Abstract
Mutations in two highly related chromatin-remodeling complexes, BRG1/BRM-associated factor (BAF) and Polybromo-associated BAF (PBAF), cause various neurodevelopmental conditions. Although BAF activity is required at multiple steps of nervous system development, the specific neural functions of PBAF remain largely unexplored. Through an unbiased genetic screen in C. elegans, we identify here critical roles for PBAF in postmitotic neuronal identity. In nerve cord motor neurons, PBAF represses gene expression by antagonizing the terminal selector-type transcription factor UNC-3 (EBF1-4). In contrast, PBAF activates gene expression in caudal motor neurons. This dichotomy in PBAF function generates distinct motor neuron identities. PBAF disruption leads to locomotion…
Abstract
Mutations in two highly related chromatin-remodeling complexes, BRG1/BRM-associated factor (BAF) and Polybromo-associated BAF (PBAF), cause various neurodevelopmental conditions. Although BAF activity is required at multiple steps of nervous system development, the specific neural functions of PBAF remain largely unexplored. Through an unbiased genetic screen in C. elegans, we identify here critical roles for PBAF in postmitotic neuronal identity. In nerve cord motor neurons, PBAF represses gene expression by antagonizing the terminal selector-type transcription factor UNC-3 (EBF1-4). In contrast, PBAF activates gene expression in caudal motor neurons. This dichotomy in PBAF function generates distinct motor neuron identities. PBAF disruption leads to locomotion defects. Genetic, genomic, and biochemical data suggest that the cell type-specific functions of PBAF in different motor neuron groups arise via its recruitment to specific gene loci by conserved transcription factors, such as MAB-9/TBX20. Altogether, our findings provide a conceptual framework to understand specific neuronal defects that arise in neurological conditions caused by mutations in broadly expressed chromatin-remodeling complexes.
Data availability
The data supporting the findings of this study are included in the figures and supporting files. The ChIP-Seq data for UNC-3 are available in the NCBI Gene Expression Omnibus (GEO) database under accession code GSE143165. The ChIP-Seq data for MAB-9, PBRM-1 and SWSN-7 are available in the ENCODE database under accession codes: ENCSR032EGI, ENCSR111IAZ, ENCSR406IHG. Single-cell RNA-Sequencing data for the SAB neurons and other C. elegans nerve cord motor neurons are deposited at the NCBI GEO under the accession code GSE234962. Source data are provided with this paper.
Code availability
The code for the SAB neuron analysis can be found here: https://doi.org/10.5281/zenodo.10567647.
References
Bota, M. & Swanson, L. W. The neuron classification problem. Brain Res. Rev. 56, 79–88 (2007).
White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1–340 (1986).
Alkaslasi, M. R. et al. Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord. Nat. Commun. 12, 2471 (2021).
Li, H. et al. Classifying Drosophila olfactory projection neuron subtypes by single-cell RNA sequencing. Cell 171, 1206–1220.e1222 (2017).
Taylor, S. R. et al. Molecular topography of an entire nervous system. Cell 184, 4329–4347.e4323 (2021).
Rosenberg, A. B. et al. Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding. Science 360, 176–182 (2018).
Konstantinides, N. et al. Phenotypic convergence: distinct transcription factors regulate common terminal features. Cell 174, 622–635.e613 (2018).
Smith, J. J. et al. A molecular atlas of adult C. elegans motor neurons reveals ancient diversity delineated by conserved transcription factor codes. Cell Rep. 43, 113857 (2024).
Ozel, M. N. et al. Coordinated control of neuronal differentiation and wiring by sustained transcription factors. Science 378, eadd1884 (2022).
Ozel, M. N. et al. Neuronal diversity and convergence in a visual system developmental atlas. Nature 589, 88–95 (2021).
Bhattacherjee, A. et al. Cell type-specific transcriptional programs in mouse prefrontal cortex during adolescence and addiction. Nat. Commun. 10, 4169 (2019).
Di Bella, D. J. et al. Molecular logic of cellular diversification in the mouse cerebral cortex. Nature 595, 554–559 (2021).
Rheaume, B. A. et al. Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes. Nat. Commun. 9, 2759 (2018).
Doe, C. Q. Temporal patterning in the Drosophila CNS. Annu. Rev. Cell Dev. Biol. 33, 219–240 (2017).
