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Comparative characterization of human accelerated regions in neurons

Nature volume 640, pages 991–999 (2025)Cite this article

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Abstract

Human accelerated regions (HARs) are conserved genomic loci that have experienced rapid nucleotide substitutions following the divergence from chimpanzees1,2. HARs are enriched in candidate regulatory regions near neurodevelopmental genes, suggesting their roles in gene regulation3. However, their target genes and functional contributions to human brain development remain largely uncharacterized. Here we elucidate the cis-regulatory functions of HARs in human and chimpanzee induced pluripotent stem (iPS) cell-induced excitatory neurons. Using genomic4 and chromatin looping information, we prioritized 20 HARs and their chimpanzee orthologues for functional characterization via single-cell CRISPR interference, and demonstrated their species-specific gene regulatory functions. Our findings reveal diverse functional outcomes of HAR-mediated cis-regulation in human neurons, including attenuated NPAS3 expression by altering the binding affinities of multiple transcription factors in HAR202 and maintaining iPS cell pluripotency and neuronal differentiation capacities through the upregulation of PUM2 by 2xHAR.319. Finally, we used prime editing to demonstrate differential enhancer activity caused by several HAR26;2xHAR.178 variants. In particular, we link one variant in HAR26;2xHAR.178 to elevated SOCS2 expression and increased neurite outgrowth in human neurons. Thus, our study sheds new light on the endogenous gene regulatory functions of HARs and their potential contribution to human brain evolution.

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Fig. 1: HARs exhibit enhancer function in excitatory neurons.
Fig. 2: Single-cell CRISPRi screens of HARs and chimpanzee orthologues for enhancer function.
Fig. 3: Loss of HAR202 enhancer function in human neurons.
Fig. 4: Enhancer function of 2xHAR.319 in the most accelerated gene desert region.
Fig. 5: HAR26;2xHAR.178 upregulates SOCS2 and enhanced neuritogenesis.

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Data availability

All datasets generated in this study (bulk RNA-seq, scRNA-seq, ATAC-seq, PLAC-seq and HyPR-seq) are available under Gene Expression Omnibus accession number GSE222113. Data can be visualized on the WashU Epigenome Browser using the following links: human (hg38; https://epigenomegateway.wustl.edu/browser/?sessionFile=https://shen-hany.s3.us-west-1.amazonaws.com/HAR_projects/Human_eg-session-SbtxhBd3Z-83c4d960-21e8-11ef-967c-b9e3f9c72d6a.json) and chimpanzee (panTro5; https://epigenomegateway.wustl.edu/browser/?sessionFile=https://shen-hany.s3.us-west-1.amazonaws.com/HAR_projects/Chimpeg-session-XqBlEkcGJ-d9a56840-21e8-11ef-9160-97ad240892ee.json).

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Acknowledgements

We thank Y. Gilad from the University of Chicago for sharing the wild-type chimpanzee C3649 and C3624 iPS cell lines; C. Karch from Washington University in St. Louis for sharing the human F12468 iPS cell line with the Ngn2 transgene; L. Gan from Cornell University for sharing the human WTC11 iPS cell line with the Ngn2 transgene; and G. Wray from Duke University for sharing the chimpanzee C3649 phased genome data. This work was supported by the US National Institutes of Health (NIH) grants U01DA052713 (to Y.S.), UM1HG009402 (to Y.S.), R21DA056293 (to Y.S.), R21HG010065 (to Y.S.), R01MH109907 (to K.S.P.), U01MH116438 (to K.S.P.) and DP2MH122400-01 (to A.A.P.), and the Schmidt Futures Foundation (to A.A.P.). H.Y. was supported by the Larry L. Hillblom Foundation Postdoctoral Fellowship. K.C.K. was supported by the UCSF Discovery Fellowship and Gladstone Institutes. A.A.P. is a New York Stem Cell Foundation Robertson Investigator and member of the Innovative Genomics Institute and the UCSF Kavli Institute for Fundamental Neuroscience. This work was made possible in part by the Center for Advanced Technology at the UCSF. This work was also supported in part by NIH grants P30DK063720 and S101S10OD021822-01 to the UCSF Parnassus Flow Cytometry Core (RRID: SCR_018206).

