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Epigenomic annotation of gene regulatory alterations during evolution of the primate brain

Nature Neuroscience volume 19, pages 494–503 (2016)Cite this article

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Abstract

Although genome sequencing has identified numerous noncoding alterations between primate species, which of those are regulatory and potentially relevant to the evolution of the human brain is unclear. Here we annotated cis-regulatory elements (CREs) in the human, rhesus macaque and chimpanzee genomes using chromatin immunoprecipitation followed by sequencing (ChIP-seq) in different anatomical regions of the adult brain. We found high similarity in the genomic positioning of rhesus macaque and human CREs, suggesting that the majority of these elements were already present in a common ancestor 25 million years ago. Most of the observed regulatory changes between humans and rhesus macaques occurred before the ancestral separation of humans and chimpanzees, leaving a modest set of regulatory elements with predicted human specificity. Our data refine previous predictions and hypotheses on the consequences of genomic changes between primate species and allow the identification of regulatory alterations relevant to the evolution of the brain.

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Figure 1: Annotation of active cis-regulatory elements in the rhesus macaque and chimpanzee brain.
Figure 2: Comparison of histone enrichment at human brain enhancers and promoters.
Figure 3: Differences in H3K27ac enrichment between human and rhesus macaque.
Figure 4: Changes in chromatin enrichment occurred primarily before the divergence of human and chimpanzee.
Figure 5: Newly introduced and specifically depleted CREs in human.
Figure 6: Functional alterations at accelerated DNA during primate evolution.

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Acknowledgements

We thank S. Levine at the MIT BioMicro Center for managing the Solexa pipeline. This work was funded by the Dutch Royal Academy of Sciences (KNAW), the Dutch Cancer Foundation (KWF) grant HUBR2012-5392 (M.P.C.) and the Parkinson's foundation (Stichting ParkinsonFonds) (M.P.C.).

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Authors and Affiliations

  1. Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands

    Marit W Vermunt, Sander C Tan, Bas Castelijns, Geert Geeven, Peter Reinink, Ewart de Bruijn, Edwin Cuppen, Wouter de Laat & Menno P Creyghton

  2. Biomedical Primate Research Center, Rijswijk, the Netherlands

    Ivanela Kondova, Stephan Persengiev & Ronald Bontrop

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  1. Marit W Vermunt
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Netherlands Brain Bank

Contributions

M.W.V. and M.P.C. conceived and designed the experiments. N.B.B. contributed human brain hemispheres. R.B., I.K. and S.P. provided nonhuman primate material from rhesus macaque and chimpanzee. M.W.V., B.C. and P.R. performed the experiments. M.W.V. and S.C.T. analyzed the data, and were supervised by M.P.C. G.G. analyzed 4C experiments, and was supervised by W.d.L. Sequencing at the Utrecht Sequencing Facility was performed by E.d.B., supervised by E.C. M.W.V. and M.P.C. wrote the manuscript.

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Correspondence to Menno P Creyghton.

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Netherlands Institute for Neuroscience, Amsterdam, the Netherlands.

Integrated supplementary information

Supplementary Figure 1 Annotation of active cis-regulatory elements in distinct brain regions.

(a) Schematic of the eight brain regions that were analyzed. Prefrontal Cortex (PFC), Precentral Gyrus (PcGm), Occipital Pole (OP), White Matter (WM), Caudate Nucleus (CN), Thalamic Nuclei (TN), Putamen (Put) and Cerebellum (CB). (b) Anatomical location of the eight regions that were sampled from two chimpanzee brains and three rhesus macaque brains. (c) Hierarchical clustering of 24 human brain samples based on normalized H3K27ac enrichment of 59,786 predicted human CREs. Color bar indicates main anatomical subdivisions. (d) PCA of 24 human brain samples on normalized H3K27ac enrichment of 59,786 human CREs.

Supplementary Figure 2 Comparison of H3K27ac enrichment in the human cortex with previously defined enhancer and promoter states.

In the upper panel, the pie chart illustrates the fraction of overlap between H3K27ac-enriched regions in the cortex and enhancer and/or promoter states defined in the dorsolateral prefrontal cortex. Bar chart further specifies exact categories that comprise more than 10%. The lower panel depicts the fraction of different chromatin states in the dorsolateral prefrontal cortex24 that is enriched for H3K27ac.

Supplementary Figure 3 Characterization of putative CREs in the brain.

(a) Selection strategy for human and rhesus macaque CREs with orthologues of comparable mappability on all three primate genomes. (b) Size distribution of all CREs in human and rhesus macaque. (c) Ratio of rhesus and human CRE length. The fraction that does not differ more than 25% in size between both species is indicated. (d) Enrichment analysis for genes within indicated modules for linked brain enhancers. (e) Percentage of genes within modules of a given tissue33 as indicated below the panels, that are linked by proximity to cortical or cerebellar brain-specific enhancers, as compared to 13 unrelated tissues. Fisher’s exact test was used to compare enrichment for brain enhancers between modules; *** P < 0.0005. (f) Venn diagram showing the overlap of putative enhancers between the main anatomical subdivisions of the brain. Brain-specific enhancers per anatomical subdivision were analyzed for linked gene ontologies using GREAT.