Holguera, I. & Desplan, C. Neuronal specification in space and time. Science 362, 176–180 (2018).
Briscoe, J., Pierani, A., Jessell, T. M. & Ericson, J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101, 435–445 (2000).
Kutejova, E., Sasai, N., Shah, A., Gouti, M. & Briscoe, J. Neural progenitors adopt specific identities by directly repressing all alternative progenitor transcriptional programs. Dev. Cell 36, 639–653 (2016).
Sagner, A. & Briscoe, J. Establishing neuronal diversity in the spinal cord: a time and a place. Development 146, dev182154 (2019). 1.
Muhr, J., Andersson, E., Persson, M., Jessell, T. M. & Ericson, J. Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104, 861–873 (2001).
Hobert, O. Terminal selectors of neuronal identity. Curr. Top. Dev. Biol. 116, 455–475 (2016).
Hobert, O. & Kratsios, P. Neuronal identity control by terminal selectors in worms, flies, and chordates. Curr. Opin. Neurobiol. 56, 97–105 (2019).
Liu, C. et al. Pet-1 is required across different stages of life to regulate serotonergic function. Nat. Neurosci. 13, 1190–1198 (2010).
Zhang, X. L., Spencer, W. C., Tabuchi, N., Kitt, M. M. & Deneris, E. S. Reorganization of postmitotic neuronal chromatin accessibility for maturation of serotonergic identity. Elife 11, e75970 (2022). 1.
Kadkhodaei, B. et al. Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons. J. Neurosci. 29, 15923–15932 (2009).
Deneris, E. & Gaspar, P. Serotonin neuron development: shaping molecular and structural identities. Wiley Interdiscip. Rev. Dev. Biol. 7, e301 (2018). 1.
Poulin, J. F., Gaertner, Z., Moreno-Ramos, O. A. & Awatramani, R. Classification of midbrain dopamine neurons using single-cell gene expression profiling approaches. Trends Neurosci. 43, 155–169 (2020).
Doan, R. N. et al. Recessive gene disruptions in autism spectrum disorder. Nat. Genet. 51, 1092–1098 (2019).
Rafati, M. et al. Identification of a novel de novo variant in OTX2 in a patient with congenital microphthalmia using targeted next-generation sequencing followed by prenatal diagnosis. Ophthalmic Genet. 43, 262–267 (2022).
Sun, C. & Chen, S. Disease-causing mutations in genes encoding transcription factors critical for photoreceptor development. Front. Mol. Neurosci. 16, 1134839 (2023).
Blackburn, P. R. et al. Novel de novo variant in EBF3 is likely to impact DNA binding in a patient with a neurodevelopmental disorder and expanded phenotypes: patient report, in silico functional assessment, and review of published cases. Cold Spring Harb. Mol. Case Stud. 3, a001743 (2017).
Chao, H. T. et al. A syndromic neurodevelopmental disorder caused by de novo variants in EBF3. Am. J. Hum. Genet. 100, 128–137 (2017).
Sleven, H. et al. De novo mutations in EBF3 cause a neurodevelopmental syndrome. Am. J. Hum. Genet. 100, 138–150 (2017).
Etchberger, J. F., Flowers, E. B., Poole, R. J., Bashllari, E. & Hobert, O. Cis-regulatory mechanisms of left/right asymmetric neuron-subtype specification in C. elegans. Development 136, 147–160 (2009).
Uchida, O., Nakano, H., Koga, M. & Ohshima, Y. The C. elegans che-1 gene encodes a zinc finger transcription factor required for specification of the ASE chemosensory neurons. Development 130, 1215–1224 (2003).
Hobert, O. Development of left/right asymmetry in the Caenorhabditis elegans nervous system: from zygote to postmitotic neuron. Genesis 52, 528–543 (2014).
Zheng, C., Jin, F. Q., Trippe, B. L., Wu, J. & Chalfie, M. Inhibition of cell fate repressors secures the differentiation of the touch receptor neurons of Caenorhabditis elegans. Development 145, dev168096 (2018). 1.
Kerk, S. Y., Kratsios, P., Hart, M., Mourao, R. & Hobert, O. Diversification of C. elegans motor neuron identity via selective effector gene repression. Neuron 93, 80–98 (2017).
Li, Y. et al. Cell context-dependent CFI-1/ARID3 functions control neuronal terminal differentiation. Cell Rep. 42, 112220 (2023).
Hsiao, H. Y., Jukam, D., Johnston, R. & Desplan, C. The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes. Dev. Biol. 381, 482–490 (2013).
Johnston, R. J. Jr. Lessons about terminal differentiation from the specification of color-detecting photoreceptors in the Drosophila retina. Ann. N. Y. Acad. Sci. 1293, 33–44 (2013).
Kratsios, P. et al. Transcriptional coordination of synaptogenesis and neurotransmitter signaling. Curr. Biol. 25, 1282–1295 (2015).
Kratsios, P., Stolfi, A., Levine, M. & Hobert, O. Coordinated regulation of cholinergic motor neuron traits through a conserved terminal selector gene. Nat. Neurosci. 15, 205–214 (2012).
Harms, F. L. et al. Mutations in EBF3 disturb transcriptional profiles and cause intellectual disability, ataxia, and facial dysmorphism. Am. J. Hum. Genet. 100, 117–127 (2017).
Kratsios, P. et al. An intersectional gene regulatory strategy defines subclass diversity of C. elegans motor neurons. Elife 6, e25751 (2017). 1.
Son, E. Y. & Crabtree, G. R. The role of BAF (mSWI/SNF) complexes in mammalian neural development. Am. J. Med. Genet. C Semin. Med. Genet. 166C, 333–349 (2014).
Sokpor, G., Xie, Y., Rosenbusch, J. & Tuoc, T. Chromatin remodeling BAF (SWI/SNF) complexes in neural development and disorders. Front. Mol. Neurosci. 10, 243 (2017).
Von Stetina, S. E., Treinin, M. & Miller, D. M. 3rd The motor circuit. Int. Rev. Neurobiol. 69, 125–167 (2006).
Cinar, H., Keles, S. & Jin, Y. Expression profiling of GABAergic motor neurons in Caenorhabditis elegans. Curr. Biol. 15, 340–346 (2005).
Li, Y. et al. Establishment and maintenance of motor neuron identity via temporal modularity in terminal selector function. Elife 9, e59464 (2020). 1.
Feng, W., Destain, H., Smith, J. J. & Kratsios, P. Maintenance of neurotransmitter identity by Hox proteins through a homeostatic mechanism. Nat. Commun. 13, 6097 (2022).
Minevich, G., Park, D. S., Blankenberg, D., Poole, R. J. & Hobert, O. CloudMap: a cloud-based pipeline for analysis of mutant genome sequences. Genetics 192, 1249–1269 (2012).
Thompson, M. Polybromo-1: the chromatin targeting subunit of the PBAF complex. Biochimie 91, 309–319 (2009).
Mathies, L. D., Kim, A. C., Soukup, E. M., Thomas, A. E. & Bettinger, J. C. PBRM-1/PBAF-regulated genes in a multipotent progenitor in Caenorhabditis elegans. G3 14, jkad297 (2024). 1.
Esposito, G., Di Schiavi, E., Bergamasco, C. & Bazzicalupo, P. Efficient and cell specific knock-down of gene function in targeted C. elegans neurons. Gene 395, 170–176 (2007).
Wang, S. et al. A toolkit for GFP-mediated tissue-specific protein degradation in C. elegans. Development 144, 2694–2701 (2017).
Hodges, C., Kirkland, J. G. & Crabtree, G. R. The many roles of BAF (mSWI/SNF) and PBAF complexes in cancer. Cold Spring Harb. Perspect. Med. 6, a026930 (2016). 1.
Bryant, P., Pozzati, G. & Elofsson, A. Improved prediction of protein-protein interactions using AlphaFold2. Nat. Commun. 13, 1265 (2022).
Mathies, L. D. et al. SWI/SNF chromatin remodeling regulates alcohol response behaviors in Caenorhabditis elegans and is associated with alcohol dependence in humans. Proc. Natl. Acad. Sci. USA 112, 3032–3037 (2015).
Large, E. E. & Mathies, L. D. Caenorhabditis elegans SWI/SNF subunits control sequential developmental stages in the somatic gonad. G3 4, 471–483 (2014).
Katsani, K. R., Mahmoudi, T. & Verrijzer, C. P. Selective gene regulation by SWI/SNF-related chromatin remodeling factors. Curr. Top. Microbiol. Immunol. 274, 113–141 (2003).
