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Activity-dependent regulome of human GABAergic neurons reveals new patterns of gene regulation and neurological disease heritability

Nature Neuroscience volume 24, pages 437–448 (2021)Cite this article

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Abstract

Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.

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Fig. 1: hGNs express RNA signatures of developing forebrain GNs.
Fig. 2: Neuronal activity-dependent gene expression of hGNs by total RNAseq.
Fig. 3: Neuronal activity-dependent gene expression of hGNs by scRNAseq.
Fig. 4: hGN activity-dependent promoters and enhancers.
Fig. 5: Genome-wide binding of the activity-dependent CREB complex.
Fig. 6: Disease heritability enrichment in hGN promoter and enhancer regions.

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Data availability

The sequencing data that support the findings of this study are available under Gene Expression Omnibus accession number GSE136656. Publicly available datasets include ENSEMBL’s Comparative Genomics Tool (https://www.ensembl.org/info/genome/compara/index.html), GenTree (http://gentree.ioz.ac.cn/index.php), HomoloGene (https://www.ncbi.nlm.nih.gov/homologene), BrainSpan atlas (http://www.brainspan.org/static/download.html), UCSC Genome Browser (https://genome.ucsc.edu/), SFARI ASD-associated genes 11-21-2018 release (https://gene-archive.sfari.org/) and the Schizophrenia Working Group of the Psychiatric Genomics Consortium (https://www.med.unc.edu/pgc/). Source data are provided with this paper.

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Acknowledgements

We thank M. Chahrour, J. L. Hecht, J. Partlow and C. A. Walsh for assistance with human tissue collection; C. C. Harwell, C. Mayer and M. T. Garcia for guidance on single-cell sequencing and cell-type identification; K. D. Hansen and A. P. Feinberg for providing additional LDSC brain-associated control category annotations; and A. N. Chang, E. E. Duffy, G. Fishell, D. Malhotra and D. Reich for helpful discussions. We acknowledge the large body of previous work that informed this study and regret omission of relevant citations due to space constraints. This work was supported by the National Institues of Health: P50MH106933 and R01NS028829 (to M.E.G.), F32NS086270 (to G.L.B.) and T32GM007753 (to E.D.); the ROADS Program funded by F. Hoffmann-La Roche (to M.E.G); and the Paul G. Allen Frontiers Group (to M.E.G). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

Author information

Author notes

  1. Ershela Durresi

    Present address: Department of Internal Medicine, NYU Langone Medical Center and NYU School of Medicine, New York, NY, USA

  2. These authors contributed equally: Gabriella L. Boulting, Ershela Durresi, Bulent Ataman, Maxwell A. Sherman.

Authors and Affiliations

  1. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Gabriella L. Boulting, Ershela Durresi, Bulent Ataman, Kevin Mei, David A. Harmin, Ava C. Carter, Daniel R. Hochbaum, Adam J. Granger, Sinisa Hrvatin, Michael R. Blanchard, Marty G. Yang, Eric C. Griffith & Michael E. Greenberg

  2. MD-PhD Program, Harvard Medical School, Boston, MA, USA

    Ershela Durresi

  3. Division of Genetics & Center for Data Sciences, Brigham and Women’s Hospital, Boston, MA, USA

    Maxwell A. Sherman

  4. Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA, USA

    Maxwell A. Sherman

  5. Department of Genetics, Stanford University School of Medicine and BASE Initiative, Betty Irene Moore Children’s Heart Center, Lucile Packard Children’s Hospital, Stanford, CA, USA

    Jesse M. Engreitz

  6. Neurobiology Imaging Facility, Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    Michael R. Blanchard

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Contributions

G.L.B., E.D., B.A. and M.E.G. conceived of the experiments. G.L.B., E.D., A.C.C., D.A.H., B.A. and K.M. performed total RNAseq. G.L.B., K.M., D.R.H. and S.H. performed scRNAseq. E.D. performed 1G ChIPseq and initial disease enrichment analysis. G.L.B. and A.C.C. performed 4G ChIPseq. G.L.B., A.C.C. and K.M. performed ATACseq. M.A.S. performed LDSC heritability enrichment analysis. K.M. performed luciferase assays, TaqMan assays and statistical analyses. B.A. performed evolutionary conservation analysis. G.L.B. and B.A. performed qPCR and immunocytochemistry with assistance from M.R.B. D.A.H. performed human BrainSpan data visualizations and correlations and statistical analyses. A.J.G. performed electrophysiology. J.M.E. performed ABC modeling. M.G.Y. assisted with IVRL comparisons. G.L.B. performed, directed or assisted with all experiments and analyses. G.L.B., M.E.G. and E.C.G. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Gabriella L. Boulting or Michael E. Greenberg.

