Rescue of deficits by Brwd1 copy number restoration in the Ts65Dn mouse model of Down syndrome

Abstract

With an incidence of ~1 in 800 births, Down syndrome (DS) is the most common chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA21 that directly contribute to cognitive deficits remain incompletely understood. Here, we found that the HSA21-encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued deficits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes.

Fig. 1. Brwd1 copy number restoration rescues synaptic and cognitive deficits in male trisomic animals.

a qPCR expression data for Brwd1 in embryonic day (E) 17.5 forebrain (FB) and adult (6-week) PFC, hippocampus (HIPP) and cerebellum (CER) from euploid vs. Ts65Dn male mice. A.U. = Arbitrary Units, with experimental (non-euploid) group averages normalized to respective euploid controls. b Schematic depicting the generation of mouse genotypes to be investigated. c qPCR expression data for Brwd1 in euploid vs. Brwd1^+/– vs. Ts65Dn vs. Ts65Dn;Brwd1^+/– E17.5 forebrain. d Deficiency of hippocampal LTP in adult male (6-week) Ts65Dn mice is rescued by Brwd1 copy number restoration. The representative traces were recorded at the end of the baseline period (dashed lines) and 60 min after induction of LTP (solid lines). Calibrations: 0.5 mV/5 ms. e Context dependent fear conditioning—displayed as % freezing in the trained context—comparing euploid vs. Brwd1^+/– vs. Ts65Dn vs. Ts65Dn;Brwd1^+/– mice. Data are presented as averages ± SEM. See Supplementary Information Materials for full caption with n’s and statistics. Source data are provided as a source data file.

Fig. 2. Rescue of aberrant gene expression in male trisomic brain by Brwd1 renormalization.

a RNA-seq heatmaps of DE genes comparing euploid vs. Ts65Dn vs. Ts65Dn;Brwd1^+/– adult male (6-week) hippocampus. Normalized RNA expression values (averaged between replicates) were used to generate z-scores for each row. b Normalized heatmaps of RNA expression values for DE genes in adult male hippocampus that display pairwise significant regulation between Ts65Dn vs. euploid, and are rescued in Ts65Dn;Brwd1^+/– vs. Ts65Dn. c Heatmap displays Jaccard index, as well as adjusted p-values, from odds ratio analyses of the overlap between DE genes from euploid vs. Ts65Dn vs. Ts65Dn;Brwd1^+/– comparisons and previously published human DS single-nuclei RNA-seq data vs. age-matched controls. d Bar graph of −log10(adj. p-val) for gene ontology (GO) processes displaying enrichment for PCGs identified in c above. See Supplementary Information Materials for full caption with n’s and statistics. Source data are provided as a source data file.

Fig. 3. BRWD1 tightly associates with the BAF complex in euploid brain.

a Schematic of mouse brain soluble nuclear protein extract (NE) preparation, density sedimentation of nuclear proteins over a 10–30% glycerol gradient, and immunoprecipitation of BAF chromatin remodeling complexes. Blue lettering indicates neuronal-specific BAF subunits. Red lettering indicates PBAF-specific subunits. b Density sedimentation of adult Brwd1^FLAG-HA brain NE over a 10–30% glycerol gradient indicates that BRWD1 predominantly associates with large protein complexes. Subunits of BAF and AP-1 complexes serve as molecular weight markers: SMARCA2/4 antibody indicates all BAF complexes including non-canonical GBAF (~1 MDa), canonical BAF (~2 MDa) and Polybromo-containing BAF (PBAF, ~3 MDa); ACTL6B and SS18L1 indicate neuronal-specific BAF complexes; c-Jun indicates AP-1 (160–440 kDa). HA signal at the expected molecular weight of BRWD1-FLAG-HA (~260 kDa) is observed in fractions containing the BAF complex. c Endogenous BRWD1-FLAG-HA interacts with BAF complexes in embryonic brain. BAF complexes were immunoprecipitated from Brwd1^FLAG-HA brain NE with antibodies against the BAF core ATPase SMARCA4, the neural progenitor subunit SS18, the neuronal subunit SS18L1 or IgG as a control. Endogenous BRWD1-FLAG-HA robustly co-immunoprecipitated with SMARCA4 and the neural progenitor subunit SS18, but less so with the neuronal subunit SS18L1 from E17.5 brain. d BAF complexes purified from adult Brwd1^FLAG-HA brain NE with antibodies against SMARCA4 or the neuronal subunit SS18L1 co-immunoprecipitate BRWD1-FLAG-HA. e The stability of the BAF:BRWD1-FLAG-HA interaction was challenged with increasing concentrations (0.25-4 M) of the denaturing agent, urea. A fraction of BRWD1 remained bound to BAF in up to 4 M urea, surpassing the stability of the dedicated BAF subunit, SMARCB1. f Quantification of urea denaturation experiments, as shown in e, with the amount of bound protein normalized to the amount of immunoprecipitated SMARCA4 (n = 3 experiments). Source data are provided as a source data file. See Supplementary Fig. 15 for uncropped blots with MW markers.

Fig. 4. Brwd1 renormalization partially rescues genomic BAF complex mistargeting in male trisomic brain.

a Heatmaps of normalized SMARCA2/4 enrichment in euploid vs. Ts65Dn vs. Ts65Dn;Brwd1^+/– adult male (6-week) hippocampus centered (±5 kb) over sites of differential SMARCA2/4 enrichment comparing Ts65Dn vs. euploid mice, separated by genomic context. b Volcano plot depicting regulation of SMARCA2/4 enriched PCGs in euploid animals displaying differential enrichment in Ts65Dn mice; gray circles = unregulated PCGs. Of the PCGs regulated with respect to Smarca2/4 enrichment, 595 are rescued (red circles) in Ts65Dn;Brwd1^+/– mice, whereas the remainder of PCGs do not display such rescue (black circles). c Relative frequency (observed/expected overlap in base pairs) of each chromatin state within significant differentially enriched sites for SMARCA2/4 (Ts65Dn vs. euploid). Chromatin states were obtained from brain regions included in the Roadmap Epigenomics Project. d Bubble plots of GO terms (burgundy) and KEGG pathways (purple) displaying enrichment for rescued differentially enriched PCGs identified in b above. e Odds ratio analysis of overlapping differentially accessible sites in Ts65Dn vs. euploid animals and Ts65Dn;Brwd1^+/– animals, separated by direction of regulation. f Odds ratio analysis of overlapping PCGs displaying rescued differential SMARCA2/4 enrichment in Ts65Dn vs. euploid animals vs. PCGs displaying differential neuronal chromatin accessibility in Ts65Dn vs. euploid mice that are either rescued, or not, in their differential accessibility in Ts65Dn;Brwd1^+/– animals. Insert numbers indicate respective p values for associations, followed by the number of PCGs overlapping per category. See Supplementary Information Materials for full caption with n’s and statistics. Source data are provided as a source data file.