Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells


In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down’s syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation.

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Fig. 1: Non-uniform probability of cPCDH-gene selection in hiPSC-derived neurons.
Fig. 2: Chromatin in hiPSCs mirrors expression in hiPSC-derived neurons across the cPCDH locus.
Fig. 3: In vitro-generated neurons inherit cPCDH-locus features from non-neuronal cells.
Fig. 4: Differences in cPCDH-locus chromatin organization between naive and primed cells.
Fig. 5: Signs of hESC-guided cPCDH-gene signatures are remarkably stable in vitro and in vivo.
Fig. 6: Two distinct types of cPCDH diversity distinguish fetal and adult brain tissues.

Data availability

The sequencing datasets generated in this study have been deposited in GEO under accession number GSE106872.


  1. 1.

    Gul, I. S., Hulpiau, P., Saeys, Y. & van Roy, F. Evolution and diversity of cadherins and catenins. Exp. Cell Res. 358, 3–9 (2017).

  2. 2.

    Hirayama, T. & Yagi, T. Regulation of clustered protocadherin genes in individual neurons. Semin. Cell Dev. Biol. 69, 122–130 (2017).

  3. 3.

    Peek, S. L., Mah, K. M. & Weiner, J. A. Regulation of neural circuit formation by protocadherins. Cell. Mol. Life Sci. 74, 4133–4157 (2017).

  4. 4.

    Mountoufaris, G., Canzio, D., Nwakeze, C. L., Chen, W. V. & Maniatis, T. Writing, reading, and translating the clustered protocadherin cell surface recognition code for neural circuit assembly. Annu. Rev. Cell Dev. Biol. 34, 471–493 (2018).

  5. 5.

    Rubinstein, R., Goodman, K. M., Maniatis, T., Shapiro, L. & Honig, B. Structural origins of clustered protocadherin-mediated neuronal barcoding. Semin. Cell Dev. Biol. 69, 140–150 (2017).

  6. 6.

    Garrett, A. M., Schreiner, D., Lobas, M. A. & Weiner, J. A. γ-Protocadherins control cortical dendrite arborization by regulating the activity of a FAK/PKC/MARCKS signaling pathway. Neuron 74, 269–276 (2012).

  7. 7.

    Kostadinov, D. & Sanes, J. R. Protocadherin-dependent dendritic self-avoidance regulates neural connectivity and circuit function. eLife 4, e08964 (2015).

  8. 8.

    Lefebvre, J. L., Kostadinov, D., Chen, W. V., Maniatis, T. & Sanes, J. R. Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 488, 517–521 (2012).

  9. 9.

    Molumby, M. J., Keeler, A. B. & Weiner, J. A. Homophilic protocadherin cell–cell interactions promote dendrite complexity. Cell Rep. 15, 1037–1050 (2016).

  10. 10.

    Mountoufaris, G. et al. Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly. Science 356, 411–414 (2017).

  11. 11.

    Wang, X., Su, H. & Bradley, A. Molecular mechanisms governing Pcdh-gamma gene expression: evidence for a multiple promoter and cis-alternative splicing model. Genes Dev. 16, 1890–1905 (2002).

  12. 12.

    Tasic, B. et al. Promoter choice determines splice site selection in protocadherin α and γ pre-mRNA splicing. Mol. Cell 10, 21–33 (2002).

  13. 13.

    Chen, W. V. & Maniatis, T. Clustered protocadherins. Development 140, 3297–3302 (2013).

  14. 14.

    Guo, Y. et al. CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice. Proc. Natl Acad. Sci. USA 109, 21081–21086 (2012).

  15. 15.

    Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015).

  16. 16.

    Kehayova, P., Monahan, K., Chen, W. & Maniatis, T. Regulatory elements required for the activation and repression of the protocadherin-α gene cluster. Proc. Natl Acad. Sci. USA 108, 17195–17200 (2011).

  17. 17.

    Monahan, K. et al. Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-α gene expression. Proc. Natl Acad. Sci. USA 109, 9125–9130 (2012).

