Abstract
Here, we construct genome-scale maps of R-loops, three-stranded nucleic acid structures comprised of a DNA/RNA hybrid and a displaced single strand of DNA, in both proliferative and differentiated zones of the human prenatal brain. We show that R-loops are abundant in the progenitor-rich germinal matrix and preferentially form at gene promoters slated for upregulated expression at later stages of differentiation, including numerous neurodevelopmental risk genes. RNase H1-mediated contraction of the genomic R-loop space in neural progenitors shifted differentiation toward the neuronal lineage and was associated with transcriptomic alterations, along with defective functional and structural neuronal connectivity in vivo and in vitro. Therefore, we conclude that R-loops are important for fine-tuning differentiation-sensitive gene expression programs of neural progenitor cells.
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Data availability
Data are available in Gene Expression Omnibus (National Library of Medicine) under GEO accession numbers GSE327314 (single cell RNA-seq), GSE324347 (bulk RNA-seq in Supplemental Material) and GSE324290 (hiPSC-NPC-neuron DRIP-seq), GSE326631 (prenatal brain DRIP-seq).
Code availability
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References
Ypsilanti AR, Pattabiraman K, Catta-Preta R, Golonzhka O, Lindtner S, Tang K, et al. Transcriptional network orchestrating regional patterning of cortical progenitors. Proc Natl Acad Sci USA. 2021;118:e2024795118.
Trevino AE, Müller F, Andersen J, Sundaram L, Kathiria A, Shcherbina A, et al. Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution. Cell. 2021;184:5053–69.e23.
Amiri A, Coppola G, Scuderi S, Wu F, Roychowdhury T, Liu F, et al. Transcriptome and epigenome landscape of human cortical development modeled in organoids. Science. 2018;362:eaat6720.
Ginno PA, Lott PL, Christensen HC, Korf I, Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol Cell. 2012;45:814–25.
Sanz LA, Hartono SR, Lim YW, Steyaert S, Rajpurkar A, Ginno PA, et al. Prevalent, dynamic, and conserved R-loop structures associate with specific epigenomic signatures in mammals. Mol Cell. 2016;63:167–78.
Sun Q, Csorba T, Skourti-Stathaki K, Proudfoot NJ, Dean C. R-loop stabilization represses antisense transcription at the arabidopsis FLC locus. Science. 2013;340:619–21.
Boque-Sastre R, Soler M, Oliveira-Mateos C, Portela A, Moutinho C, Sayols S, et al. Head-to-head antisense transcription and R-loop formation promotes transcriptional activation. Proc Natl Acad Sci USA 2015;112:5785–90.
Arab K, Karaulanov E, Musheev M, Trnka P, Schäfer A, Grummt I, et al. GADD45A binds R-loops and recruits TET1 to CpG island promoters. Nat Genet. 2019;51:217–23.
Proudfoot NJ. Transcriptional termination in mammals: stopping the RNA polymerase II juggernaut. Science. 2016;352:aad9926.
Huertas P, Aguilera A. Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment and transcription-associated recombination. Mol Cell. 2003;12:711–21.
Li X, Manley JL. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell. 2005;122:365–78.
Herrera-Moyano E, Mergui X, GarcÃa-Rubio ML, Barroso S, Aguilera A. The yeast and human FACT chromatin-reorganizing complexes solve R-loop-mediated transcription-replication conflicts. Genes Dev. 2014;28:735–48.
Yan P, Liu Z, Song M, Wu Z, Xu W, Li K, et al. Genome-wide R-loop Landscapes during cell differentiation and reprogramming. Cell Rep. 2020;32:107870.
Pruss GJ, Manes SH, Drlica K. Escherichia coli DNA topoisomerase I mutants: increased supercoiling is corrected by mutations near gyrase genes. Cell. 1982;31:35–42.
Reyes A, Melchionda L, Nasca A, Carrara F, Lamantea E, Zanolini A, et al. RNASEH1 mutations impair mtDNA replication and cause adult-onset mitochondrial encephalomyopathy. Am J Hum Genet. 2015;97:186–93.
Bugiardini E, Poole OV, Manole A, Pittman AM, Horga A, Hargreaves I, et al. Clinicopathologic and molecular spectrum of RNASEH1-related mitochondrial disease. Neurol Genet. 2017;3:e149.
