Fig. 4: ERVK solo-LTRs function as regulatory elements in the brain of the zebra finch.

a In zebra finch, the number of genes located within 2 kb, 5 kb, and 10 kb flanking regions of the regulatory elements overlap with 5535 ERVK solo-LTRs. b Significantly enriched GO terms of 1640 genes on molecular function were visualized by REVIGO tool. c Volcano plot shows DEGs between the brain tissues of zebra finch and chicken among 12,028 orthologous genes. Horizontal red dashed line indicates the cutoff of Benjamini-Hochberg adjusted p-value = 0.05, and the vertical black dashed lines indicate a 2-fold change. d Genomic collinearity plot of ITGA2 gene showed that the solo-LTRs residue overlapped with H3K27ac signal in zebra finch. Silhouettes of the zebra finch and chicken are from https://www.phylopic.org/. e Fluorescence in situ hybridization (FISH) microscope photographs of the brain of a zebra finch. Expression signals of ITGA2 and SCG10 were indicated in red and cyan, respectively. The SCG10 gene, which encodes a neuron-specific stathmin protein, was used as control. The blue signal, obtained from 4’ 6-diamidino-2-phenylindole (DAPI) staining, shows the location of the nucleus. The HVC, RA, and Area X regions are labeled according to the schematic diagram, which was modified based on the data retrieved from the ZEBrA database (Oregon Health & Science University, Portland, OR 97239; http://www.zebrafinchatlas.org)²⁹. f Relative ITGA2 expression to the DAPI signal revealed that ITGA2 was highly expressed in the zebra finch compared to the chicken. The ITGA2 and DAPI signals were measured using the “Analytical Particle” function of the software FIJI⁸², with ten random samples (n = 10) taken from regions throughout the whole brain. g Dual-luciferase reporter assay in the UMNSAH/DF-1 cell line demonstrated the potential cis-regulation activity of the 405 bp ERVK solo-LTRs insertion located upstream of the ITGA2 gene in the zebra finch. Relative luciferase activity was determined with n = 9 biologically independent repetitions for each experimental group. Horizontal lines indicate the mean (± s.d.) in (f, g). Welch’s two-tailed t-test was conducted using GraphPad Prism (p-value: **** < 0.0001) in (f, g). Source data are provided as a Source Data file.