  18. 18.

    Ribich, S., Tasic, B. & Maniatis, T. Identification of long-range regulatory elements in the protocadherin-α gene cluster. Proc. Natl Acad. Sci. USA 103, 19719–19724 (2006).

  19. 19.

    Jiang, Y. et al. The methyltransferase SETDB1 regulates a large neuron-specific topological chromatin domain. Nat. Genet. 49, 1239–1250 (2017).

  20. 20.

    El Hajj, N., Dittrich, M. & Haaf, T. Epigenetic dysregulation of protocadherins in human disease. Semin. Cell Dev. Biol. 69, 172–182 (2017).

  21. 21.

    Woodruff, G. et al. Defective transcytosis of APP and lipoproteins in human iPSC-derived neurons with familial Alzheimer’s disease mutations. Cell Rep. 17, 759–773 (2016).

  22. 22.

    Woodruff, G. et al. The presenilin-1 ΔE9 mutation results in reduced γ-secretase activity, but not total loss of PS1 function, in isogenic human stem cells. Cell Rep. 5, 974–985 (2013).

  23. 23.

    Mikkelsen, T. S. et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560 (2007).

  24. 24.

    Mertens, J. et al. Directly reprogrammed human neurons retain aging-associated transcriptomic signatures and reveal age-related nucleocytoplasmic defects. Cell Stem Cell 17, 705–718 (2015).

  25. 25.

    Cacchiarelli, D. et al. Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency. Cell 162, 412–424 (2015).

  26. 26.

    Kilens, S. et al. Parallel derivation of isogenic human primed and naive induced pluripotent stem cells. Nat. Commun. 9, 360 (2018).

  27. 27.

    O’Leary, T. et al. Tracking the progression of the human inner cell mass during embryonic stem cell derivation. Nat. Biotechnol. 30, 278–282 (2012).

  28. 28.

    Warrier, S. et al. Transcriptional landscape changes during human embryonic stem cell derivation. Mol. Hum. Reprod. 24, 543–555 (2018).

  29. 29.

    Theunissen, T. W. et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15, 471–487 (2014).

  30. 30.

    Ji, X. et al. 3D chromosome regulatory landscape of human pluripotent cells. Cell Stem Cell 18, 262–275 (2016).

  31. 31.

    Theunissen, T. W. & Jaenisch, R. Mechanisms of gene regulation in human embryos and pluripotent stem cells. Development 144, 4496–4509 (2017).

  32. 32.

    Yagi, T. Molecular codes for neuronal individuality and cell assembly in the brain. Front. Mol. Neurosci. 5, 45 (2012).

  33. 33.

    Wada, T., Wallerich, S. & Becskei, A. Stochastic gene choice during cellular differentiation. Cell Rep. 24, 3503–3512 (2018).

  34. 34.

    Trujillo, C. A. et al. Complex oscillatory waves emerging from cortical organoids model early human brain network development. Cell Stem Cell 25, 558–569 (2019).

  35. 35.

    Brown, J. P. et al. Transient expression of doublecortin during adult neurogenesis. J. Comp. Neurol. 467, 1–10 (2003).

  36. 36.

    Stiles, J. & Jernigan, T. L. The basics of brain development. Neuropsychol. Rev. 20, 327–348 (2010).

  37. 37.

    Frank, M. et al. Differential expression of individual gamma-protocadherins during mouse brain development. Mol. Cell. Neurosci. 29, 603–616 (2005).

  38. 38.

    Li, Y. et al. Synaptic and nonsynaptic localization of protocadherin-γC5 in the rat brain. J. Comp. Neurol. 518, 3439–3463 (2010).

  39. 39.

    Zou, C., Huang, W., Ying, G. & Wu, Q. Sequence analysis and expression mapping of the rat clustered protocadherin gene repertoires. Neuroscience 144, 579–603 (2007).

  40. 40.

    Bohaciakova, D. et al. A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors. Stem Cell Res. Ther. 10, 83 (2019).

