Five autism-associated transcriptional regulators target shared loci proximal to brain-expressed genes.

Many autism spectrum disorder (ASD)-associated genes act as transcriptional regulators (TRs). Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify the regulatory targets of ARID1B, BCL11A, FOXP1, TBR1, and TCF7L2, ASD-associated TRs in the developing human and mouse cortex. These TRs shared substantial overlap in the binding sites, especially within open chromatin. The overlap within a promoter region, 1-2,000 bp upstream of the transcription start site, was highly predictive of brain-expressed genes. This signature was observed in 96 out of 102 ASD-associated genes. In vitro CRISPRi against ARID1B and TBR1 delineated downstream convergent biology in mouse cortical cultures. After 8 days, NeuN+ and CALB+ cells were decreased, GFAP+ cells were increased, and transcriptomic signatures correlated with the postmortem brain samples from individuals with ASD. We suggest that functional convergence across five ASD-associated TRs leads to shared neurodevelopmental outcomes of haploinsufficient disruption.

Massively parallel reporter assays and mouse transgenic assays provide complementary information about neuronal enhancer activity.

Genetic studies find hundreds of thousands of noncoding variants associated with psychiatric disorders. Massively parallel reporter assays (MPRAs) and in vivo transgenic mouse assays can be used to assay the impact of these variants. However, the relevance of MPRAs to in vivo function is unknown and transgenic assays suffer from low throughput. Here, we studied the utility of combining the two assays to study the impact of non-coding variants. We carried out an MPRA on over 50,000 sequences derived from enhancers validated in transgenic mouse assays and from multiple fetal neuronal ATAC-seq datasets. We also tested over 20,000 variants, including synthetic mutations in highly active neuronal enhancers and 177 common variants associated with psychiatric disorders. Variants with a high impact on MPRA activity were further tested in mice. We found a strong and specific correlation between MPRA and mouse neuronal enhancer activity including changes in neuronal enhancer activity in mouse embryos for variants with strong MPRA effects. Mouse assays also revealed pleiotropic variant effects that could not be observed in MPRA. Our work provides a large catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays.

AlphaMissense is better correlated with functional assays of missense impact than earlier prediction algorithms.

Missense variants that alter a single amino acid in the encoded protein contribute to many human disorders but pose a substantial challenge in interpretation. Though these variants can be reliably identified through sequencing, distinguishing the clinically significant ones remains difficult, such that "Variants of Unknown Significance" outnumber those classified as "Pathogenic" or "Likely Pathogenic." Numerous in silico approaches have been developed to predict the functional impact of missense variants to inform clinical interpretation, the latest being AlphaMissense, which uses artificial intelligence methods trained on predicted protein structure. To independently assess the performance of AlphaMissense and 38 other predictors of missense severity, we compared predictions to data from multiplexed assays of variant effect (MAVE). MAVE experiments generate almost every possible individual amino acid change in a gene and measure their functional impact using a high-throughput assay. Assessing 17,696 variants across five genes (DDX3X, MSH2, PTEN, KCNQ4, and BRCA1), we find that AlphaMissense is consistently one of the top five algorithms based on correlation with functional impact and is the best-correlated algorithm for two genes. We conclude that AlphaMissense represents the current best-in-class predictor by this metric; however, the improvement over other algorithms is modest. We note that multiple missense predictors, including AlphaMissense, appear to overcall variants as pathogenic despite minimal functional impact and that substantially more high-quality training data, including consistently analyzed patient cohorts and MAVE analyses, are required to improve accuracy.

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements.

CRISPR-based gene activation (CRISPRa) is a promising therapeutic approach for gene therapy, upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis- regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to candidate cis- regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically and potently upregulating target genes, including autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans- acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.

Celf4 controls mRNA translation underlying synaptic development in the prenatal mammalian neocortex.

Abnormalities in neocortical and synaptic development are linked to neurodevelopmental disorders. However, the molecular and cellular mechanisms governing initial synapse formation in the prenatal neocortex remain poorly understood. Using polysome profiling coupled with snRNAseq on human cortical samples at various fetal phases, we identify human mRNAs, including those encoding synaptic proteins, with finely controlled translation in distinct cell populations of developing frontal neocortices. Examination of murine and human neocortex reveals that the RNA binding protein and translational regulator, CELF4, is expressed in compartments enriched in initial synaptogenesis: the marginal zone and the subplate. We also find that Celf4/CELF4-target mRNAs are encoded by risk genes for adverse neurodevelopmental outcomes translating into synaptic proteins. Surprisingly, deleting Celf4 in the forebrain disrupts the balance of subplate synapses in a sex-specific fashion. This highlights the significance of RNA binding proteins and mRNA translation in evolutionarily advanced synaptic development, potentially contributing to sex differences.

Alterations in Retrotransposition, Synaptic Connectivity, and Myelination Implicated by Transcriptomic Changes Following Maternal Immune Activation in Nonhuman Primates.

BACKGROUND: Maternal immune activation (MIA) is a proposed risk factor for multiple neuropsychiatric disorders, including schizophrenia. However, the molecular mechanisms through which MIA imparts risk remain poorly understood. A recently developed nonhuman primate model of exposure to the viral mimic poly:ICLC during pregnancy shows abnormal social and repetitive behaviors and elevated striatal dopamine, a molecular hallmark of human psychosis, providing an unprecedented opportunity for studying underlying molecular correlates. METHODS: We performed RNA sequencing across psychiatrically relevant brain regions (prefrontal cortex, anterior cingulate, hippocampus) and primary visual cortex for comparison from 3.5- to 4-year-old male MIA-exposed and control offspring-an age comparable to mid adolescence in humans. RESULTS: We identify 266 unique genes differentially expressed in at least one brain region, with the greatest number observed in hippocampus. Co-expression networks identified region-specific alterations in synaptic signaling and oligodendrocytes. Although we observed temporal and regional differences, transcriptomic changes were shared across first- and second-trimester exposures, including for the top differentially expressed genes-PIWIL2 and MGARP. In addition to PIWIL2, several other regulators of retrotransposition and endogenous transposable elements were dysregulated following MIA, potentially connecting MIA to retrotransposition. CONCLUSIONS: Together, these results begin to elucidate the brain-level molecular processes through which MIA may impart risk for psychiatric disease.

Translational derepression of Elavl4 isoforms at their alternative 5' UTRs determines neuronal development.

Neurodevelopment requires precise regulation of gene expression, including post-transcriptional regulatory events such as alternative splicing and mRNA translation. However, translational regulation of specific isoforms during neurodevelopment and the mechanisms behind it remain unknown. Using RNA-seq analysis of mouse neocortical polysomes, here we report translationally repressed and derepressed mRNA isoforms during neocortical neurogenesis whose orthologs include risk genes for neurodevelopmental disorders. We demonstrate that the translation of distinct mRNA isoforms of the RNA binding protein (RBP), Elavl4, in radial glia progenitors and early neurons depends on its alternative 5' UTRs. Furthermore, 5' UTR-driven Elavl4 isoform-specific translation depends on upstream control by another RBP, Celf1. Celf1 regulation of Elavl4 translation dictates development of glutamatergic neurons. Our findings reveal a dynamic interplay between distinct RBPs and alternative 5' UTRs in neuronal development and underscore the risk of post-transcriptional dysregulation in co-occurring neurodevelopmental disorders.