Rusecka JM, Kierdaszuk B, Stępniak I, Rydzanicz M, Stawiński P, Gambin T, et al. Ataxia and oculomotor apraxia caused by a large-scale deletion in the senataxin gene. J Appl Genet. 2025. https://doi.org/10.1007/s13353-025-01001-2.
Skourti-Stathaki K, Proudfoot NJ, Gromak N. Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol Cell. 2011;42:794–805.
Grunseich C, Wang IX, Watts JA, Burdick JT, Guber RD, Zhu Z, et al. Senataxin mutation reveals how R-loops promote transcription by blocking DNA methylation at gene promoters. Mol Cell. 2018;69:426–37.e7.
Groh M, Lufino MM, Wade-Martins R, Gromak N. R-loops associated with triplet repeat expansions promote gene silencing in friedreich ataxia and fragile X syndrome. PLoS Genet. 2014;10:e1004318.
Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015;525:56–61.
Loomis EW, Sanz LA, Chédin F, Hagerman PJ. Transcription-associated R-loop formation across the human FMR1 CGG-repeat region. PLoS Genet. 2014;10:e1004294.
Chédin F, Hartono SR, Sanz LA, Vanoosthuyse V. Best practices for the visualization, mapping, and manipulation of R-loops. EMBO J. 2021;40:e106394.
Dutrow N, Nix DA, Holt D, Milash B, Dalley B, Westbroek E, et al. Dynamic transcriptome of Schizosaccharomyces pombe shown by RNA-DNA hybrid mapping. Nat Genet. 2008;40:977–86.
Hu Z, Zhang A, Storz G, Gottesman S, Leppla SH. An antibody-based microarray assay for small RNA detection. Nucleic Acids Res. 2006;34:e52.
Konig F, Schubert T, Langst G. The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences. PLoS One. 2017;12:e0178875.
Chen X, Liu W, Wang Q, Wang X, Ren Y, Qu X, et al. Structural visualization of transcription initiation in action. Science. 2023;382:eadi5120.
Frick DN, Richardson CC. DNA primases. Annu Rev Biochem. 2001;70:39–80.
GarcÃa-Rubio ML, Pérez-Calero C, Barroso SI, Tumini E, Herrera-Moyano E, Rosado IV, et al. The fanconi anemia pathway protects genome integrity from R-loops. PLoS Genet. 2015;11:e1005674.
Kotsantis P, Silva LM, Irmscher S, Jones RM, Folkes L, Gromak N, et al. Increased global transcription activity as a mechanism of replication stress in cancer. Nat Commun. 2016;7:13087.
Graf M, Bonetti D, Lockhart A, Serhal K, Kellner V, Maicher A, et al. Telomere length determines TERRA and R-loop regulation through the cell cycle. Cell. 2017;170:72–85.e14.
Crossley MP, Bocek MJ, Hamperl S, Swigut T, Cimprich KA. qDRIP: a method to quantitatively assess RNA-DNA hybrid formation genome-wide. Nucleic Acids Res. 2020;48:e84.
Sanz LA, Castillo-Guzman D, Chedin F., Mapping R-loops and RNA:DNA hybrids with S9.6-based immunoprecipitation methods. J Vis Exp, 2021.
Wulfridge P, Sarma K. Intertwining roles of R-loops and G-quadruplexes in DNA repair, transcription and genome organization. Nat Cell Biol. 2024;26:1025–36.
Wanrooij PH, Uhler JP, Shi Y, Westerlund F, Falkenberg M, Gustafsson CM. A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop. Nucleic Acids Res. 2012;40:10334–44.
Miller JA, Ding SL, Sunkin SM, Smith KA, Ng L, Szafer A, et al. Transcriptional landscape of the prenatal human brain. Nature. 2014;508:199–206.
Liu J, Wu X, Zhang H, Pfeifer GP, Lu Q. Dynamics of RNA polymerase II pausing and bivalent histone H3 methylation during neuronal differentiation in brain development. Cell Rep. 2017;20:1307–18.
Cascante A, Klum S, Biswas M, Antolin-Fontes B, Barnabé-Heider F, Hermanson O. Gene-specific methylation control of H3K9 and H3K36 on neurotrophic BDNF versus astroglial GFAP genes by KDM4A/C regulates neural stem cell differentiation. J Mol Biol. 2014;426:3467–77.
Zu X, Yu L, Sun Y, Tian J, Liu F, Sun Q, et al. Global mapping of ZBTB7A transcription factor binding sites in HepG2 cells. Cell Mol Biol Lett. 2010;15:260–71.
