Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism

Abstract

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.

Keywords: BAF; activity dependent; autism; mouse model; recessive.

Fig. 1.

Biallelic mutations in ACTL6B cause recessive autism. (A) Q-Q plot showing the observed/expected number of mutations for all coding genes in the SRAC. ACTL6B and CD36 were significantly mutated. (B) Q-Q plot showing the observed/expected number of mutations for all coding genes in a genetically matched non-ASD recessive neurodevelopmental cohort, where ACTL6B was not found to be enriched. (C) ACTL6B encodes a tissue-restricted subunit of the neuronal (nBAF) BAF complex. Representation of the multisubunit nBAF complex containing ubiquitously expressed subunits (gray), a core ATPase subunit (dark blue), and neuronal-specific subunits (yellow) including ACTL6B in bold. The balls on a string represent nucleosomes. (D) Recessive ASD inheritance with ACTL6B mutations in six independent consanguineous families. Double lines, first cousin status; squares, males; circles, females; slash-through, mortality; black fill, ASD. Missense variants (green) and truncating variants (blue). Obligate carriers depicted with dot at center of symbol.

Fig. 2.

Patient mutations destabilize ACTL6B protein and cause a loss-of-function “perfect dendritic retargeting” phenotype in the fly olfactory system. (A) Patient missense mutant proteins were less stable than wild-type ACTL6B when expressed in HEK293T cells; n = 2 for T175P and G417W; n = 4 for wild type, L154F, and G393R. (B) Endogenous ACTL6B protein was decreased in human neurons derived from iPSCs of two affected individuals (ACTL6B^L154F/L154F) in family 2703 relative to the wild-type control; n = 3 replicates. Neurofilament light chain, a mature neuronal marker that confirms differentiation status. (C) Missense mutant proteins were less stable than wild-type ACTL6B when expressed for 2 wk in cultured striatal neurons from Actl6b^−/− E18.5 mice; n = 2. (D) Missense mutant proteins showed decreased recovery following coimmunoprecipitation with SMARCA4 from striatal neurons shown in C; n = 1 (see similar results in cortical neurons, human ESCs, and HEK293Ts shown in SI Appendix, Fig. S4 B–E). (E) MARCM (mosaic analysis with a repressible cell marker) was used to fluorescently label and replace fly Bap55 with human ACTL6B alleles in olfactory projection neurons (PNs). PNs always project a dendrite to the dorsolateral glomerulus (DL1) in the posterior antennal lobe. Clones were induced in GH146-GAL4 flies at a time when DL1 PNs were born. Replacement of the fly ortholog with human wild-type ACTL6B but not patient variants (L154F or G393R) rescued the perfect dendritic retargeting phenotypes in single-neuron clones. Arrows, cell bodies; Ncad, neuropil marker. (Scale bar, 20 µm.) (F) Quantification of E where single PNs were correctly targeted to DL1 or mistargeted to DA4l or DM6; n = animals per condition.

Fig. 3.

Loss of ACTL6B causes corpus callosum hypoplasia in humans and mice. (A) Human brain MRI scans of control compared with ACTL6B mutated subjects. (Top row) Midline sagittal. White arrows: corpus callosum hypoplasia. (Bottom row) Axial images at the level of the foramen of Monroe showing paucity of white matter compared with control. (B) Adult mouse coronal brain slices from wild type, Actl6b^+/−, or Actl6b^−/− were stained with antibodies to neurofilament light chain (NFL) to visualize callosal axon tracks. Representative images of male wild-type, Actl6b^+/−, and Actl6b^−/− brain slices. Actl6b^−/− corpus callosum was visibly thinner, indicated with a white arrow. (C) Quantification of relative callosal thickness showing significantly thinner callosum in knockouts. Blinded measurements from n = 2 slices were averaged for each animal and compared for n = 4 mice per genotype. (D) Quantification of relative thickness of the somatosensory cortex in slices used for C showing no significant difference. Significance was assessed by ordinary one-way ANOVA with Tukey’s correction for multiple comparisons; F_(2,9) = 10.73 for C; F_(2,9) = 0.62 for D. Error bars indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Actl6b^−/− mice exhibit autism-related behaviors. (A) Social interaction and control tests: relative interaction time between the adult test mouse and a juvenile mouse (3–5 wk) or novel object. Open-field test measured activity over 20 min as total distance traveled. (B) Schematic of three-chamber sociability assay: Test mice may enter the zone with a novel object or the zone with a novel juvenile mouse. Social preference scores were calculated from time spent in each zone using the formula shown. (C) Box plots showing male and female littermates of Actl6b^+/− × Actl6b^+/− crosses with gene dosage-dependent impairment in social interaction with a juvenile mouse but not with (D) a control novel object. (E) Actl6b^+/− and Actl6b^−/− mice of both sexes showed defects in sociability, which were most severe in female knockouts. (F) Single, repeated photobeam breaks or “stereotypic counts” represented by a small red box in an open field. Male Actl6b^−/− mice showed repetitive movements indicated from increased stereotypic counts over 120 min. (G) Diagram of Barnes maze test of spatial memory: an elevated, white, circular open field containing 12 holes, with one “target” escape hole leading to a comfortable cage. Visual cues provided the mouse with a frame of reference for the location of the target hole. Male Actl6b^−/− mice spent less time around the target hole, indicating impaired memory as to the location of the target hole. (H) Actl6b^−/− mice of both sexes showed increased activity, not observed in Actl6b^+/−. Tests shown in C, E, and H were conducted at Stanford University on a cohort of mice highly backcrossed to the C57BL/6 background; mice (^+/+, ^+/−, ^−/−): n = 9, 12, 7 males, n = 11, 12, 10 females for C; n = 10, 13, 6 males, n = 11, 12, 9 females for D; n = 10, 12, 7 males, n = 11, 12, 9 females for E; n = 10, 13, 6 males, n = 11, 11, 5 females for H. Values for each test in this cohort were as follows: F_{(2,30)Females} = 79.87 and F_(2,25)Males = 59.49 for C; F_{(2,29)Females} = 0.29 and F_(2,26)Males = 0.74 for D; F_{(2,29)Females} = 6.82 and F_(2,26)Males = 4.83 for E; and F_{(2,24)Females} = 13.44 and F_(2,26)Males = 8.60 for H. Stereotypic counts in F and spatial memory in G were assessed at Fujita Health University on adult male mice that were the F₁ offspring of a cross between Actl6b^+/− 129S6/SvEv × Actl6b^+/− C57BL/6. Mice (^+/+, ^−/−): n = 22, 21 in F, and n = 22, 14 in G. Significance was calculated for B, E, and H using a one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison post hoc. Significance for F and G was calculated using Student’s t test: t₄₁ = 8.68 in F and t₃₄ = 3.99 in G. Whiskers indicate 10th and 90th percentiles. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

