being highly quantitative, the use of mouse to compare human and chimpanzee variants, low throughput, and relatively high cost. Adapted with permission from [23]. The study also performed in vitro luciferase reporter assays and showed HANCS426 interacts with the dosage-sensitive CUX1 promoter. b Massively parallel reporter assays (MPRAs) enable thousands of reporter constructs to be tested as a library (top) in which each HAR variant is associated with a unique DNA barcode (such as 20-bp sequence). RNA sequencing of barcodes (bottom) provides a quantitative estimate of the activity of each HAR variant. MPRAs are high throughput, allowing thousands of HARs and variants thereof to be tested, and they are quantitative, enabling detection of single nucleotide differences with moderate effects on expression. Current weaknesses of the technology include being limited to HARs or HAR segments less than 200 bp and being restricted to testing in cell lines or mouse tail vein assays