Figure 4. Reintroduced alleles restore regulatory functions lost in Eurasians.

(a) Conceptual model of restored regulatory function from Neanderthal admixture. Here, A is a cis-acting regulatory allele that is exclusively found on introgressed haplotypes (gray box) in modern Europeans (EUR). Allele A is also present in sub-Saharan Yoruba individuals (YRI) where it is not in LD with Neanderthal-derived alleles (NDAs). If this allele has similar cis-regulatory activity in both populations, it suggests that the reintroduced allele influences gene regulation independent of the associated NDAs. (b) Two examples of genes (SDSL and HDHD5) with consistent expression differences (in RPKM) associated with RA eQTLs in EUR and the corresponding allele in YRI LCLs. The RAs are present only on introgressed haplotypes in EUR, and the NDAs associated with the RAs are not present in YRI. This suggests that these RAs restore lost gene regulatory functions in EUR. (c) Schematic of the HDHD5 locus highlighting the locations of one NDA (orange) and four RA eQTLs (blue) in the introgressed haplotype and the different combinations of these alleles present in EUR, YRI, and Neanderthals. (d) Luciferase activity driven by constructs carrying different combinations of alleles present in the HDHD5 locus. A reporter construct with the EUR version of this sequence without introgression (EUR-EUR) drove significant expression above baseline (~2.0x vector with no insert, P < 0.01, t-test). We compared this activity to constructs synthesized to carry the RAs with the associated NDA (NDA-RA), the RAs without the NDA (EUR-RA), and the NDA without the RAs (NDA-EUR). This last combination never segregated in human populations, but is included here for context. As expected from the eQTL data, constructs lacking RAs drive significantly stronger expression (~2x baseline) than constructs containing RAs (~1x baseline; two-tailed t-test, P < 0.01 (**) and P < 0.001 (***)). The regulatory effect of the RAs are independent of the NDA in introgressed EUR haplotypes. (e) Regulatory activity in a massively parallel reporter assay (MPRA) for the four HDHD5 RA eQTLs reveals that rs71312076 has significant regulatory effects (RA:EUR allelic skew=2.122, P=6.6e−3, FDR=0.034) in the non-introgressed EUR background sequence.