The functional and evolutionary impacts of human-specific deletions in conserved elements

Abstract

Conserved genomic sequences disrupted in humans may underlie uniquely human phenotypic traits. We identified and characterized 10,032 human-specific conserved deletions (hCONDELs). These short (average 2.56 base pairs) deletions are enriched for human brain functions across genetic, epigenomic, and transcriptomic datasets. Using massively parallel reporter assays in six cell types, we discovered 800 hCONDELs conferring significant differences in regulatory activity, half of which enhance rather than disrupt regulatory function. We highlight several hCONDELs with putative human-specific effects on brain development, including HDAC5, CPEB4, and PPP2CA. Reverting an hCONDEL to the ancestral sequence alters the expression of LOXL2 and developmental genes involved in myelination and synaptic function. Our data provide a rich resource to investigate the evolutionary mechanisms driving new traits in humans and other species.

Fig. 1.. hCONDELs are dispersed in noncoding genomic regions that are enriched for developmental function.

(A) hCONDEL identification strategy. (B) Distribution of hCONDEL lengths (in base pairs). (C) Overlap with genomic annotation. (D) Chromosomal distribution of hCONDELs. (E) Enrichment z score of hCONDELs in tissue-specific H3K27ac-CREs. (F) hCONDEL gene ontology enrichments include gene regulation (yellow), neurodevelopment (blue), and development (mauve). (G) Enrichment log P value of hCONDEL association with neurological GWAS (t test P < 0.01 for all bars). (H) Distribution of hCONDEL ages by most recent common ancestor.

Fig. 2.. Identification of hCONDELs with species-specific activity perturb TF-binding motifs.

(A) MPRA characterization strategy. (B) Identification of hCONDELs with significant (BH adjusted P < 0.05) species-specific activity. Regulatory activity for chimpanzee sequence x axis versus orthologous human sequence (y axis) showing significant human loss (red) and gain (green). Illustrative SK-N-SH data are plotted. (C) Species activity correlated with predicted TF alteration score [difference in log-likelihood (base 2) in human versus chimpanzee sequence motif match]. Data from the cell type with the most significant MPRA-measured effect are shown. (D) Breakdown of regulatory activity and TF-binding differences categorized into activators (teal) and repressors (red), with either improved (solid line) or diminished (dashed line) motif prediction.

Fig. 3.. PPP2CA-associated hCONDEL induces species-specific regulatory changes.

(A) Genome track of hCONDEL position. Strand-specific CAGE, H3K27ac, H3K4me3, and TF chromatin immunoprecipitation signals are depicted along with conservation. (B) Vertebrate sequences aligned to the hCONDEL position with perturbed TF motif. (C) MPRA result plotting human (blue) and chimpanzee (yellow) sequence activities. Error bars indicate SD of chimpanzee and human activity. (D) hCONDEL H3K27ac signal between human and rhesus macaque. (E) hCONDEL luciferase assay result (two-sided t test P = 0.0014). Boxes indicate the median (thick line), 25th percentile (bottom end of box), and 75th percentile (top end of box); whiskers indicate ±interquartile range. (F) qPCR results for canonical and alternative isoform of PPP2CA from CRISPR mutagenesis of human sequence surrounding hCONDEL (two-sided t test P = 1.9 × 10⁻³). Bar height is the mean from three biological replicates. Error bars, SD.

Fig. 4.. hCONDEL at LOXL2 induces transcriptomic changes related to myelination and calcium signaling.

(A) Genome track of hCONDEL position in LOXL2, including H3K27ac and DNase I hypersensitive site signals from SK-N-SH and conservation scores. (B) Sequence alignment at hCONDEL with perturbed TF motif (top) and deleted conserved base (red). (C) MPRA result for LOXL2-associated hCONDEL (skew and BH adjusted P). Error bars indicate SD of chimpanzee/human activity. (D) UMAP of SK-N-SH–edited cells, with species genotype labeling for human (yellow) or chimpanzee reference (blue). (E) LOXL2 expression of SK-N-SH cells bearing the chimpanzee versus human base (Wilcoxon rank-sum test P value).(F) Volcano plot for most differentially expressed genes comparing SK-N-SH cells bearing the chimpanzee versus human sequence (genes with BH adjusted P < 0.1 highlighted in green). (G) Highlighted GO enrichments of differentially expressed genes from (F).