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. 2020 May 18;11:2476. doi: 10.1038/s41467-020-16129-4

Fig. 4. Evolutionary trade-offs of constitutive NRF2 activity in feather development genes.

Fig. 4

a Glutathione S-transferase A2 (GSTA2) is shared among gene sets associated with: (1) NRF2-regulation in mice; (2) plumage coloration in Neoaves; and (3) the Zebra finch song system32, 36, 37. Avian GSTA2 is a target of positive selection (p < 10−17; M8 PAML, Supplementary Table 9). GSTA2 sites targeted by natural selection (dN/dS = 3.50) are located within the glutathione (GSH)-binding site (PDB ID: 2WJU) and display Neoaves-specific amino acid variants. b Loss of an NRF2 antioxidant response regulatory element (ARE) in Neoaves (Woodcock, N = 2 biologically independent animals; Sparrow, N = 3 biologically independent animals; Supplementary Table 8) is consistent with a significant decrease in GSTA2 expression relative to that of Chicken [N = 3 biologically independent animals]. **p = 0.005, ***p = 0.0012, respectively, one-sided t test. Data are presented as mean values. c KEAP1 knockout mice die from starvation shortly after birth from hyperkeratosis of the gastrointestinal tract, likely through overexpression of α-keratins and loricrins in squamous cells38. d AREs were detected upstream of β-keratins across avian genomes (Supplementary Table 7). Skin chromatin immunoprecipitation (ChIP) with anti-NRF2 antibody revealed NRF2 occupancy of AREs for feather β-keratins (grey) and loricrin (black) identified within the Chicken genome. e AREs for feather β-keratins are markedly deceased in the Zebra finch genome (Supplementary Table 7) and display a trend toward decreased NRF2 occupancy in ChIP analysis of the skin obtained from two highly diverged wild Neoaves species (common yellow throat Geothlypis trichas and Black-billed cuckoo Coccyzus erythropthalmus). “Female Common Yellowthroat” (CC BY-SA 4.0) Sandhillcrane https://commons.wikimedia.org/wiki/File:Female_Common_Yellowthroat.jpg, no changes were made. “Black-Billed Cuckoo” (C) 2008 Wolfgang Wander (GFDL). Source data are provided as a Source data file. All error bars represent standard error.