Pereira JD, Sansom SN, Smith J, Dobenecker MW, Tarakhovsky A, Livesey FJ. Ezh2, the histone methyltransferase of PRC2, regulates the balance between self-renewal and differentiation in the cerebral cortex. Proc Natl Acad Sci USA 2010;107:15957–62.
Huang Y, Myers SJ, Dingledine R. Transcriptional repression by REST: recruitment of Sin3A and histone deacetylase to neuronal genes. Nat Neurosci. 1999;2:867–72.
Baldelli P, Meldolesi J., The transcription repressor REST in adult neurons: physiology, pathology, and diseases. eNeuro, 2015;2.
Koopmans F, van Nierop P, Andres-Alonso M, Byrnes A, Cijsouw T, Coba MP, et al. SynGO: an evidence-based, expert-curated knowledge base for the synapse. Neuron. 2019;103:217–34.e4.
Huang G, Chen S, Chen X, Zheng J, Xu Z, Doostparast Torshizi A, et al. Uncovering the functional link between SHANK3 deletions and deficiency in neurodevelopment using iPSC-derived human neurons. Front Neuroanat. 2019;13:23.
Li Y, Jia X, Wu H, Xun G, Ou J, Zhang Q, et al. Genotype and phenotype correlations for SHANK3 de novo mutations in neurodevelopmental disorders. Am J Med Genet A. 2018;176:2668–76.
Zhang G, Li S, Yang L, Wang M, Chen G, Zhu D. [Analysis of NOVA2 gene variant in a child with neurodevelopmental disorder with or without autistic features and/or structural brain abnormalities]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2023;40:213–6.
Mattioli F, Hayot G, Drouot N, Isidor B, Courraud J, Hinckelmann MV, et al. De novo frameshift variants in the neuronal splicing factor NOVA2 result in a common C-terminal extension and cause a severe form of neurodevelopmental disorder. Am J Hum Genet. 2020;106:438–52.
Brennand K, Savas JN, Kim Y, Tran N, Simone A, Hashimoto-Torii K, et al. Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia. Mol Psychiatry. 2015;20:361–8.
Hoffman GE, Hartley BJ, Flaherty E, Ladran I, Gochman P, Ruderfer DM, et al. Transcriptional signatures of schizophrenia in hiPSC-derived NPCs and neurons are concordant with post-mortem adult brains. Nat Commun. 2017;8:2225.
Hausen P, Stein H. Ribonuclease H. An enzyme degrading the RNA moiety of DNA-RNA hybrids. Eur J Biochem. 1970;14:278–83.
Cerritelli SM, Frolova EG, Feng C, Grinberg A, Love PE, Crouch RJ. Failure to produce mitochondrial DNA results in embryonic lethality in rnaseh1 null mice. Molecular Cell. 2003;11:807–15.
Wahba L, Amon JD, Koshland D, Vuica-Ross M. RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability. Mol Cell. 2011;44:978–88.
Ohle C, Tesorero R, Schermann G, Dobrev N, Sinning I, Fischer T. Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell. 2016;167:1001–13.e7.
Lockhart A, Pires VB, Bento F, Kellner V, Luke-Glaser S, Yakoub G, et al. RNase H1 and H2 are differentially regulated to process RNA-DNA hybrids. Cell Rep. 2019;29:2890–900.e5.
Skourti-Stathaki K, Torlai Triglia E, Warburton M, Voigt P, Bird A, Pombo A. R-loops enhance polycomb repression at a subset of developmental regulator genes. Mol Cell. 2019;73:930–45.e4.
Wu H, Lima WF, Crooke ST. Investigating the structure of human RNase H1 by site-directed mutagenesis. J Biol Chem. 2001;276:23547–53.
Brown TA, Tkachuk AN, Clayton DA. Native R-loops persist throughout the mouse mitochondrial DNA genome. J Biol Chem. 2008;283:36743–51.
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.
Chen PB, Chen HV, Acharya D, Rando OJ, Fazzio TG. R loops regulate promoter-proximal chromatin architecture and cellular differentiation. Nat Struct Mol Biol. 2015;22:999–1007.
Kabeche L, Nguyen HD, Buisson R, Zou L. A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation. Science. 2018;359:108–14.
Vanoosthuyse V. Strengths and weaknesses of the current strategies to map and characterize R-loops. Noncoding RNA. 2018;4:9.
