Three-dimensional genome rewiring in loci with human accelerated regions

[INTRODUCTION] Human accelerated regions (HARs) are evolutionarily conserved sequences that acquired an unexpectedly high number of nucleotide substitutions in the human genome since divergence from our common ancestor with chimpanzees. Prior work has established that many HARs are gene regulatory enhancers that function during embryonic development, particularly in neurodevelopment, and that most HARs show signatures of positive selection. However, the events that caused the sudden change in selective pressures on HARs remain a mystery.[RATIONALE] Because HARs acquired many substitutions in our ancestors after millions of years of extreme constraint across diverse mammals, we reasoned that their conserved roles in regulating development of the brain and other organs must have changed during human evolution. One mechanism that could drive such a functional shift is enhancer hijacking, whereby the target gene repertoire of a noncoding sequence is changed through alterations in three-dimensional genome folding. The regulatory information encoded in a hijacked enhancer would likely need to change to avoid deleterious expression of the altered target gene while also possibly supporting modified expression patterns. Structural variants—large genomic insertions, deletions, and rearrangements—are the greatest sources of sequence differences between the human and chimpanzee genomes, and they have the potential to affect how a region of the genome folds and localizes in the nucleus. We therefore hypothesized that some HARs were generated through enhancer hijacking triggered by nearby human-specific structural variants (hsSVs).[RESULTS] We leveraged an alignment of hundreds of mammalian genomes plus a Nextflow pipeline that we wrote for automating the detection of lineage-specific accelerated regions to identify 312 high-confidence HARs (zooHARs). Through massively parallel reporter assays and machine learning integration of hundreds of epigenomic datasets, we showed that many zooHARs function as neurodevelopmental enhancers and that their human substitutions alter transcription factor binding sites, consistent with previous studies. We further mapped zooHARs to specific cell types and tissues using single-cell open chromatin and gene expression data, and we found that they represent a more diverse set of neurodevelopmental processes than a parallel set of chimpanzee accelerated regions. To test the enhancer hijacking hypothesis, we first examined the three-dimensional neighborhoods of zooHARs using publicly available chromatin capture (Hi-C) data, finding a significant enrichment of zooHARs in domains with hsSVs. This motivated us to use deep learning to predict how hsSVs changed genome folding in the human versus the chimpanzee genomes. We found that 30% of zooHARs occur within 500 kb of an hsSV that substantially alters local chromatin interactions, and we confirmed this association in Hi-C data that we generated in human and chimpanzee neural progenitor cells. Finally, we showed that chromatin domains containing zooHARs and hsSVs are enriched for genes differentially expressed in human versus chimpanzee neurodevelopment.[CONCLUSION] The origin of many HARs may be explained by human-specific structural variants that altered three-dimensional genome folding, causing evolutionarily conserved enhancers to adapt to different target genes and regulatory domains.This study received support from a Discovery Fellowship (K.C.K.), National Institute of Mental Health grants R01MH109907 and U01MH116438 (N.A., K.S.P., and K.C.K.), National Institute of Mental Health grant DP2MH122400-01 (A.P. and T.F.), National Institute of Human Genome Research grant R01HG008742 (E.K.), Gladstone Institutes (K.S.P.), the Schmidt Futures Foundation (A.P. and T.F.), the Shurl and Kay Curci Foundation (A.P. and T.F.), and a Swedish Research Council Distinguished Professor Award (K.L.-T.).Peer reviewe

Identifying and Dissecting Regulatory Elements that Drive Drug Response and Human Evolution

Gene regulation is known to contribute to the wide diversity of biological differences between cell types, individuals, and species. Enhancers are regulatory elements that determine when, where, and how much a protein-coding gene is expressed in every tissue. They contain short motifs called transcription factor binding sites and function through chromatin remodeling and DNA looping to activate transcription of their target genes. Due to their role in activating gene expression across tissues and developmental timepoints, disruption in enhancer function can lead to disease and morphological differences between species. By characterizing enhancers we can learn how genetic changes in non-coding DNA alter gene function and ultimately use this knowledge to diagnose and treat disease.Using RNA-sequencing and chromatin immunoprecipitation (ChIP) sequencing, I identified genome-wide antibiotic-induced changes in gene expression and regulation in HepG2 cells, a human liver cell line. More specifically, I found 209 genes responsive to penicillin-streptomycin (PenStrep), a commonly used cell culture antibiotic cocktail, and 9,514 H3K27ac peaks that were PenStrep-responsive. I also performed a massively parallel reporter assay (MPRA) to quantify enhancer activity of conserved DNA elements that have rapidly evolved in humans called human accelerated regions (HARs) in human and chimpanzee iPSC-derived neural and glial progenitor cells. This method allowed us to detect novel brain enhancers with species-specific function and dissect the regulatory architecture of these enhancers. Our results showed that the cis features or sequence level changes were greater drivers of differences in enhancer activity than the trans environment, or cell species and cell stage, that these sequences were tested in. My research sheds insight on the regulatory code driving drug response to common antibiotics, as well as the uniquely human patterns in early neurodevelopment

Machine learning dissection of human accelerated regions in primate neurodevelopment.

Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 human accelerated regions (HARs), finding 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in chimpanzee and human neural progenitor cells. The species-specific enhancer activity of HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. This suggests that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly

Three-dimensional genome rewiring in loci with human accelerated regions

Human accelerated regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with an automated pipeline and an alignment of 241 mammalian genomes. Combining deep learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs