Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism Wendy Wenderskia,b,c,d, Lu Wange,f,g,1, Andrey Krokhotina,b,c,d,1, Jessica J. Walshh, Hongjie Lid,i, Hirotaka Shojij, Shereen Ghoshe,f,g, Renee D. Georgee,f,g, Erik L. Millera,b,c,d, Laura Eliasa,b,c,d, Mark A. Gillespiek, Esther Y. Sona,b,c,d, Brett T. Staahla,b,c,d, Seung Tae Baeke,f,g, Valentina Stanleye,f,g, Cynthia Moncadaa,b,c,d, Zohar Shiponya,b,c,d, Sara B. Linkerl, Maria C. N. Marchettol, Fred H. Gagel, Dillon Chene,f,g, Tipu Sultanm, Maha S. Zakin, Jeffrey A. Ranishk, Tsuyoshi Miyakawaj, Liqun Luod,i, Robert C. Malenkah, Gerald R. Crabtreea,b,c,d,2, and Joseph G. Gleesone,f,g,2 aDepartment of Pathology, Stanford Medical School, Palo Alto, CA 94305; bDepartment of Genetics, Stanford Medical School, Palo Alto, CA 94305; cDepartment of Developmental Biology, Stanford Medical School, Palo Alto, CA 94305; dHoward Hughes Medical Institute, Stanford University, Palo Alto, CA 94305; eDepartment of Neuroscience, University of California San Diego, La Jolla, CA 92037; fHoward Hughes Medical Institute, University of California San Diego, La Jolla, CA 92037; gRady Children’s Institute of Genomic Medicine, University of California San Diego, La Jolla, CA 92037; hNancy Pritztker Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford Medical School, Palo Alto, CA 94305; iDepartment of Biology, Stanford University, Palo Alto, CA 94305; jDivision of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University, 470-1192 Toyoake, Aichi, Japan; kInstitute for Systems Biology, Seattle, WA 98109; lLaboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037; mDepartment of Pediatric Neurology, Institute of Child Health, Children Hospital Lahore, 54000 Lahore, Pakistan; and nClinical Genetics Department, Human Genetics and Genome Research Division, National Research Centre, 12311 Cairo, Egypt Edited by Arthur L. Beaudet, Baylor College of Medicine, Houston, TX, and approved March 9, 2020 (received for review May 20, 2019) Synaptic activity in neurons leads to the rapid activation of genes Although present in all cells, BAF complexes orchestrate cell involved in mammalian behavior. ATP-dependent chromatin type-specific functions through combinatorial assembly of ∼15 remodelers such as the BAF complex contribute to these responses subunits from the products of 29 genes (5). During neural and are generally thought to activate transcription. However, the “ ” mechanisms keeping such early activation genes silent have NEUROSCIENCE been a mystery. In the course of investigating Mendelian recessive Significance autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally Autism is a complex neurodevelopmental disorder whose named BAF53b). Accordingly, ACTL6B was the most significantly causative mechanisms are unclear. Taking advantage of a mutated gene in the Simons Recessive Autism Cohort. At least 14 unique cohort with recessively inherited autism, we identified subunits of the nBAF complex are mutated in autism, collectively six families with biallelic mutation of the neuronal-specific making it a major contributor to autism spectrum disorder (ASD). subunit of the BAF complex, ACTL6B (also known as BAF53b). Patient mutations destabilized ACTL6B protein in neurons and Relative to all other genes, ACTL6B was the most statistically rerouted dendrites to the wrong glomerulus in the fly olfactory significant mutated gene in the recessive autism cohort. We system. Humans and mice lacking ACTL6B showed corpus callosum describe autism-relevant phenotypes in human brain organoids hypoplasia, indicating a conserved role for ACTL6B in facilitating and in mouse and fly models. We foresee the outcomes from neural connectivity. Actl6b knockout mice on two genetic back- this study will be the following: 1) a link between neuronal grounds exhibited ASD-related behaviors, including social and activity-dependent transcriptional repression and autism; 2) a memory impairments, repetitive behaviors, and hyperactivity. Sur- characterization of mouse and fly models to study ACTL6B prisingly, mutation of Actl6b relieved repression of early response mutant autism; and 3) an understanding the role of ACTL6B genes including AP1 transcription factors (Fos, Fosl2, Fosb, and and nBAF complexes in neuronal transcriptional regulation. Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with Author contributions: W.W., J.J.W., H.L., H.S., E.L.M., L.E., F.H.G., T.M., L.L., R.C.M., G.R.C., early response transcription factor activity. ACTL6B loss is thus and J.G.G. designed research; W.W., L.W., A.K., J.J.W., H.L., H.S., S.G., L.E., M.A.G., E.Y.S., B.T.S., C.M., S.B.L., M.C.N.M., T.S., M.S.Z., and J.G.G. performed research; W.W., L.W., an important cause of recessive ASD, with impaired neuron- S.T.B., V.S., Z.S., F.H.G., J.A.R., T.M., L.L., R.C.M., G.R.C., and J.G.G. contributed new re- specific chromatin repression indicated as a potential mechanism. agents/analytic tools; W.W., L.W., A.K., J.J.W., H.L., H.S., R.D.G., M.A.G., E.Y.S., B.T.S., S.T.B., V.S., C.M., Z.S., D.C., and J.G.G. analyzed data; W.W., G.R.C., and J.G.G. wrote the paper; A.K. helped to write a section of the paper; and S.G. contributed preliminary autism | mouse model | recessive | BAF | activity dependent data that helped inform our decision to switch to the brain organoid system. Competing interest statement: G.R.C. is a founder of Foghorn Therapeutics. utism spectrum disorder (ASD) represents a heterogeneous This article is a PNAS Direct Submission. Agroup of neurodevelopmental disorders that are character- This open access article is distributed under Creative Commons Attribution-NonCommercial- ized by social deficits and restricted or repetitive behaviors, and NoDerivatives License 4.0 (CC BY-NC-ND). ∼ affect 1% of children worldwide (1, 2). The heritability of ASD Data deposition: The exome-sequencing data from individuals in this study have been is estimated to be ∼80% (3), implicating genetic mutation as the deposited in the Database of Genotypes and Phenotypes (dbGaP), https://www.ncbi.nlm. prominent cause of autism. Indeed, exome-sequencing studies nih.gov/gap/ (accession no. phs000288.v2.p2) and are available upon reasonable request. have identified hundreds of genetic mutations that substantially The raw RNA- and ATAC-sequencing data have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE147056). increase ASD risk (4). Among the most frequently mutated genes The processed RNA-sequencing dataset is included as Dataset S1 and ATAC-sequencing in ASD are subunits of the mammalian SWI/SNF (BAF) ATP- dataset as Dataset S2. dependent chromatin remodeling complex (4). BAF complexes 1L.W. and A.K. contributed equally to this work. facilitate dynamic changes in gene expression by controlling DNA 2To whom correspondence may be addressed. Email: [email protected] or accessibility to the transcriptional machinery (5). To accomplish this, [email protected]. BAF complexes mobilize nucleosomes, evict polycomb repressive This article contains supporting information online at https://www.pnas.org/lookup/suppl/ complexes, and recruit type II topoisomerases that decatenate doi:10.1073/pnas.1908238117/-/DCSupplemental. DNA (6–9). First published April 20, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1908238117 PNAS | May 5, 2020 | vol. 117 | no. 18 | 10055–10066 Downloaded by guest on October 1, 2021 development, exit from the cell cycle is accompanied by BAF Appendix, Fig. S1C) (25). Individuals with recessive ASD-like subunit exchange: neural progenitor (np) subunits ACTL6A phenotypes linked to biallelic ACTL6B mutations have also (BAF53a), DPF2/PHF10 (BAF45a/d), and SS18 are exchanged been identified in recent case studies (29, 40, 41). Cumulatively, for neuron-specific (n) subunits ACTL6B (BAF53b), DPF1/3 these findings implicate mutations in ACTL6B as an important (BAF45b/c), and SS18L1 (CREST), respectively (10–13). Subunit cause of recessive autism. exchange is critical for neuronal function, as genetic deletion of Focusing on ACTL6B, we more closely examined the patient either ACTL6B or SS18L1 impairs activity-dependent dendritic phenotype and sought to understand how mutations in this gene arborization (14, 15). Furthermore, expression of the two microRNAs, give rise to autism. Six families representing 4.4% of the SRAC miR9* and miR124, which control BAF subunit switching, is sufficient cohort demonstrated homozygous variants in ACTL6B with fully to convert fibroblasts into neurons (13). penetrant recessive inheritance (Fig. 1D and SI Appendix, Table Recent findings link mutations in nearly every constitutive S1). Variants were not present in ExAC or gnomAD databases, member of the BAF complex to ASD or intellectual disability (ID), nor in ethnically matched controls from the Greater Middle East including syndromic forms such as Coffin–Siris and Nicolaides– – Variome or the cohort of 256 controls (42, 43), indicating that Baraitser syndromes (16 18). Implicated subunits include the fol- they are rare variants. Subjects exhibited nonverbal autism with ARID1B ARID1A lowing (protein/gene): BAF250b/ , BAF250a/ , stereotypies, as well as ID, developmental delay, hyperactivity, BAF200/ARID2,BCL11A/BCL11A,BRG1/SMARCA4,BRM/ and mild spasticity (SI Appendix, Table S2 and Movie S1). There SMARCA2, BAF155/SMARCC1, BAF170/SMARCC2, BAF45a/ were no syndromic features that would have identified these PHF10, BAF45d/DPF2 BAF47/SMARCB1, BAF57/SMARCE1, SI Ap- BAF53a/ACTL6A, BAF53b/ACTL6B, BAF60a/SMARCD1, and patients as unique a priori from other SRAC subjects ( β-actin/ACTB (17, 19–29). BAF mutant forms of ASD share over- pendix, Table S2 and Supplementary Text). Epilepsy was seen in lapping clinical features such as corpus callosal hypoplasia, epilepsy, all subjects, presenting at 2 wk to 5 y of age, and generally ID, lack of speech, craniofacial abnormalities, developmental de- responded to anticonvulsant medication.