Xu X, Wells AB, O’Brien DR, Nehorai A, Dougherty JD. Cell type-specific expression analysis to identify putative cellular mechanisms for neurogenetic disorders. J Neurosci. 2014;34:1420–31.
Flaherty E, Zhu S, Barretto N, Cheng E, Deans PJM, Fernando MB, et al. Neuronal impact of patient-specific aberrant NRXN1alpha splicing. Nat Genet. 2019;51:1679–90.
Basu SN, Kollu R, Banerjee-Basu S. AutDB: a gene reference resource for autism research. Nucleic Acids Res. 2009;37:D832–6.
Zhou X, Feliciano P, Shu C, Wang T, Astrovskaya I, Hall JB, et al. Integrating de novo and inherited variants in 42,607 autism cases identifies mutations in new moderate-risk genes. Nat Genet. 2022;54:1305–19.
Helsmoortel C, Vulto-van Silfhout AT, Coe BP, Vandeweyer G, Rooms L, van den Ende J, et al. A SWI/SNF-related autism syndrome caused by de novo mutations in ADNP. Nat Genet. 2014;46:380–4.
Arnett AB, Rhoads CL, Hoekzema K, Turner TN, Gerdts J, Wallace AS, et al. The autism spectrum phenotype in ADNP syndrome. Autism Res. 2018;11:1300–10.
Ka M, Kim WY. ANKRD11 associated with intellectual disability and autism regulates dendrite differentiation via the BDNF/TrkB signaling pathway. Neurobiol Dis. 2018;111:138–52.
Gallagher D, Voronova A, Zander MA, Cancino GI, Bramall A, Krause MP, et al. Ankrd11 is a chromatin regulator involved in autism that is essential for neural development. Dev Cell. 2015;32:31–42.
Tian C, Paskus JD, Fingleton E, Roche KW, Herring BE. Autism spectrum disorder/intellectual disability-associated mutations in trio disrupt neuroligin 1-mediated synaptogenesis. J Neurosci. 2021;41:7768–78.
Parenti I, Leitão E, Kuechler A, Villard L, Goizet C, Courdier C, et al. The different clinical facets of SYN1-related neurodevelopmental disorders. Front Cell Dev Biol. 2022;10:1019715.
Fassio A, Patry L, Congia S, Onofri F, Piton A, Gauthier J, et al. SYN1 loss-of-function mutations in autism and partial epilepsy cause impaired synaptic function. Hum Mol Genet. 2011;20:2297–307.
Wang Z, Li J, Zhang T, Lu T, Wang H, Jia M, et al. Family-based association study identifies SNAP25 as a susceptibility gene for autism in the Han Chinese population. Prog Neuropsychopharmacol Biol Psychiatry. 2021;105:109985.
Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. Nature. 2014;511:421–7.
Pardiñas AF, Holmans P, Pocklington AJ, Escott-Price V, Ripke S, Carrera N, et al. Common schizophrenia alleles are enriched in mutation-intolerant genes and in regions under strong background selection. Nat Genet. 2018;50:381–9.
Wu Y, Li X, Liu J, Luo XJ, Yao YG. SZDB2.0: an updated comprehensive resource for schizophrenia research. Hum Genet. 2020;139:1285–97.
Mullins N, Forstner AJ, O’Connell KS, Coombes B, Coleman JRI, Qiao Z, et al. Genome-wide association study of more than 40,000 bipolar disorder cases provides new insights into the underlying biology. Nat Genet. 2021;53:817–29.
Epi25 Collaborative. Ultra-rare genetic variation in the epilepsies: a whole-exome sequencing study of 17,606 individuals. Am J Hum Genet. 2019;105:267–82.
Stahl EA, Raychaudhuri S, Remmers EF, Xie G, Eyre S, Thomson BP, et al. Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet. 2010;42:508–14.
Luhmann HJ, Sinning A, Yang JW, Reyes-Puerta V, Stüttgen MC, Kirischuk S, et al. Spontaneous neuronal activity in developing neocortical networks: from single cells to large-scale interactions. Front Neural Circuits. 2016;10:40.
Durbec P, Franceschini I, Lazarini F, Dubois-Dalcq M. In vitro migration assays of neural stem cells. Methods Mol Biol. 2008;438:213–25.
Taniguchi Y, Young-Pearse T, Sawa A, Kamiya A. In utero electroporation as a tool for genetic manipulation in vivo to study psychiatric disorders: from genes to circuits and behaviors. Neuroscientist. 2012;18:169–79.