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Peer review information Nature Neuroscience thanks Andrew Jaffe and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 The gene expression profile of hGNs is similar to that of developing human telencephalic GABAergic neurons.

a. Image from DIV14 1G hGN culture immunostained for neural progenitor marker protein NESTIN, post-mitotic neuronal marker protein MAP2 showing the majority of hGN cells expressing MAP2 and very few express NESTIN. Representative image of 4 independent experiments. b. Whole-cell patch clamp measurements of DIV14-15 hGN cells reveals immature neuronal firing and membrane properties. Frequency and amplitude of the spontaneous inhibitory post-synaptic currents (sIPSCs), which confirm that hGNs form synapses in these culture conditions. c. mRNA expression levels of regional and developmental marker genes in unstimulated 1G and 4 G hGN cultures by total RNAseq (1G: n = 6; 4G: n = 4; both: box center = mean; box minima/maxima = mean±1 SE; whiskers minima/maxima = mean±1 SD). Adapted from Straccia M., et al., 2015. d. DIV14 4G hGN cultures immunostained for NESTIN and MAP2. The majority of hGN cells express MAP2 and very few express NESTIN in hGNs from genotypes 1434 and 1501. No NESTIN-positive cells were detected in hGN cultures from genotypes 1505 and CW20049. For 1434: representative image of 4 independent experiments. For other three genotypes: images from one independent experiment.

Extended Data Fig. 2 Single-cell RNA-sequencing of hGNs reveals developing ventral forebrain cell types.

a. – f. UMAP visualizations of the entire scRNAseq data set with individual cells colored purple if expression of a given marker gene was detected. g. – j. Brainspan human developmental brain expression data for marker genes within different brain regions. Loess curves interpolated through the means of available data at each age, with error bands on either side interpolated through values, at each age, 1.96 standard errors above or below the mean.

Extended Data Fig. 3 Activity-dependent gene expression patterns detected by total RNAseq.

a. mRNA expression level changes measured by total RNAseq of 4 G hGNs at 15 minutes (n = 4), 1 hour (n = 3), 2 hours (n = 4), and 4 hours (n = 4) after membrane depolarization compared to expression in unstimulated cultures (n = 4) represented by MA-plot. Genes with a significantly different gene expression level and a minimum fold-change magnitude of 1.5 after depolarization are marked in red. Genes having inducible expression fall above y=0 and example gene names are labeled. b. Time courses of previously known activity-inducible gene transcript levels in unstimulated and stimulated 1 G hGNs. Means±s.e.m. Timepoints at which the transcript was significantly induced compared to unstimulated cultures are marked with an asterisk. c. qPCR Fold-induction of mRNA transcripts in 1 G hGNs cultures of genes detected to be inducible by total and single cell RNAseq. Sensitivity limitations of qPCR prevent detection of significant differences when a gene’s expression is modestly induced in a minority of the cells (for example MMP1 and SHANK3). d. The percentage of 1 G and 4 G hGN inducible genes whose gene products have known subcellular localizations. Early inducible genes (15 min. - 1 hr.) have a greater percentage of nuclear localized products than gene transcripts induced at later stimulation timepoints, when the percentage of induced genes in the membrane-bound or secreted category increases.

Extended Data Fig. 4 Activity-dependent gene expression patterns detected by single-cell RNAseq.

a. One biological replicate (rep1) of unstimulated (0 h), 1 hour, and 2 hour membrane depolarized hGN cultures, and two biological replicates (rep2 and 3) of unstimulated, 1 hour, 2 hour, and 4 hour membrane depolarized hGN cultures contributed to all cell clusters identified by single cell RNA sequencing. Replicate – timepoint contribution is calculated for each cluster in the table below b. Selected genes previously known to have inducible expression are also inducible in hGNs after membrane depolarization, as measured by single-cell RNAseq. This includes genes with induction in many hGN cell types (for example FOS, LINC00473, NPAS4) and genes showing hGN cell type-restricted induction (for example BDNF, NPTX2). MMP1 expression was identified to be activity-inducible in this study by total RNAseq, and single-cell RNAseq shows that it is only induced in cluster 9 striatal-like neurons of hGN cultures. The magnitude of fold-change of mRNA levels in each cell cluster at different timepoints after membrane depolarization compared to unstimulated mRNA levels is indicated by dot color. The percentage of cells in each cluster from which expression was detected is represented by the size of the dot. Only timepoints with significantly induced mRNA levels (compared to unstimulated) are shown.