  41. 41.

    Toyoda, S. et al. Developmental epigenetic modification regulates stochastic expression of clustered protocadherin genes, generating single neuron diversity. Neuron 82, 94–108 (2014).

  42. 42.

    Miller, J. A. et al. Transcriptional landscape of the prenatal human brain. Nature 508, 199–206 (2014).

  43. 43.

    Juhasova, J. et al. Time course of spinal doublecortin expression in developing rat and porcine spinal cord: implication in in vivo neural precursor grafting studies. Cell. Mol. Neurobiol. 35, 57–70 (2015).

  44. 44.

    El Hajj, N. et al. Epigenetic dysregulation in the developing Down syndrome cortex. Epigenetics 11, 563–578 (2016).

  45. 45.

    Horvath, S. et al. Accelerated epigenetic aging in Down syndrome. Aging Cell 14, 491–495 (2015).

  46. 46.

    Esumi, S. et al. Monoallelic yet combinatorial expression of variable exons of the protocadherin-α gene cluster in single neurons. Nat. Genet. 37, 171–176 (2005).

  47. 47.

    Smith, A. Formative pluripotency: the executive phase in a developmental continuum. Development 144, 365–373 (2017).

  48. 48.

    Takashima, Y. et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254–1269 (2014).

  49. 49.

    Borgel, J. et al. Targets and dynamics of promoter DNA methylation during early mouse development. Nat. Genet. 42, 1093–1100 (2010).

  50. 50.

    Hasegawa, S. et al. The protocadherin-α family is involved in axonal coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb in mouse. Mol. Cell. Neurosci. 38, 66–79 (2008).

  51. 51.

    Hasegawa, S. et al. Distinct and cooperative functions for the protocadherin-α, -β and -γ clusters in neuronal survival and axon targeting. Front. Mol. Neurosci. 9, 155 (2016).

  52. 52.

    Hasegawa, S. et al. Clustered protocadherins are required for building functional neural circuits. Front. Mol. Neurosci. 10, 114 (2017).

  53. 53.

    Katori, S. et al. Protocadherin-α family is required for serotonergic projections to appropriately innervate target brain areas. J. Neurosci. 29, 9137–9147 (2009).

  54. 54.

    Prasad, T. & Weiner, J. A. Direct and indirect regulation of spinal cord Ia afferent terminal formation by the γ-protocadherins. Front. Mol. Neurosci. 4, 54 (2011).

  55. 55.

    Suo, L., Lu, H., Ying, G., Capecchi, M. R. & Wu, Q. Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase. J. Mol. Cell Biol. 4, 362–376 (2012).

  56. 56.

    Chen, W. V. et al. Pcdhαc2 is required for axonal tiling and assembly of serotonergic circuitries in mice. Science 356, 406–411 (2017).

  57. 57.

    Yamagishi, T. et al. Molecular diversity of clustered protocadherin-α required for sensory integration and short-term memory in mice. Sci. Rep. 8, 9616 (2018).

  58. 58.

    Gore, A. et al. Somatic coding mutations in human induced pluripotent stem cells. Nature 471, 63–67 (2011).

  59. 59.

    Young, J. E. et al. Elucidating molecular phenotypes caused by the SORL1 Alzheimer’s disease genetic risk factor using human induced pluripotent stem cells. Cell Stem Cell 16, 373–385 (2015).

  60. 60.

    Cowan, C. A. et al. Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–1356 (2004).

  61. 61.

    Ying, Q.-L. et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

  62. 62.

    Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl Acad. Sci. USA 90, 8424–8428 (1993).

  63. 63.

    Guo, G. et al. Klf4 reverts developmentally programmed restriction of ground state pluripotency. Development 136, 1063–1069 (2009).

  64. 64.

    Yuan, S. H. et al. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS ONE 6, e17540 (2011).

  65. 65.

    Shi, Y., Kirwan, P. & Livesey, F. J. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat. Protoc. 7, 1836–1846 (2012).

  66. 66.

