Homo sapiens-Specific Binding Site Variants within Brain Exclusive Enhancers Are Subject to Accelerated Divergence across Human Population

Abstract

Empirical assessments of human accelerated noncoding DNA frgaments have delineated presence of many cis-regulatory elements. Enhancers make up an important category of such accelerated cis-regulatory elements that efficiently control the spatiotemporal expression of many developmental genes. Establishing plausible reasons for accelerated enhancer sequence divergence in Homo sapiens has been termed significant in various previously published studies. This acceleration by including closely related primates and archaic human data has the potential to open up evolutionary avenues for deducing present-day brain structure. This study relied on empirically confirmed brain exclusive enhancers to avoid any misjudgments about their regulatory status and categorized among them a subset of enhancers with an exceptionally accelerated rate of lineage specific divergence in humans. In this assorted set, 13 distinct transcription factor binding sites were located that possessed unique existence in humans. Three of 13 such sites belonging to transcription factors SOX2, RUNX1/3, and FOS/JUND possessed single nucleotide variants that made them unique to H. sapiens upon comparisons with Neandertal and Denisovan orthologous sequences. These variants modifying the binding sites in modern human lineage were further substantiated as single nucleotide polymorphisms via exploiting 1000 Genomes Project Phase3 data. Long range haplotype based tests laid out evidence of positive selection to be governing in African population on two of the modern human motif modifying alleles with strongest results for SOX2 binding site. In sum, our study acknowledges acceleration in noncoding regulatory landscape of the genome and highlights functional parts within it to have undergone accelerated divergence in present-day human population.

Fig. 1.

—Schematic display of the carried out steps in the work design.

Fig. 2.

—271 Human brain specific VISTA enhancers: Test for positive selection using branch specific Wong and Nielson method with foreground branch human. (a) Y-axis contains P-values. X-axis contains a total of 271 Enhancers. Each enhancer was compared and analyzed with conserved intron 5 of human FHL1 gene. 86/271 enhancers significantly indicated signals of positive selection (enhancers under the bar = P value < 0.05). (b) Previously collected 86 enhancers in (a) were subjected to a robust analysis. Each enhancer was compared and analyzed with a locus specific intronic proxy from a nearby gene. This analysis contracted the previous findings to a number of 15 enhancers that were persistent in showing signals of positive selection (enhancers under the bar = P value < 0.05). (c) The resultant 15 enhancers were checked for human unique TFBSs on comparison with nonhuman primates (chimp, gorilla, macaque, and orangutan). Fifteen corresponding TFBSs were unique to human in nine of the enhancers with signals of positive selection. The asterisk mark on the bars indicates modern human specific variant in the TFBSs.

Fig. 3.

—Human accelerated enhancers with H. sapiens-unique transcription factor binding sites. (a) Human enhancer hs1210 (shown in brown) was shortlisted to be an enhancer under positive selection when compared with MEIS1 introns with a resultant P value of 0.03. In this figure, an aligned patch within human forebrain enhancer hs1210 has been shown with an existing transcription factor binding site of SOX2. The region also showed a novel substitution within the binding site of SOX2 (TAGACA*ACAATGGAT) in the modern human lineage, unlike the consistent nucleotide observed for archaic humans, primates and nonprimate mammals (TAGACT*ACAATGGAT). (b) Human enhancer hs563 (shown in brown) was shortlisted to be under positive selection when compared with a non-coding non repetitive sequence with a resultant P value of 0.03. In this figure, an aligned patch within human hindbrain enhancer hs563 has been shown with the existing transcription factor binding motif of RUNX1/RUNX3. The region also showed a novel substitution within the binding site of RUNX1/RUNX3 (TGTGGT*) in the modern human lineage, unlike the consistent nucleotide observed for archaic humans, primates and nonprimate mammals (TGTGGG*). (c) Human enhancer hs304 (shown in brown) was shortlisted to be under positive selection when compared with a noncoding non repetitive sequence with a resultant P value of 0.04. In this figure, an aligned patch has been shown with the existing transcription factor binding site of FOS/JUND. The region also showed a novel substitution within the binding site of FOS/JUND (T*GACTCA) in the modern human lineage, unlike the consistent nucleotide observed for archaic humans, primates, and nonprimate mammals (C*GACTCA).

Fig. 4.

—EHH plots and bifurcation diagrams of SNPs rs4452126 and rs11897580 belonging to forebrain expressing VISTA enhancer hs1210 in the African population. (a) EHH plot for SNP rs4452126 has a clear demarcation for derived allele T in terms of positive selection. EHH = 1 indicates all haplotypes carrying either ancestral or derived state of the allele are matching upto this point. Bifurcation diagram of the derived variant of the allele confirms the deduction with a clearly long haplotype and absolutely no branching at the nodes upto 10.8 kb region. (b) EHH plot for SOX2 TFBS modifying allele A of SNP rs11897580 also harbors evidence to be selected under positive selection compared with the ancestral allele T for a 10.8 kb region. Bifurcation diagram uncovers little branching at the nodes interpreting for lesser recombination events and hence longer haplotypes for the derived allele compared with the ancestral variant T, especially for a 2.5 kb region [chr2: 66762480–66764997] containing six SNPs (table 3).

Fig. 5.

—EHH plots and bifurcation diagrams for African population depicting SNPs rs2498442 and rs6477258 within VISTA enhancers hs563 and hs304, respectively. (a) SNP rs2498442 within enhancer hs563 expressing in the hindbrain tissue. African Population shows a more pronounced EHH plot with the RUNX1/RUNX3 TFBS modifying derived allele T (shown in green) covering more area under the curve in the downstream region than the ancestral allele G (shown in red). Bifurcation diagram spanning a 10.25 kb region (shown in green) has lesser branching showing lesser recombination events and making of longer haplotypes with the derived allele whereas ancestral allele has relatively more branching and shorter haplotypes in the same region. (b) SNP rs6477258 within enhancer hs304 expressing in the midbrain/forebrain tissue. EHH plot for FOS/JUND TFBS modifying derived allele T (shown in green) indicates greater area coverage in Africa on both sides when compared with the ancestral allele C (shown in red). Corresponding bifurcation diagram for Africa also reveal longer haplotype with lesser recombination events shown as branching at the nodes for TFBS modifying allele T than the ancestral allele C for a 4 kb region.