ACTL6B suppresses the activity-responsive transcriptional program in resting neurons. (A) Experimental design: Primary E16.5/DIV7 cortical cultures from (n = 5) wild-type or (n = 7) Actl6b^−/− littermates were treated for 2 h with TTX/APV to silence action potentials and represent the “resting” neuronal state. RNA was collected to measure transcription by RNA-seq and DNA was transposed with Tn5 to measure chromatin accessibility by ATAC-seq. (B) MA plot of transcriptional changes in resting Actl6b^−/− (KO) neurons relative to wild type (WT). Differentially expressed genes showing FDR < 5% and absolute log₂ fold change >0.5 were defined as “significant” and highlighted in red. Gene names are shown for the top 30 most significant genes. (C) Transcription factor coexpression analysis was performed on the significantly up- or down-regulated genes in resting Actl6b^−/− (KO) neurons. Up-regulated genes commonly coexpress with early response transcription factors such as Jun, Nr4a2, and Fosb, indicating possible activity-dependent transcription factor activity. (D) Heat maps showing log₂ fold changes for genes that were both significantly differentially expressed in resting Actl6b^−/− (KO) neurons and in wild-type (WT) neurons that were stimulated for 1 or 6 h with 55 mM KCl. Early response genes showed altered expression after 1-h KCl stimulation in wild type (SI Appendix, Fig. S10A) and late response genes showed altered expression after 6-h KCl stimulation in wild type (SI Appendix, Fig. S10B). Representative genes from each group are labeled. Transcriptional changes in resting Actl6b^−/− neurons significantly correlated with activity-induced responses in wild type. (E) mRNA expression of activity-responsive genes in the biological samples used for RNA-seq, measured by RT-qPCR. AP1 transcription factors Fos, Fosb, Fosl2, and their late response target gene Vgf were significantly increased in resting Actl6b^−/− neurons. (F) mRNA expression of activity-responsive genes in 28-d-old cerebral brain organoids cultured from induced pluripotent cells of an unaffected father (ACTL6B^L154F/+) and his two affected children (ACTL6B^L154F/L154F) in family 2703, measured by RT-qPCR; n = 3 technical replicates per individual. AP1 transcription factors FOS, FOSB, FOSL2, JUN, and their late response target gene VGF were significantly increased in affected human brain organoids. (G) Chromatin accessibility was assayed by ATAC-seq as described in A, and HOMER de novo motif analysis was performed on the significantly increased or decreased sites in Actl6b^−/− (KO) neurons. Sites with increased chromatin accessibility were selectively enriched for the AP1 transcription factor binding motif, indicated by FRA1. (H) Summary model: Autism mutant ACTL6B “B” proteins are unstable and rapidly degraded, leading to retention of the nonneuronal homolog ACTL6A “A” in the nBAF complex. The loss of ACTL6B relieves transcriptional repression on early response transcription factors but increases repression on repetitive elements in resting neurons. mRNAs encoding early response transcription factors, particularly those in the AP1 family, are translated into proteins that regulate the expression of late response neuronal genes. Multisubunit nBAF complexes containing ubiquitously expressed subunits (gray), a core ATPase subunit (dark blue), neuronal-specific subunits (yellow), and neural-progenitor subunit ACTL6A (purple). AP1 transcription factor proteins are shown in pink. Balls on a string indicate nucleosomes. The arrow represents transcriptional activation; T represents transcriptional repression. Dashed lines in H indicate that the mechanism of repression may be direct or indirect. Significance was calculated by Spearman rank correlation in D, individual Student’s t tests in E, and two-way analysis of variance (ANOVA) in F. Error bars indicate SEM. *P < 0.05; **P < 0.01; ***P < 0.001.