Author information

Author notes

  1. These authors contributed equally: Xiekui Cui, Han Yang

Authors and Affiliations

  1. Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA

    Xiekui Cui, Han Yang, Charles Cai, Cooper Beaman, Xiaoyu Yang, Hongjiang Liu, Xingjie Ren, Zachary Amador, Ian R. Jones, Zhen Ye, Katherine S. Pollard & Yin Shen

  2. Pharmaceutical Sciences and Pharmacogenomics Graduate Program, University of California, San Francisco, San Francisco, CA, USA

    Ian R. Jones & Kathleen C. Keough

  3. Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA

    Kathleen C. Keough & Katherine S. Pollard

  4. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA

    Meng Zhang

  5. Department of Radiation Oncology, University of California, San Francisco, San Francisco, CA, USA

    Meng Zhang

  6. Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research, Univeristy of California, San Francisco, San Francisco, CA, USA

    Tyler Fair & Alex A. Pollen

  7. Department of Neurology, University of California, San Francisco, San Francisco, CA, USA

    Tyler Fair, Alex A. Pollen & Yin Shen

  8. Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA

    Armen Abnousi, Shreya Mishra & Ming Hu

  9. Department of Epidemiology and Biostatistics and Bakar Institute for Computational Health Sciences, University of California, San Francisco, San Francisco, CA, USA

    Katherine S. Pollard

  10. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Katherine S. Pollard

  11. Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA

    Yin Shen

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  1. Xiekui Cui
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Contributions

Y.S. and X.C. conceived the study. Y.S. supervised the study. X.C. and H.Y. generated the inducible Ngn2 chimpanzee iPS cell lines (C3649 and C3624). X.C., H.Y. and X.Y. generated the human 1 (WTC11), human 2 (F12468) and chimpanzee 1 (C3649) dCas9-KRAB iPS cell lines. T.F. made the chimpanzee 2 (C3624) dCas9-KRAB iPS cell line. X.C. performed PLAC-seq with the assistance of C.C. and I.R.J., bulk RNA-seq with the assistance of H.Y. and Z.Y., and ATAC-seq. X.C. performed HyPR-seq with the support of X.Y. X.C. performed validation using CRISPRi and CRISPR deletion with the assistance of X.R. X.C., K.C.K. and X.R. designed gRNAs for HARs CRISPRi. X.C. and H.Y. carried out prime editing with the assistance of X.R. X.C. performed CRISPRa experiments. X.C. also carried out allelic expression imbalance analysis with the assistance of Z.A., performed neuritogenesis-related morphological quantification assay, conducted ChIP experiments and carried out immunostaining experiments with the assistance of X.Y. H.Y. performed scRNA-seq and analysis. H.Y., C.C., X.Y. and H.L. performed the PLAC-seq, bulk RNA-seq, ATAC-seq and HyPR-seq data analyses under the supervision of Y.S. M.Z. mapped initial RNA-seq and supported the data analysis of the function of PUM2 related to neurogenesis. A.A. and S.M. supported the PLAC-seq and TAD-related data analysis. A.A.P., K.S.P. and M.H. also contributed to the supervision of the experiments. X.C., H.Y. and Y.S. analysed and interpreted the data. Y.S., X.C., H.Y. and C.B. prepared the manuscript with input from all other authors.

Corresponding author

Correspondence to Yin Shen.

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Nature thanks Yijun Ruan, Gabriel Santpere and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Characterizing human and chimpanzee iPSC-induced excitatory neurons.

a, Immunostaining of neuronal markers in excitatory neurons. Bar = 10 μM. b, Comparison of the expression of marker genes for neural lineage, forebrain, midbrain, hindbrain, and pluripotency in human and chimpanzee iPSC-induced neurons. c, Comparison of the expression of marker genes for excitatory neurons in human and chimpanzee iPSC-induced neurons. d, UMAP of scRNA-seq datasets of human and chimpanzee iPSC-induced neurons, color-coded by the cluster or expression levels of the neuronal marker genes (TUBB3 and STMN2).

Extended Data Fig. 2 Differential gene expression analyses between human and chimpanzee neurons.

a, Principal component analyses of the RNA-seq data of human and chimpanzee iPSC-induced neurons mapped to human or chimpanzee assembly. b, Volcano plot displaying differentially expressed genes (FDR < 0.05, Log2(fold change) > 1) between human and chimpanzee neurons mapped to human or chimpanzee assembly. P-values were calculated by DESeq2 based on a model using the negative binomial distribution with FDR adjustment. c, Gene Ontology enriched terms for differentially expressed genes between the two species. P-values of enrichment analyses were conducted with the enrichGO with Benjamini-Hochberg adjustment.