Dykhuizen, E. C. et al. BAF complexes facilitate decatenation of DNA by topoisomerase IIalpha. Nature 497, 624–627 (2013).
Pan, J. et al. The ATPase module of mammalian SWI/SNF family complexes mediates subcomplex identity and catalytic activity-independent genomic targeting. Nat. Genet. 51, 618–626 (2019).
Richmond, E. & Peterson, C. L. Functional analysis of the DNA-stimulated ATPase domain of yeast SWI2/SNF2. Nucleic Acids Res. 24, 3685–3692 (1996).
van der Vaart, A., Godfrey, M., Portegijs, V. & van den Heuvel, S. Dose-dependent functions of SWI/SNF BAF in permitting and inhibiting cell proliferation in vivo. Sci. Adv. 6, eaay3823 (2020).
Sawa, H., Kouike, H. & Okano, H. Components of the SWI/SNF complex are required for asymmetric cell division in C. elegans. Mol. Cell 6, 617–624 (2000).
Pocock, R. et al. Neuronal function of Tbx20 conserved from nematodes to vertebrates. Dev. Biol. 317, 671–685 (2008).
Gerstein, M. B. et al. Integrative analysis of the Caenorhabditis elegans genome by the modENCODE project. Science 330, 1775–1787 (2010).
Humphreys, I. R. et al. Computed structures of core eukaryotic protein complexes. Science 374, eabm4805 (2021).
Rennie, M. L., Arkinson, C., Chaugule, V. K. & Walden, H. Cryo-EM reveals a mechanism of USP1 inhibition through a cryptic binding site. Sci. Adv. 8, eabq6353 (2022).
Rennie, M. L. & Oliver, M. R. Emerging frontiers in protein structure prediction following the AlphaFold revolution. J. R. Soc. Interface 22, 20240886 (2025).
Weeratunga, S. et al. Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2. J. Biol. Chem. 300, 105541 (2024).
Javer, A., Ripoll-Sanchez, L. & Brown, A. E. X. Powerful and interpretable behavioural features for quantitative phenotyping of Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 373, 20170375 (2018). 1.
Yemini, E., Jucikas, T., Grundy, L. J., Brown, A. E. & Schafer, W. R. A database of Caenorhabditis elegans behavioral phenotypes. Nat. Methods 10, 877–879 (2013).
Tolstenkov, O. et al. Functionally asymmetric motor neurons contribute to coordinating locomotion of Caenorhabditis elegans. Elife 7, e34997 (2018). 1.
Lessard, J. et al. An essential switch in subunit composition of a chromatin remodeling complex during neural development. Neuron 55, 201–215 (2007).
Yoo, A. S., Staahl, B. T., Chen, L. & Crabtree, G. R. MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. Nature 460, 642–646 (2009).
Braun, S. M. G. et al. BAF subunit switching regulates chromatin accessibility to control cell cycle exit in the developing mammalian cortex. Genes Dev. 35, 335–353 (2021).
Staahl, B. T. et al. Kinetic analysis of npBAF to nBAF switching reveals exchange of SS18 with CREST and integration with neural developmental pathways. J. Neurosci. 33, 10348–10361 (2013).
Tuoc, T. C. et al. Chromatin regulation by BAF170 controls cerebral cortical size and thickness. Dev. Cell 25, 256–269 (2013).
Narayanan, R. et al. Loss of BAF (mSWI/SNF) complexes causes global transcriptional and chromatin state changes in forebrain development. Cell Rep. 13, 1842–1854 (2015).
Tuoc, T. et al. Ablation of BAF170 in developing and postnatal dentate gyrus affects neural stem cell proliferation, differentiation, and learning. Mol. Neurobiol. 54, 4618–4635 (2017).
Matsumoto, S. et al. Brg1 is required for murine neural stem cell maintenance and gliogenesis. Dev. Biol. 289, 372–383 (2006).
Vogel-Ciernia, A. et al. The neuron-specific chromatin regulatory subunit BAF53b is necessary for synaptic plasticity and memory. Nat. Neurosci. 16, 552–561 (2013).
Kim, B. et al. Neuronal activity-induced BRG1 phosphorylation regulates enhancer activation. Cell Rep. 36, 109357 (2021).
Wu, J. I. et al. Regulation of dendritic development by neuron-specific chromatin remodeling complexes. Neuron 56, 94–108 (2007).