Saito A, Taniguchi Y, Rannals MD, Merfeld EB, Ballinger MD, Koga M, et al. Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1. Mol Psychiatry. 2016;21:1449–59.
Zhu X, Nedelcovych MT, Thomas AG, Hasegawa Y, Moreno-Megui A, Coomer W, et al. JHU-083 selectively blocks glutaminase activity in brain CD11b(+) cells and prevents depression-associated behaviors induced by chronic social defeat stress. Neuropsychopharmacology. 2019;44:683–94.
Gourley SL, Olevska A, Warren MS, Taylor JR, Koleske AJ. Arg kinase regulates prefrontal dendritic spine refinement and cocaine-induced plasticity. J Neurosci. 2012;32:2314–23.
Skalska L, Beltran-Nebot M, Ule J, Jenner RG. Regulatory feedback from nascent RNA to chromatin and transcription. Nat Rev Mol Cell Biol. 2017;18:331–7.
Santos-Pereira JM, Aguilera A. R loops: new modulators of genome dynamics and function. Nat Rev Genet. 2015;16:583–97.
Aguilera A, Garcia-Muse T. R loops: from transcription byproducts to threats to genome stability. Mol Cell. 2012;46:115–24.
Rios M, Lambe EK, Liu R, Teillon S, Liu J, Akbarian S, et al. Severe deficits in 5-HT2A -mediated neurotransmission in BDNF conditional mutant mice. J Neurobiol. 2006;66:408–20.
Belotserkovskii BP, Hanawalt PC. Topology and kinetics of R-loop formation. Biophys J. 2022;121:3345–57.
Pan X, Huang LF. Multi-omics to characterize the functional relationships of R-loops with epigenetic modifications, RNAPII transcription and gene expression. Brief Bioinform. 2022;23:bbac238.
Pezone A, Zuchegna C, Tramontano A, Romano A, Russo G, de Rosa M, et al. RNA stabilizes transcription-dependent chromatin loops induced by nuclear hormones. Sci Rep. 2019;9:3925.
Salas-Armenteros I, Pérez-Calero C, Bayona-Feliu A, Tumini E, Luna R, Aguilera A. Human THO-Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability. EMBO J. 2017;36:3532–47.
Marchetto MC, Belinson H, Tian Y, Freitas BC, Fu C, Vadodaria K, et al. Altered proliferation and networks in neural cells derived from idiopathic autistic individuals. Mol Psychiatry. 2017;22:820–35.
Kim EC, Patel J, Zhang J, Soh H, Rhodes JS, Tzingounis AV, et al. Heterozygous loss of epilepsy gene KCNQ2 alters social, repetitive and exploratory behaviors. Genes Brain Behav. 2020;19:e12599.
Brennand KJ, Simone A, Jou J, Gelboin-Burkhart C, Tran N, Sangar S, et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature. 2011;473:221–5.
Tran NN, Ladran IG, Brennand KJ. Modeling schizophrenia using induced pluripotent stem cell-derived and fibroblast-induced neurons. Schizophr Bull. 2013;39:4–10.
Roussos P, Mitchell AC, Voloudakis G, Fullard JF, Pothula VM, Tsang J, et al. A role for noncoding variation in schizophrenia. Cell Rep. 2014;9:1417–29.
Topol A, Tran NN, Brennand KJ. A guide to generating and using hiPSC derived NPCs for the study of neurological diseases. J Vis Exp, 2015: e52495.
Roberts RW, Crothers DM. Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition. Science. 1992;258:1463–6.
Schneider CA, Rasband WS, Eliceiri KW. NIH image to imageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.
Peter CJ, Saito A, Hasegawa Y, Tanaka Y, Nagpal M, Perez G, et al. In vivo epigenetic editing of Sema6a promoter reverses transcallosal dysconnectivity caused by C11orf46/Arl14ep risk gene. Nat Commun. 2019;10:4112.
Saxena A, Wagatsuma A, Noro Y, Kuji T, Asaka-Oba A, Watahiki A, et al. Trehalose-enhanced isolation of neuronal sub-types from adult mouse brain. Biotechniques. 2012;52:381–5.
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20.
Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–9.
Amemiya HM, Kundaje A, Boyle AP. The ENCODE blacklist: identification of problematic regions of the genome. Sci Rep. 2019;9:9354.
Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26:841–2.
Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 2010;38:576–89.
Yu G, Wang LG, He QY. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015;31:2382–3.