Extended Data Fig. 5 hGN activity-dependent enhancers and promoters.

hGNs from four independent iPSC donors (4G) were cultured in parallel and H3K27ac ChIPseq was performed in unstimulated cultures, at 15 minutes, and two hours after membrane depolarization for each of them in duplicate (technical replicates). Using each of the four genotypes as biological replicates, differential H3K27ac enrichment analysis was performed to identify promoters and enhancers that underwent histone modification changes in response to neuronal depolarization. a. MA-plots representing all 4G H3K27ac peaks identified with those in red having increased significantly in H3K27ac and those in blue having decreased significantly at 15 minutes or 2 hours. b. The total number of reproducible 4G H3K27ac peaks that were called across the four genotypes of hGNs and the subset that were significantly inducible. As with the 1G H3K27ac peaks, 4G H3K27ac peaks are highly represented in the in vivo reference list, and most inducible peaks were inducible at either 15 min or 2 hours but not at both time points. c. Proportions of H3K27ac peaks that intersect a TSS (promoter regions) or do not intersect a TSS (enhancer regions). As with the 1G H3K27ac peaks, 4G 15 minute inducible H3K27ac peaks are significantly enriched for promoter regions. d. As with the 1G H3K27ac peaks, the CREB binding sequence is the most enriched sequence motif within 15 min 4G inducible H3K27ac regions, and the AP-1 motif is the most enriched within 2 hour 4G inducible H3K27ac regions. e. 1G inducible H3K27ac promoter and enhancer regions were cloned into the pGL4.11 (promoter regions) or NUED2 (enhancer regions) plasmids and transfected into embryonic mouse cortical cultures. Luciferase assays were performed to test for these sequences’ ability to drive luciferase reporter gene expression in response to membrane depolarization. 15/15 constructs that show significant reporter expression induction compared to the corresponding empty control plasmid condition are indicated with an asterisk. n = 2–10 independent replicates (neuronal dissection, plasmid transfection, and luminometer reading); box and whisker plot showing median and interquartile range. f. Pearson correlations showing similar levels of H3K27ac induction between hGNs from independent genetic donors of the 4G data set. g. Hierarchical clustering of H3K27ac induction across all 4G samples.

Extended Data Fig. 6 Detection of CREB, pCREB, and CRTC1.

a. 15 minutes after membrane depolarization is the timepoint at which the greatest number of hGNs immunostain positive using the phosphorylated ser133 CREB antibody used for ChIPseq. Median and interquartile range of counts from six images for each condition from a single immunostaining event of one differentiation lot of 1G neurons. b. Western blot for CRTC1 and GAPDH from hGN cell lysates after 6 or 9 days of non-targeting (NT) or CRTC1-targeting siRNA blotted with the antibody that was used for CRTC1 ChIPseq. Blot displays the two independent experiments performed for this quantification. 9 day treatments were used for siRNA treatments in this study, which resulted in an average 64.1% protein knockdown. c. Western blot of hGN cell lysates using the CREB antibody used for CREB ChIPseq. Image is representative of two independent experiments. d. Immunostaining for CRTC1 in hGN cultures before (unstimulated) and 15 minutes after membrane depolarization. Membrane depolarization causes CRTC1 to translocate to the nucleus. Scale bar = 100 µm. Images are representative of 3 independent experiments.