Extended Data Fig. 3 Epigenomic features of human and chimpanzee iPSC-induced neurons.

a, Histograms showing the number of interactions for promoters in human and chimpanzee neurons. b, Cumulative distribution function (CDF) plots of chromatin interaction distances in human and chimpanzee neurons. c, Summary of the percentage of chromatin interactions corresponding to promoters with distal regions (XOR) and promoters with promoters (AND). d, Comparison of shared interactions between human and chimpanzee neurons. e, Bootstrap distribution of shared interaction ratios calculated between shuffled interactions (shuffled 1000 times) and human or chimpanzee interactions. The red dashed line indicates the actual shared interaction ratio between human and chimpanzee interactions. f, Comparison of shared accessible peaks between human and chimpanzee neurons. g, Bootstrap distribution of shared accessible peak ratios calculated between shuffled accessible peaks (shuffled 1000 times) and human or chimpanzee accessible peaks. The red dashed line indicates the actual shared accessible peaks ratio. h, Numbers of accessible peaks overlapping with H3K4me3-associated interactions in human and chimpanzee neurons. i, Bootstrap distribution of shared accessible peaks ratios related to H3K4me3-marked interaction calculated between shuffled accessible peaks (shuffled 1000 times) and human or chimpanzee accessible peaks related to H3K4me3-marked interactions. The red dashed line indicates the actual shared accessible peak ratios related to H3K4me3-associated interactions. j, Bar graph of the proportion of interactions in each study related to HAR sequences. k, Venn diagram shows the overlapping chromatin interactions with HARs between different studies24,25. l, Gene Ontology enriched terms for HAR- and HAR ortholog-interacting genes. P-values of enrichment analyses were conducted with the enrichGO with Benjamini-Hochberg adjustment. m, Linear regression analysis of gene expression associated with HARs and their orthologs in humans and chimpanzees. Coefficient values for both gene groups and OCNSs were calculated based on Meyer et al.55. Group I includes 374 and 377 genes interacting with accessible HARs or HAR orthologs, Group II includes 346 and 274 genes interacting with non-accessible HARs or HAR orthologs, and Group III includes 999 and 974 genes close to HARs or HAR orthologs but without interaction in human and chimpanzee neurons, respectively. n, Human and chimpanzee allele frequencies for 20,020 variants across 3,257 HARs based on the gnomAD database71.

Extended Data Fig. 4 Establishment of dCas9-KRAB lines and targeted single-cell CRISPRi screening of HARs and their chimpanzee orthologs in neurons.

a, Normal karyotyping results of iPSCs with inducible Ngn2 from the AAVS1 locus and stably expressing dCas9-KRAB from the CLYBL safe-harbor locus in human and chimpanzee, respectively. b, qPCR following bulk CRISPRi in human and chimpanzee neurons. Three independent differentiations per condition were used. All values represent the mean ± SD. c, The WashU Comparative Epigenome Browser illustrating the chromatin interaction of HARs or their chimpanzee orthologs in human (hg38, yellow) and chimpanzee (panTro5, blue) neurons, respectively. Two gRNAs targeting HAR26;2xHAR.178 are shown as examples. d, Probes 1-5 targeting the gene PLXNC1 are shown. The process of probe hybridization and the PCR amplification template containing the target gene’s transcript information are shown. e, CDF plots showing the number of cells recovered for each gRNA in single-cell CRISPRi experiments. The mean number of cells recovered per guide is 62 for human neurons and 81 for chimpanzee neurons. f, Heatmap displaying the read count distributions for each gRNA in each cell. Each line on the y-axis represents a single cell. Colors represent the number of cells with each gRNA based on read counts.

Extended Data Fig. 5 Validation of functional HARs and their target genes.

The WashU Comparative Epigenome Browser displaying regions with HARs and their orthologs. Quantitative effects of HAR on a, ARL8B, b, ONECUT2, and c, TBC1D5 expression. P values were calculated based on the two-sided two-sample t-test and adjusted by Benjamini-Hochberg multiple testing correction. The median, upper and lower quantiles are shown by circle and bar. Each dot represents one single cell. n is indicated in Supplementary Table 4d. The results of bulk CRISPRi followed by qPCR in human and chimpanzee neurons from three biological replicates are summarized on the right. P values were calculated based on the two-sided t-test.