    Shi, Y., Kirwan, P., Smith, J., Robinson, H. P. C. & Livesey, F. J. Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nat. Neurosci. 15, 477–486 (2012).

  67. 67.

    Fong, L. K. et al. Full-length amyloid precursor protein regulates lipoprotein metabolism and amyloid-β clearance in human astrocytes. J. Biol. Chem. 293, 11341–11357 (2018).

  68. 68.

    Crook, J. M. et al. The generation of six clinical-grade human embryonic stem cell lines. Cell Stem Cell 1, 490–494 (2007).

  69. 69.

    Mann, D. L. et al. Origin of the HIV-susceptible human CD4+ cell line H9. AIDS Res. Hum. Retroviruses 5, 253–255 (1989).

  70. 70.

    Hefferan, M. P. et al. Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation. PLoS ONE 7, e42614 (2012).

  71. 71.

    Kakinohana, O. et al. Region-specific cell grafting into cervical and lumbar spinal cord in rat: a qualitative and quantitative stereological study. Exp. Neurol. 190, 122–132 (2004).

  72. 72.

    Almenar-Queralt, A. et al. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation. J. Biol. Chem. 288, 35222–35236 (2013).

  73. 73.

    Benner, C. et al. Decoding a signature-based model of transcription cofactor recruitment dictated by cardinal cis-regulatory elements in proximal promoter regions. PLoS Genet. 9, e1003906 (2013).

  74. 74.

    Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).

  75. 75.

    Johnson, W. E., Li, C. & Rabinovic, A. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8, 118–127 (2007).

  76. 76.

    Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

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We thank M. G. Rosenfeld and W. Dillmann for providing a nurturing research environment to perform this study, and the many colleagues who helped us to conduct or understand our research (in alphabetical order): A. Becskei, A. Gamliel, T. Haaf, A. Holder, K. Jepsen, E. Kothari, S. Linker, C. Marchetto, S. Marsala, J. Mertens, I. Narvaiza, F. Neri, D. Meluzzi, A. Muotri, P. Negraes, K. Ohgi, M. Parast, S. Sathe, D. Skowronska-Krawczyk, C. Trujillo, R. Tsunemoto, R. Van der Kant, G. Yeo; the team at the UCSD Human Embryonic Stem Cell Core Facility for reagents and technical assistance, and the ENCODE and BrainSpan Consortia for sharing data. Special thanks to H. Garcia Garcia in representation of the many deceased anonymous donors who altruistically donate their bodies for the advancement of science; these donors have made our work possible. Q.M. was supported by the postdoctoral fellowship from the American Cancer Society. C.A. was supported by the postdoctoral fellowship Sara Borrell. Study supported by the US Department of Defense (DoD) (AZ140064) to A.A.-Q., the Sanford Stem Cell Clinical Center (SANPORC) to M.M. and NIH/NIA (1RF1AG048083-01 and 5P50AG005131-34) to L.S.B.G. The Department of Medicine, School of Medicine (UCSD) supported I.G.-B.

Author information

A.A.-Q. and I.G.-B. conceived and designed experiments. A.A.-Q., H.S.K., M.N., R.S.C., A.G.C., B.K., L.K.F., J.E.Y. and I.G.-B. performed experiments. D.M., Q.M., C.A., S.P.D., C.M., N.E.H, M.D. and I.G.B. performed computational analyses. G.W., D.B., M.H.-P. and T.T. generated essential reagents. A.A.-Q., M.M., L.S.B.G. and I.G.-B. supervised the research. A.A.-Q. and I.G.-B. wrote the manuscript.

Correspondence to Angels Almenar-Queralt or Ivan Garcia-Bassets.

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M.M. is the scientific founder of Neurgain Technologies and has an equity interest in the company. In addition, M.M. serves as a consultant to Neurgain Technologies and receives compensation for these services. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.

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Almenar-Queralt, A., Merkurjev, D., Kim, H.S. et al. Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells. Nat Genet 51, 1691–1701 (2019).

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