Carroll TS, Liang Z, Salama R, Stark R, de Santiago I. Impact of artifact removal on ChIP quality metrics in ChIP-seq and ChIP-exo data. Front Genet. 2014;5:75.
RamÃrez F, Dündar F, Diehl S, Grüning BA, Manke T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 2014;42:W187–91.
Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ, et al. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature. 2012;481:389–93.
Ge SX, Jung D, Yao R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics. 2020;36:2628–9.
Gel B, DÃez-Villanueva A, Serra E, Buschbeck M, Peinado MA, Malinverni R. regioneR: an R/Bioconductor package for the association analysis of genomic regions based on permutation tests. Bioinformatics. 2016;32:289–91.
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.
Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30.
Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM 3rd, et al. Comprehensive integration of single-cell data. Cell. 2019;177:1888–902.e21.
Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284–7.
Zhao ZH, Zheng G, Wang T, Du KJ, Han X, Luo WJ, et al. Low-level gestational lead exposure alters dendritic spine plasticity in the hippocampus and reduces learning and memory in rats. Sci Rep. 2018;8:3533.
de la Torre-Ubieta L, Stein JL, Won H, Opland CK, Liang D, Lu D, et al. The dynamic landscape of open chromatin during human cortical neurogenesis. Cell. 2018;172:289–304.e18.
Acknowledgements
We thank all members of the Akbarian and Brennand laboratories for constructive comments and discussions. Bibi Kassim, Natalie Barretto, Samuel Powell, Esther Cheng, Yuto Hasegawa, and Sasha Layne for laboratory assistance. The New York Genome Center, Kristin Beaumont and the Mount Sinai Genomics Core Facility, and Prashant Singh at the Roswell Park Genomics Shared Resource for sequencing support. John Greally, Julie Nadel, Achla Gupta, and the Albert Einstein College of Medicine Proteomics Core for assistance with S9.6 antibody generation and purification. Zhiping Weng, Kaili Fan, Kiran Girdhar, and Gabriel Hoffman for productive discussions. We also thank Frédéric Chédin and Lionel Sanz for generously sharing reagents and the DRIP-seq protocol.
Funding
This work was supported by National Institute of Mental Health PsychENCODE grants 1U01DA048279 and R01MH106056 (SA and KJB), R01DA054526 (SA), R56MH101454 (KJB), RF1DA048810 (NMT), R01DA060630 (AK), R01AG065168 (AK), P50MH136297 (AK), and a predoctoral Ruth L. Kirschstein fellowship F31MH121062 (EAL).
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Experiments conducted by EAL (hiPSC differentiations, RNASEH1 cloning and lentiviral construction/transduction, DRIP-seq, RNA-seq, MEA, ICC, qPCR, Migration assay, WB, dot blot), AS and AF (IUE, spine density and Sholl analyses, in vivo IHC/qPCR), BJ (FISH), AH (qPCR, optical intensity and image analyses), MBF (hiPSC-neuron differentiation and ICC), KJB (hiPSC/NPC generation), and NMT (prenatal brain dissection). Bioinformatic analysis conducted by EAL (DRIP-seq, RNA-seq, scRNA-seq), APJ (scRNA-seq), CD, SEG, JEE, ME, and WL Research supervised by SA, KJB, NMT, AK, BT, and LS Study and experiments conceived by EAL, SA, and KJB Figures designed by EAL and SA Paper written by EAL, SA, and KJB, with contributions from all co-authors.
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All methods were performed in accordance with the relevant guidelines and regulations. All human induced pluripotent stem cell work was conducted under the oversight of the Institutional Review Board (IRB) (HS13-Â00500) at the Icahn School of Medicine at Mount Sinai, with informed consent from all participants. Brain tissue after death was collected at the Mount Sinai Pathology Department in accordance with the institutional policies and regulations, along with next-of-kin consent for the de-identified tissue to be used for research purposes and approved under IRB STUDY-18-00983. All animal experiments were performed in the Johns Hopkins University Brain Science Institute’s Behavioral Core according to the University’s Animal Care and Use Committee’s guidelines and approved under Protocol Number MO23M218.
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LaMarca, E.A., Saito, A., Plaza-Jennings, A. et al. R-loop landscapes in the developing human brain are linked to neural differentiation and cell type-specific transcription. Transl Psychiatry (2026). https://doi.org/10.1038/s41398-026-04009-2
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DOI: https://doi.org/10.1038/s41398-026-04009-2