Extended Data Fig. 6 Enhancer function of HARs in the most accelerated NPAS3 locus.

a, Left, NPAS3 expression in human and chimpanzee neurons by RT-qPCR assay. Right, NPAS3 expression in human and chimpanzee neurons based on bulk RNA-seq data. n = 8 biological replicates. b, Quantitative effects of HAR96, 2xHAR.142, 2xHAR.122, and HAR144 on NPAS3 expression in human and chimpanzee neurons. P values were calculated based on the two-sided two-sample t-test and adjusted by Benjamini-Hochberg multiple testing correction. The median, upper and lower quantiles are shown by circle and bar. Each dot represents one single cell. n is indicated in Supplementary Table 4d. c, NPAS3 expression following CRISPRi of 2xHAR.142 and its ortholog in human and chimpanzee neurons. d, Characteristics of the transcription factor motifs overlapping with variants in HAR202 versus its chimpanzee ortholog. Colors represent TF enrichment scores based on JASPAR. Strand information and TF motifs related to neurogenesis are shown. h, human; c, chimpanzee. e, Summary of STAT1, 3, 5 A and 5B and BCL6 transcription levels in human and chimpanzee neurons according to bulk RNA-seq. Bar = 10 Kb. f, Anti-BCL6, MEIS2 and PAX5 ChIP-qPCR assay in neurons. P values were calculated based on the two-sided t-test in a, c, and f. Three independent biological replicates per condition were used for each experiment in c and f. All values represent the mean ± SD in a, c, and f.

Extended Data Fig. 7 Prime Editing of HAR202 in human neurons.

a, The genomic locations of HAR202 and an adjacent allelic SNP in NPAS3 intron are shown. The selected region for NPAS3 allelic expression assay is highlighted in gray. b, Multiple alignments of selected vertebrate species and measurements of evolutionary conservation for HAR202. White bars indicate human-specific variants from the ancestral sequence. c, Sanger sequencing verification of the HAR202 prime editing to the chimpanzee orthologous sequence. d, Sanger sequencing verification of the allelic SNP for NPAS3. e, Schematic representation of the cis-regulatory relationship between HAR202 and NPAS3.

Extended Data Fig. 8 Characterization of HAR’s enhancer function in the most accelerated gene desert region.

a, Chromatin accessibilities of 2xHAR.319 and HAR63 in developing human cortex according to ATAC-seq data from Song et al.25. Bar = 1 Kb. b, Quantitative effects of HAR63, 2xHAR.319, 2xHAR.155 and HAR103 on LAPTM4A and RHOB expression in human and chimpanzee neurons. P values were calculated based on the two-sided two-sample t-test and adjusted by Benjamini-Hochberg multiple testing correction. The median, upper and lower quantiles are shown by circle and bar. Each dot represents one single cell. n is indicated in Supplementary Table 4d. c, Sanger sequencing verification of 2xHAR.319, HAR63, and their orthologs’ CRISPR deletion in human and chimpanzee iPSC clones. d, LAPTM4A and RHOB expression upon CRISPR deletion of 2xHAR.319 or HAR63 and their orthologs in neurons. Three individual clones of wildtype and HAR deletion were used. e, Characteristics of TF motifs overlapping with variants in human 2xHAR.319 versus its chimpanzee ortholog. Colors represent enrichment scores based on the P values from JASPAR. Strand information and TF motifs related to neurogenesis are shown. The motifbreakR result for SP1 is shown as an example. f, CRISPRa of PUM2 followed by RT-qPCR in 2xHAR.319 deleted iPSCs. Three independent biological replicates per condition were used. P values were calculated based on the two-sided two-sample t-test in d and f. All values represent the mean ± SD in d and f.

Extended Data Fig. 9 HAR26;2xHAR.178 and candidate regulatory target genes.

a, Interactions of HAR26;2xHAR.178 with four protein-coding genes linked to neuronal functions and psychiatric disorders in humans; their genomic locations, chromatin accessibilities, and transcription levels in human and chimpanzee are also shown. b, Expression of SOCS2, PLXNC1, NDUFA12, and NR2C1 in iPSC-induced neurons. Four independent differentiation per condition were used. P values were calculated based on the two-sided two-sample t-test. All values represent the mean ± SD. c, Brain tissue-level RNA-seq analysis for SOCS2, PLXNC1, NDUFA12, and NR2C1 in human and chimpanzee based on Zhu et al.46. The full names of brain regions are shown on the right. Boxplot indicates the median and interquartile range; whiskers mark the 5th and 95th percentiles, with outliers shown as circles. n is indicated in Zhu et al.46 with 36 human, 5 chimpanzee and 26 matched macaque brain specimens corresponding to fetal to adulthood stages of human. Adapted from ref. 72, Springer Nature Ltd. d, SOCS2 expression in the reconstructed human and primate cerebral organoids47. Left SPRING plots highlight the SOCS2 expression in the NPCs and neurons of different regions in the human, chimpanzee, and macaque organoids. Right SPRING plots demonstrate the expression of SOCS2 in the human, chimpanzee, and macaque 2-4 months organoids.

