Enhancer Function and Evolutionary Roles of Human Accelerated Regions

Abstract

Human accelerated regions (HARs) are the fastest-evolving sequences in the human genome. When HARs were discovered in 2006, their function was mysterious due to scant annotation of the noncoding genome. Diverse technologies, from transgenic animals to machine learning, have consistently shown that HARs function as gene regulatory enhancers with significant enrichment in neurodevelopment. It is now possible to quantitatively measure the enhancer activity of thousands of HARs in parallel and model how each nucleotide contributes to gene expression. These strategies have revealed that many human HAR sequences function differently than their chimpanzee orthologs, though individual nucleotide changes in the same HAR may have opposite effects, consistent with compensatory substitutions. To fully evaluate the role of HARs in human evolution, it will be necessary to experimentally and computationally dissect them across more cell types and developmental stages.

Keywords: enhancer; epigenetics; evolution; human accelerated region; machine learning; reporter assay.

Figure 1

Human accelerated regions have acquired many nucleotide substitutions (red) in the human genome since their divergence from the common ancestor with chimpanzees, but they are highly conserved in other vertebrates. This sequence signature suggests a constrained function during vertebrate evolution that was lost or changed in humans.

Figure 2

Human accelerated regions (HARs) are marked with dozens of epigenetic features. This histogram shows the number of epigenetic marks overlapping HARs (71). Less than 20% of HARs (134/713) overlap no peaks, and the top 10% of HARs overlap more than 40 peaks each. This analysis focuses on 5% irreproducible discovery rate peak calls from primary tissues. Including peaks from cell lines and/or less conservative peak calls would increase the number of overlaps.

Figure 3

Deep learning analysis of human variants in human accelerated regions (HARs). All single-nucleotide polymorphisms (SNPs) included on the SNP Database (dbSNP) that overlap with a HAR were scored for their effects on tissue-specific enhancer state predictions using the model Sei (9). This analysis includes all SNPs in all HARs tested in three massively parallel reporter assay (MPRA) studies (22, 66, 71). (a) Increases (red) and decreases (blue) in predicted enhancer state (rows) for all SNPs (columns). (b) Distribution of effects in panel a. Many SNPs in HARs have effect sizes greater than those of known human disease variants [vertical dashed lines represent the median of all SNPs in the Human Gene Mutation Database (HGMD), as reported in Reference 9]. (c) Example of a SNP (rs1325354597) in HARsv2_2635 (22) where the minor allele is predicted to substantially decrease the brain enhancer state and the CTCF state. This variant overlaps an annotated candidate regulatory element (ENCODE cCRE) and motifs of CTCF and NR2F2 as well as other neurological transcription factors (8). The SNP deletes an important nucleotide (T) in the CTCF motif. Consistent with CTCF’s role in loop extrusion, this genomic element has a significant chromatin loop with the promoter of the transcription factor NEUROD6 in cells carrying the major allele (63).

Figure 4

Comparing enhancer activity between human and chimpanzee human accelerated region (HAR) sequences. Massively parallel reporter assay studies involve cloning HAR sequences into reporter vectors along with barcodes that uniquely identify each tested sequence. These vectors are inserted into cell lines, such as neural progenitor cells, using molecular tools such as lentiviruses. They randomly insert into the cell line’s genome. HAR enhancer activity is measured with RNA sequencing of the transcribed barcodes. By associating each tested sequence with many barcodes, activity can be averaged across genomic integration points, providing a robust measurement.

Figure 5

Massively parallel reporter assay (MPRA) studies converge on some of the same active and differentially active human accelerated regions (HARs). The 441 HARs that have been tested in three MPRA studies were compared for consistency of results [Uebbing et al. (66), Girskis et al. (22), Whalen et al. (71)]. (a) Counts of HARs that were active in one, two, or all three studies. (b) Counts of HARs where the human and chimpanzee alleles were differentially active in one, two, or all three studies.