Parrish, J. Z., Kim, M. D., Jan, L. Y. & Jan, Y. N. Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites. Genes Dev. 20, 820–835 (2006).
Bachmann, C. et al. mSWI/SNF (BAF) complexes are indispensable for the neurogenesis and development of embryonic olfactory epithelium. PLoS Genet. 12, e1006274 (2016).
Weinberg, P., Flames, N., Sawa, H., Garriga, G. & Hobert, O. The SWI/SNF chromatin remodeling complex selectively affects multiple aspects of serotonergic neuron differentiation. Genetics 194, 189–198 (2013).
Waldhauser, V., Baroti, T., Frob, F. & Wegner, M. PBAF subunit Pbrm1 selectively influences the transition from progenitors to pre-myelinating cells during oligodendrocyte development. Cells 12, 1556 (2023). 1.
Xu, Y. et al. Inactivation of BRD7 results in impaired cognitive behavior and reduced synaptic plasticity of the medial prefrontal cortex. Behav. Brain Res. 286, 1–10 (2015).
Ruijtenberg, S. & van den Heuvel, S. G1/S inhibitors and the SWI/SNF complex control cell-cycle exit during muscle differentiation. Cell 162, 300–313 (2015).
Poole, R. J., Flames, N. & Cochella, L. Neurogenesis in Caenorhabditis elegans. Genetics 228, iyae116 (2024). 1.
Glenwinkel, L. et al. In silico analysis of the transcriptional regulatory logic of neuronal identity specification throughout the C. elegans nervous system. Elife 10, e64906 (2021). 1.
Reilly, M. B. et al. Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification. PLoS Genet. 18, e1010372 (2022).
Jafari, S. et al. Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression. PLoS Biol. 10, e1001280 (2012).
Eade, K. T., Fancher, H. A., Ridyard, M. S. & Allan, D. W. Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system. PLoS Genet. 8, e1002501 (2012).
Cheng, L. et al. Lmx1b, Pet-1, and Nkx2.2 coordinately specify serotonergic neurotransmitter phenotype. J. Neurosci. 23, 9961–9967 (2003).
Kitt, M. M. et al. An adult-stage transcriptional program for survival of serotonergic connectivity. Cell Rep. 39, 110711 (2022).
Ng, Y. H. et al. Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics. Stem Cell Rep. 16, 1763–1776 (2021).
Stott, S. R. et al. Foxa1 and foxa2 are required for the maintenance of dopaminergic properties in ventral midbrain neurons at late embryonic stages. J. Neurosci. 33, 8022–8034 (2013).
O’Meara, M. M., Zhang, F. & Hobert, O. Maintenance of neuronal laterality in Caenorhabditis elegans through MYST histone acetyltransferase complex components LSY-12, LSY-13 and LIN-49. Genetics 186, 1497–1502 (2010).
Bordet, G., Couillault, C., Soulavie, F., Filippopoulou, K. & Bertrand, V. PRC1 chromatin factors strengthen the consistency of neuronal cell fate specification and maintenance in C. elegans. PLoS Genet. 18, e1010209 (2022).
Padeken, J., Methot, S. P. & Gasser, S. M. Establishment of H3K9-methylated heterochromatin and its functions in tissue differentiation and maintenance. Nat. Rev. Mol. Cell Biol. 23, 623–640 (2022).
Toskas, K. et al. PRC2-mediated repression is essential to maintain identity and function of differentiated dopaminergic and serotonergic neurons. Sci. Adv. 8, eabo1543 (2022).
Lipinski, M. et al. KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain. Nat. Commun. 11, 2588 (2020).
Lipinski, M. et al. CBP is required for establishing adaptive gene programs in the adult mouse brain. J. Neurosci. 42, 7984–8001 (2022).
Kaeser, M. D., Aslanian, A., Dong, M. Q., Yates, J. R. 3rd & Emerson, B. M. BRD7, a novel PBAF-specific SWI/SNF subunit, is required for target gene activation and repression in embryonic stem cells. J. Biol. Chem. 283, 32254–32263 (2008).
Kakarougkas, A. et al. Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin. Mol. Cell 55, 723–732 (2014).
Soshnikova, N. V. et al. PHF10 subunit of PBAF complex mediates transcriptional activation by MY