Supplementary Figure 4 Positional conservation of enhancers and promoters in the brain across primate evolution.

(a) Upper panels show the fraction of enhancers and promoters in cerebellum (CB) and prefrontal cortex (PFC) that is enriched, as defined by peak calling, in both human and rhesus macaque (blue box) or one species only (red box). Species-specific CREs are further subdivided using DESeq2 into significantly differentially enriched (DE higher in human, purple; DE lower in human, light brown) and not DE (grey). Lower panels show normalized RNA read counts, indicated for human and rhesus macaque by silhouettes, for genes close to DE and not DE enhancers. Dissimilarity between distributions was calculated using a Wilcoxon rank-sum test; *** P < 0.0005, ** P < 0.005, * P < 0.05, # P > 0.05. (b) Fraction of enhancers (left) and promoters (right) for each brain region that is positionally conserved between human and rhesus macaque (blue and grey). (c) Average PhastCons scores for all nucleotides within the indicated elements. (d) Fraction of reads within CREs that was mappable onto the other genome for (not) positionally conserved CREs. (e) Upper panels show the fraction of enhancers and promoters in CB and PFC that is enriched, as defined by peak calling, in both human and rhesus macaque (blue box) or one species only (red box). Shared CREs (blue) were further subdivided into DE, higher in human (purple), DE, lower in human (light brown) and not DE (grey) using DESeq2. Lower panels show normalized RNA read counts, indicated for human and rhesus macaque by the silhouettes, for genes close to (not) differentially enriched enhancers. Dissimilarity between distributions was calculated using a Wilcoxon rank-sum test; *** P < 0.0005, * P < 0.05, # P > 0.05.

Supplementary Figure 5 Differential H3K27ac enrichment corresponds to changes in gene expression.

(a) Heatmaps and boxplots for the eight brain regions illustrate (the ratio of) average read counts (normalized for number of reads in peaks) of replicates in human and rhesus macaque. Upper panels in purple depict DE enhancers, higher in human. Middle panels in light brown are DE enhancers, lower in human. Lower panels in grey represent enhancers that are not DE. (b) Percentage of human promoters per brain region that is (not) DE between human and rhesus macaque as defined by DESeq2 (≥ twofold change, FDR < 0.01). The number of analyzed promoters per subdivision is indicated in the color bar below the graph. (c) Boxplot showing the comparison of normalized RNA read counts from human and rhesus macaque brain samples as indicated by the silhouettes. Displayed are the closest genes to a (not) DE enhancer in the prefrontal cortex (PFC). Dissimilarity between distributions was calculated using a Wilcoxon rank-sum test; # P > 0.05. (d) Comparison of normalized RNA read counts for genes with (no) differential H3K27ac enrichment at their promoters (shown for CB and PFC). Dissimilarity between distributions was calculated using a Wilcoxon rank-sum test; *** P < 0.0005, ** P < 0.005, * P < 0.05, # P > 0.05. (e) Ratio of enhancer length between rhesus macaque and human for (not) DE enhancers. (f) Left panels show a RPM normalized ChIP-seq read distribution (axis limit 5 RPM) for H3K27ac at a genomic region spanning 115kb containing the DFNB31 gene. Eight tracks represent the different anatomical brain regions that were analyzed in both human (HS1) and rhesus macaque (RM1) as indicated by the silhouettes. Blue shade highlights the repurposed enhancer and promoter. The right panel displays RPKM normalized RNA sequencing reads for DFNB31 relative to the human cortex.

Supplementary Figure 6 Changes in active chromatin at predicted CREs occurred mainly before the human-chimpanzee separation.

(a) t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis for all human, chimpanzee and rhesus macaque brain samples using normalized H3K27ac enrichment at 60,702 predicted human and rhesus CREs with orthologues on all three genomes. (b) Upper panels show scaled H3K27ac enrichment of (not) DE enhancers as indicated between human and rhesus macaque in prefrontal cortex (PFC). In purple, DE enhancers, higher in human. In light brown, DE enhancers, lower in human. In grey, not DE enhancers. Lower panels display normalized RNA read counts from human, rhesus macaque and chimpanzee brain samples for genes close to (not) DE enhancers. Dissimilarity between distributions was calculated using a Wilcoxon rank-sum test; * P < 0.05, # P > 0.05. (c) Fraction of (not) DE enhancers in cortical and subcortical samples defined between human and rhesus macaque with a twofold difference in normalized read counts (mean of replicates) between human and the indicated species.

