ASCL1 Is Involved in the Pathogenesis of Schizophrenia by Regulation of Genes Related to Cell Proliferation, Neuronal Signature Formation, and Neuroplasticity.

Schizophrenia (SZ) is a common psychiatric neurodevelopmental disorder with a complex genetic architecture. Genome-wide association studies indicate the involvement of several transcription factors, including ASCL1, in the pathogenesis of SZ. We aimed to identify ASCL1-dependent cellular and molecular mechanisms associated with SZ. We used Capture-C, CRISPR/Cas9 systems and RNA-seq analysis to confirm the involvement of ASCL1 in SZ-associated pathogenesis, establish a mutant SH-SY5Y line with a functional ASCL1 knockout (ASCL1-del) and elucidate differentially expressed genes that may underlie ASCL1-dependent pathogenic mechanisms. Capture-C confirmed the spatial interaction of the ASCL1 promoter with SZ-associated loci. Transcriptome analysis showed that ASCL1 regulation may be through a negative feedback mechanism. ASCL1 dysfunction affects the expression of genes associated with the pathogenesis of SZ, as well as bipolar and depressive disorders. Genes differentially expressed in ASCL1-del are involved in cell mitosis, neuronal projection, neuropeptide signaling, and the formation of intercellular contacts, including the synapse. After retinoic acid (RA)-induced differentiation, ASCL1 activity is restricted to a small subset of genes involved in neuroplasticity. These data suggest that ASCL1 dysfunction promotes SZ development predominantly before the onset of neuronal differentiation by slowing cell proliferation and impeding the formation of neuronal signatures.

Cellular Models in Schizophrenia Research.

Schizophrenia (SZ) is a prevalent functional psychosis characterized by clinical behavioural symptoms and underlying abnormalities in brain function. Genome-wide association studies (GWAS) of schizophrenia have revealed many loci that do not directly identify processes disturbed in the disease. For this reason, the development of cellular models containing SZ-associated variations has become a focus in the post-GWAS research era. The application of revolutionary clustered regularly interspaced palindromic repeats CRISPR/Cas9 gene-editing tools, along with recently developed technologies for cultivating brain organoids in vitro, have opened new perspectives for the construction of these models. In general, cellular models are intended to unravel particular biological phenomena. They can provide the missing link between schizophrenia-related phenotypic features (such as transcriptional dysregulation, oxidative stress and synaptic dysregulation) and data from pathomorphological, electrophysiological and behavioural studies. The objectives of this review are the systematization and classification of cellular models of schizophrenia, based on their complexity and validity for understanding schizophrenia-related phenotypes.

A modified protocol of Capture-C allows affordable and flexible high-resolution promoter interactome analysis.

Large-scale epigenomic projects have mapped hundreds of thousands of potential regulatory sites in the human genome, but only a small proportion of these elements are proximal to transcription start sites. It is believed that the majority of these sequences are remote promoter-activating genomic sites scattered within several hundreds of kilobases from their cognate promoters and referred to as enhancers. It is still unclear what principles, aside from relative closeness in the linear genome, determine which promoter(s) is controlled by a given enhancer; however, this understanding is of great fundamental and clinical relevance. In recent years, C-methods (chromosome conformation capture-based methods) have become a powerful tool for the identification of enhancer-promoter spatial contacts that, in most cases, reflect their functional link. Here, we describe a new hybridisation-based promoter Capture-C protocol that makes use of biotinylated dsDNA probes generated by PCR from a custom pool of long oligonucleotides. The described protocol allows high-resolution promoter interactome description, providing a flexible and cost-effective alternative to the existing promoter Capture-C modifications. Based on the obtained data, we propose several tips on probe design that could potentially improve the results of future experiments.