Extended Data Fig. 7 Disease heritability enrichment in hGN promoter and enhancer regions.

a. Heritability enrichments (top) and p-values of heritability enrichment (bottom) of the 1G hGN H3K27ac promoter and enhancer regions across neurological and non-neurological disorders. b. p-values of heritability enrichment of 4G hGN H3K27ac promoter and enhancer regions across neurological and non-neurological disorders. c. Heritability enrichments (left) and p-values of heritability enrichment (right) of the inducible and constitutive H3K27ac enhancer regions across neurological diseases for both 4G (top) and 1G (bottom), showing significant enrichment for only constitutive enhancer regions. d. p-values of heritability enrichment of 4G (top) hGN inducible and constitutive enhancers (central 500 bp of 4G ATACseq peaks within 4G H3K27ac enhancer regions) across neurological disorders, and (bottom) 1G heritability enrichments (left) and p-values of heritability enrichment (right), showing significant enrichment for only constitutive enhancers. e. p-values of heritability enrichment of 4G (left) hGN inducible and constitutive promoters across neurological disorders, and 1G heritability enrichments (center) and p-values of heritability enrichment (right), showing significant ASD and BP heritability enrichment in inducible promoters but not constitutive promoters. f. Heritability enrichment (top) p-values of heritability enrichment (bottom) of the inducible and constitutive 1G (left) and 4G (right) H3K27ac promoter regions across neurological diseases. g. Proportions of each hGN scRNAseq cluster’s inducible gene list that have inducible promoters by 1G H3K27ac ChIPseq. Proportions are calculated at multiple gene expression fold-change cut-off thresholds. Cluster 10 (striatal spiny projection neuron-like cells) inducible genes have the highest proportion of SFARI ASD-associated genes, regardless of gene expression fold-change cut-off threshold. h. LDSC heritability enrichments were calculated using summary statistics from two schizophrenia (SCZ) GWASs: Psychiatric Genomics Consortium (PGC), 2014 and Pardiñas, et al., 2018. The latter incorporates 40.5 K cases compared to 37 K in the 2014 PGC study and uses fewer controls (63 K vs 113 K). i. LDSC heritability enrichment calculations were repeated for all neurological disorders in parallel with Alzheimer’s Disease (AD), using the summary statistics from Lambert, J., Ibrahim-Verbaas, C., Harold, D. et al., Nature Genetics, 2013. Please note that the GWASs for the other neurological disorders included in our analyses were uniformly processed by the PGC whereas the AD GWAS was processed differently, which may contribute to the relatively large error bars associated with the AD analysis. All LDSC Plots are color coded according to Fig. 6a. Each heritability enrichment value is provided in bar plot (+/- std. error). p-values of heritability enrichment are multi-test corrected and heritability enrichment bars with p = or > 0.05 are cross-hatched. Exact p-values are provided in Supplementary Data 14.

Extended Data Fig. 8 ABC model predictions of inducible enhancer-gene associations.

a. The number of inducible genes and H3K27ac enhancer regions, as defined by this study (1G), predicted to interact by the Activity-by-Contact (ABC) model described in Fulco et al., Nat. Genetics, 2019. b. An example of one ASD-associated and neuronal activity-dependent gene locus, KCNQ3, as visualized in the IGV genome browser, with chromatin data presented in this resource (1G tracks below) and ABC model predictions of enhancer associations with the KCNQ3 promoter (arcs above). These include enhancer associations that remain constitutively predicted across all stimulation conditions (purple), those that are potentially transient but not necessarily activity-dependent (yellow), and those that are likely to be activity-dependent (red). In this case, at 15 minutes and 2 hours after neuronal depolarization, the KCNQ3 promoter is predicted to interact with an activity-dependent intronic enhancer (shaded), which increases in H3K27ac enrichment and becomes associated with the activated CREB-complex after depolarization. See Supplementary Data 13 for the full list of ABC model predicted enhancer-gene associations.

Supplementary information

Supplementary Information

Supplementary Tables 1–3.

Reporting Summary

Supplementary Data 1–5 and 7–10

Differential gene expression levels for depolarization-inducible and decreased hGN genes. Each tab describes different subsets of genes.

Supplementary Data 6

All differential gene expression output tables from Monocle analysis of hGN scRNAseq data.

Supplementary Data 11 and 12

Differential enrichment output tables from EdgeR and DESeq2 analysis of hGN H3K27ac ChIPseq data

Supplementary Data 13

Activity-by-contact (ABC) model predictions of 1G enhancer–gene interactions associated with Extended Data Fig. 8.

Supplementary Data 14

LDSC P values associated with Fig. 6 and Extended Data Fig. 7.

Source data

Source Data Fig. 1

Unprocessed western blot images.

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Boulting, G.L., Durresi, E., Ataman, B. et al. Activity-dependent regulome of human GABAergic neurons reveals new patterns of gene regulation and neurological disease heritability. Nat Neurosci 24, 437–448 (2021). https://doi.org/10.1038/s41593-020-00786-1

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