Extended Data Fig. 10 HAR26;2xHAR.178 regulated genes are linked to neuritogenesis.

a, NDUFA12 and NR2C1 expression after perturbing HAR26;2xHAR.178by CRISPRi in neurons. Three independent biological replicates per condition were used. b, Sanger sequencing verification of chimpanzee HAR26;2xHAR.178 orthologous sequence after prime editing. c, Allelic expression assay for NDUFA12 and NR2C1 in humanized chimpanzee neurons. Three independent replicates from WT and edited clones are shown. d, Sanger sequencing verification of double prime edited HAR26;2xHAR.178 ortholog. The wildtype sequence (panTro5) of chimpanzee HAR26;2xHAR.178 ortholog was shown on top with variant 1-4 labeled. e, RT-qPCR analysis of SOCS2 and NDUFA12 expression in double prime edited chimpanzee neurons. Three independent double-edited chimpanzee clones were used as biological replicates. f, mRNA levels of NR2F1 and TFAP2A in human and chimpanzee neurons. Bar = 1 Kb. g, Knockdown of SOCS2 by CRISPRi (n = 6 replicates of 2 independent differentiations). h, Representative images of immunostaining of neuronal markers TUJ1 in wildtype and PUM2 knockdown neurons. Bar = 50 µM. i, Quantification of neurite numbers in wildtype and SOCS2 knockdown neurons with the Neurite Tracer-Sholl analysis. Data is presented as the average number of processes per neuron that intersect at a given radius centered on the soma (mean ± SEM). n = 15 cells for each sample. j, Biological replication with neurons from another PE1 edited clone for the quantification of neurite numbers. Data is presented for human, humanized chimpanzee, and wildtype chimpanzee neurons, as the average number of processes per neuron that intersect at a given radius centered on the soma (mean ± SEM). n = 15 cells for each sample. k, Representative immunostaining images of GFP-labeling of humanized (PE1, PE1 + PE4, and PE1 + PE2) chimpanzee neurons compared with wildtype chimpanzee and human neurons. Bar = 10 µM. l, Quantification of neurite numbers in humanized chimpanzee, wildtype chimpanzee, and human neurons with the Neurite Tracer-Sholl analysis. Data is presented as the average number of processes per neuron that intersect at a given radius centered on the soma (mean ± SEM; the differences of neurite outgrowth were calculated by comparing: 1) human with wildtype chimpanzee, PE1 chimpanzee with wildtype chimpanzee, PE1 + PE4 chimpanzee with wildtype chimpanzee, PE1 + PE2 chimpanzee with wildtype chimpanzee; 2) PE1 + PE4 chimpanzee with PE1 chimpanzee, PE1 + PE2 chimpanzee with PE1 chimpanzee. Only comparison between wildtype human and wildtype chimpanzee (P = 8.88 × 10−16), PE1 chimpanzee and wildtype chimpanzee (P = 4.18 × 10−11), PE1 + PE2 chimpanzee and wildtype chimpanzee (P = 0.03) exhibited significant differences were labeled. Other comparison groups yielded non-significant differences were not labeled. n = 15 cells for each sample. P values were calculated based on the two-sided two-sample t-test in a, c, e, g, i, j, and l. Boxplot indicates the median and interquartile range; whiskers mark the 5th and 95th percentiles for c. All values represent the mean ± SD in a, e, and g. n.s: not significant.

Supplementary information

Reporting Summary

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Supplementary Table 1

Differentially gene expressed and Gene Ontology analyses between human and chimpanzee iPSC-induced excitatory neurons.

Supplementary Table 2

HAR and their chimpanzee ortholog interacting genes in neurons.

Supplementary Table 3

Prioritized HARs and their putative target genes for single-cell CRISPRi experiments.

Supplementary Table 4

Single-cell CRISPRi and targeted scRNA-seq experiments.

Supplementary Table 5

Transcription factor binding motifs analysis of HARs and HAR orthologs.

Supplementary Table 6

scRNA-seq, bulk RNA-seq, ATAC-seq, and PLAC-seq data processing metrics.

Supplementary Table 7

List of primers and oligos used in this study.

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Cui, X., Yang, H., Cai, C. et al. Comparative characterization of human accelerated regions in neurons. Nature 640, 991–999 (2025). https://doi.org/10.1038/s41586-025-08622-x

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