The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC’s long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a “mixed” neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the “neuronal” genes are bound by both SOX2 and AP1.

Non-coding variation in complex human disease has been well established by genome-wide association studies, and is thought to involve regulatory elements, such as enhancers, whose variation affects the expression of the gene responsible for the disease. The regulatory elements often lie far from the gene they regulate, or within introns of genes differing from the regulated gene, making it difficult to identify the gene whose function is affected by a given enhancer variation. Enhancers are connected to their target gene promoters via long-range physical interactions (loops). In our study, we re-mapped, onto the human genome, more than 10,000 enhancers connected to promoters via long-range interactions, that we had previously identified in mouse brain-derived neural stem cells by RNApolII-ChIA-PET analysis, coupled to ChIP-seq mapping of DNA/chromatin regions carrying epigenetic enhancer marks. These interactions are thought to be functionally relevant. We discovered, in the human genome, thousands of DNA regions syntenic with the interacting mouse DNA regions (enhancers and connected promoters). We further annotated these human regions regarding their overlap with sequence variants (single nucleotide polymorphisms, SNPs; copy number variants, CNVs), that were previously associated with neurodevelopmental disease in humans. We document various cases in which the genetic variant, associated in humans to neurodevelopmental disease, affects an enhancer involved in long-range interactions: SNPs, previously identified by genome-wide association studies to be associated with schizophrenia, bipolar disorder, and intelligence, are located within our human syntenic enhancers, and alter transcription factor recognition sites. Similarly, CNVs associated to autism spectrum disease and other neurodevelopmental disorders overlap with our human syntenic enhancers. Some of these enhancers are connected (in mice) to homologs of genes already associated to the human disease, strengthening the hypothesis that the gene is indeed involved in the disease. Other enhancers are connected to genes not previously associated with the disease, pointing to their possible pathogenetic involvement. Our observations provide a resource for further exploration of neural disease, in parallel with the now widespread genome-wide identification of DNA variants in patients with neural disease.

DNA sequence variants (single nucleotide polymorphisms or variants, SNPs/SNVs; copy number variants, CNVs) associated to neurodevelopmental disorders (NDD) and traits often map on putative transcriptional regulatory elements, including, in particular, enhancers. However, the genes controlled by these enhancers remain poorly defined. Traditionally, the activity of a given enhancer, and the effect of its possible alteration associated to the sequence variants, has been thought to influence the nearest gene promoter. However, the obtainment of genome-wide long-range interaction maps in neural cells chromatin challenged this view, showing that a given enhancer is very frequently not connected to the nearest promoter, but to a more distant one, skipping genes in between. In this Perspective, we review some recent papers, who generated long-range interaction maps (by HiC, RNApolII ChIA-PET, Capture-HiC, or PLACseq), and overlapped the identified long-range interacting DNA segments with DNA sequence variants associated to NDD (such as schizophrenia, bipolar disorder and autism) and traits (intelligence). This strategy allowed to attribute the function of enhancers, hosting the NDD-related sequence variants, to a connected gene promoter lying far away on the linear chromosome map. Some of these enhancer-connected genes had indeed been already identified as contributive to the diseases, by the identification of mutations within the gene’s protein-coding regions (exons), validating the approach. Significantly, however, the connected genes also include many genes that were not previously found mutated in their exons, pointing to novel candidate contributors to NDD and traits. Thus, long-range interaction maps, in combination with DNA variants detected in association with NDD, can be used as “pointers” to identify novel candidate disease-relevant genes. Functional manipulation of the long-range interaction network involving enhancers and promoters by CRISPR-Cas9-based approaches is beginning to probe for the functional significance of the identified interactions, and the enhancers and the genes involved, improving our understanding of neural development and its pathology.

Non-coding variation in complex human disease has been well established by genome-wide association studies, and it is thought to involve regulatory elements, such as enhancers, whose variation affects the expression of the gene responsible for the disease. The regulatory elements often lie far from the gene they regulate, or within introns of genes differing from the regulated gene, making it difficult to identify the gene whose function is affected by a given enhancer variation.Enhancers are connected to their target gene promoters via long-range physical interactions (loops). In our study, we re-mapped, onto the human genome, more than 10,000 enhancers connected to promoters via long-range interactions, that we had previously identified in mouse brain-derived neural stem cells by RNApolII-ChIA-PET analysis, coupled to ChIP-seq mapping of DNA/chromatin regions carrying epigenetic enhancer marks. These interactions are thought to be functionally relevant. We discovered, in the human genome, thousands of DNA regions syntenic with the interacting mouse DNA regions (enhancers+connected promoters). We further annotated these human regions regarding their overlap with sequence variants (single nucleotide polymorphisms, SNPs; copy number variants, CNVs), that were previously associated with neurodevelopmental disease in human. We document various cases in which the genetic variant, associated in human to neurodevelopmental disease, affects an enhancer involved in long-range interactions: SNPs, previously identified by genome-wide association studies to be associated with schizophrenia, bipolar disorder, and intelligence, are located within our human syntenic enhancers, and alter transcription factors recognition sites. Similarly, CNVs associated to autism spectrum disease and other neurodevelopmental disorders overlap with our human syntenic enhancers. Some of these enhancers are connected (in mouse) to homologs of genes already associated to the human disease, strengthening the hypothesis that the gene is indeed involved in the disease. Other enhancers are connected to genes not previously associated with the disease, pointing to their possible pathogenetic involvement.Our observations provide a resource for further exploration of neural disease, in parallel with the now widespread genome-wide identification of DNA variants in patients with neural disease.