Supplementary Figure 7 Newly introduced and specifically depleted CREs in humans.

(a) RPM normalized ChIP-seq read distribution (axis limit 5 RPM) for H3K27ac at a genomic region spanning ~173kb. Eight tracks represent the different anatomical brain regions that were analyzed in HS1, Ch1 and RM1 as indicated by the silhouettes. Colored boxes highlight CREs that are significantly higher in human (purple, 3 putative enhancers and 1 promoter), significantly lower in human (light brown, 1 enhancer) and gained in great apes (yellow, 1 enhancer in the subcortical structures). (b) Same analysis as (a) for a human-specific depletions. (c) Left graph shows distributions of human enhancers over the subdivisions of the brain. Fractions of shared enhancers between subdivisions are plotted in diagonal stripes that are colored according to the different subdivisions. This is shown for all predicted human enhancers and for newly introduced enhancers in comparison to rhesus macaque (random sampling, P < 0.001). Right graph shows fraction of new enhancers per (shared) anatomical subdivision of the original set of all enhancers. (d) Similar analysis as in (c) for human-specific depletions.

Supplementary Figure 8 Chromosome conformation capture for a CRE at accelerated DNA.

In green, enrichment for H3K27ac in the human cerebellum for a genomic region containing CADM1. In blue, circular chromosome conformation capture (4C-seq) using the genomic region containing HAR87 as a viewpoint (exact location indicated by green arrow) in human cerebellum. The second CADM1 enhancer is illustrated with a green arrow. Black boxes represent genes. Interaction peaks with both the second CADM1 enhancer and the CADM1 promoter are indicated using a red arrow.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1743 kb)

Supplementary Methods Checklist (PDF 1657 kb)

Supplementary Table 1

Background information on the brain donors used in this study. (XLSX 29 kb)

Supplementary Table 2

GEO Sample IDs, anatomical location, alignment summary, number of called peaks, FRiP score and read length for each sample. (XLSX 26 kb)

Supplementary Table 3

List of the H3K27ac-enriched regions in human. (XLSX 3110 kb)

Supplementary Table 4

List of the H3K27ac-enriched regions in chimpanzee. (XLSX 3053 kb)

Supplementary Table 5

List of the H3K27ac-enriched regions in rhesus macaque. (XLSX 3202 kb)

Supplementary Table 6

List of the 60702 human and rhesus macaque CREs with mappability on the three genomes. (XLSX 6261 kb)

Supplementary Table 7

Differentially enriched CREs between human and rhesus macaque. Enhancers higher in human. (XLSX 1253 kb)

Supplementary Table 8

Differentially enriched CREs between human and rhesus macaque. Enhancers lower in human. (XLSX 1071 kb)

Supplementary Table 9

Differentially enriched CREs between human and rhesus macaque. Promoters higher in human. (XLSX 184 kb)

Supplementary Table 10

Differentially enriched CREs between human and rhesus macaque. Promoters lower in human. (XLSX 96 kb)

Supplementary Table 11

Gene Ontology output for genes close to a DE higher and DE lower in human enhancer. (XLSX 13 kb)

Supplementary Table 12

Differentially enriched CREs between human and chimpanzee. Enhancers higher in human. (XLSX 277 kb)

Supplementary Table 13

Differentially enriched CREs between human and chimpanzee. Enhancers lower in human. (XLSX 275 kb)

Supplementary Table 14

Differentially enriched CREs between human and chimpanzee. Promoters higher in human. (XLSX 33 kb)

Supplementary Table 15

Differentially enriched CREs between human and chimpanzee. Promoters lower in human. (XLSX 28 kb)

Supplementary Table 16

Gene Ontology output for genes close to DE enhancers of human versus rhesus and chimpanzee. (XLSX 14 kb)

Supplementary Table 17

List of newly enriched enhancers in human. (XLSX 109 kb)

Supplementary Table 18

List of depleted enhancers in human. (XLSX 97 kb)

Supplementary Table 19

List of newly enriched promoters in human. (XLSX 13 kb)

Supplementary Table 20

List of depleted promoters in human. (XLSX 12 kb)

Supplementary Table 21

List of CREs that overlap a human accelerated region, with specification of whether the region is differentially enriched. (XLSX 55 kb)

Supplementary Data 1

This file provides raw read counts for all CREs per species. (ZIP 8662 kb)

Supplementary Data 2

This file provides raw and normalized read counts for the 60702 CREs with comparable sequence content in the three primate genomes. (ZIP 34430 kb)

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Vermunt, M., Tan, S., Castelijns, B. et al. Epigenomic annotation of gene regulatory alterations during evolution of the primate brain. Nat Neurosci 19, 494–503 (2016). https://doi.org/10.1038/nn.4229

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