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To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase‐scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here, we show that very specific long‐range interactions are anchored by cohesin/CTCF sites, but not cohesin‐only or CTCF‐only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post‐mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin‐dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin‐bound and non‐bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programmes within them. Chromosomal compartmentalization has been recognized as important for genome function. High‐resolution techniques such as Hi‐C, ChIP‐ and 4C‐seq offer novel insights into cohesin's dynamic role in shaping the nuclear architecture.

Glioblastomas are hierarchically organised tumours driven by glioma stem cells that retain partial differentiation potential. Glioma stem cells are maintained in specialised microenvironments, but whether, or how, they undergo lineage progression outside of these niches remains unclear. Here we identify the white matter as a differentiative niche for glioblastomas with oligodendrocyte lineage competency. Tumour cells in contact with white matter acquire pre-oligodendrocyte fate, resulting in decreased proliferation and invasion. Differentiation is a response to white matter injury, which is caused by tumour infiltration itself in a tumoursuppressive feedback loop. Mechanistically, tumour cell differentiation is driven by selective white matter upregulation of SOX10, a master regulator of normal oligodendrogenesis. SOX10 overexpression or treatment with myelination-promoting agents that upregulate endogenous SOX10, mimic this response, leading to niche-independent pre-oligodendrocyte differentiation and tumour suppression in vivo. Thus, glioblastoma recapitulates an injury response and exploiting this latent programme may offer treatment opportunities for a subset of patients. Glioma stem cells (GSCs) retain the ability to partially differentiate, but it is unclear how the brain microenvironment may influence this response. Here the authors show that glioblastoma cells infiltrating into the white matter acquire pre-oligodendrocyte-like fate in a process that mimics myelin repair and results in tumour suppression

Adult neural stem cells (NSCs) must tightly regulate quiescence and proliferation. Single-cell analysis has suggested a continuum of cell states as NSCs exit quiescence. Here we capture and characterize in vitro primed quiescent NSCs and identify LRIG1 as an important regulator. We show that BMP-4 signaling induces a dormant non-cycling quiescent state (d-qNSCs), whereas combined BMP-4/FGF-2 signaling induces a distinct primed quiescent state poised for cell cycle re-entry. Primed quiescent NSCs (p-qNSCs) are defined by high levels of LRIG1 and CD9, as well as an interferon response signature, and can efficiently engraft into the adult subventricular zone (SVZ) niche. Genetic disruption of Lrig1 in vivo within the SVZ NSCs leads an enhanced proliferation. Mechanistically, LRIG1 primes quiescent NSCs for cell cycle re-entry and EGFR responsiveness by enabling EGFR protein levels to increase but limiting signaling activation. LRIG1 is therefore an important functional regulator of NSC exit from quiescence. How neural stem cells can transition between states of proliferation and quiescence is unclear. Here, the authors identify Lrig1 as a specific marker for the primed quiescent state and demonstrate that Lrig1 maintains cells in a quiescent state via modulation of the EGFR pathway.

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions. The oncohistone H3.3-K27M decreases chromatin accessibility and H3K27ac at some active enhancers and downregulates nearby neurodevelopmental genes, while increasing transcriptional repression of a subset of PRC2-bound neurodevelopment genes.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers. Despite increasing knowledge of the genetic and epigenetic changes that underlie tumour initiation and growth, the prognosis for GBM patients remains dismal. Genome analysis has failed to lead to success in the clinic. Fresh approaches are needed that can stimulate new discoveries across all levels: cell-intrinsic mechanisms (transcriptional/epigenetic and metabolic), cell-cell signalling, niche and microenvironment, systemic signals, immune regulation, and tissue-level physical forces. GBMs are inherently extremely challenging: tumour detection occurs too late, and cells infiltrate widely, hiding in quiescent states behind the blood-brain barrier. The complexity of the brain tissue also provides varied and complex microenvironments that direct cancer cell fates. Phenotypic heterogeneity is therefore superimposed onto pervasive genetic heterogeneity. Despite this bleak outlook, there are reasons for optimism. A myriad of complementary, and increasingly sophisticated, experimental approaches can now be used across the research pipeline, from simple reductionist models devised to delineate molecular and cellular mechanisms, to complex animal models required for preclinical testing of new therapeutic approaches. No single model can cover the breadth of unresolved questions. This Review therefore aims to guide investigators in choosing the right model for their question. We also discuss the recent convergence of two key technologies: human stem cell and cancer stem cell culture, as well as CRISPR/Cas tools for precise genome manipulations. New functional genetic approaches in tailored models will likely fuel new discoveries, new target identification and new therapeutic strategies to tackle GBM.

Deregulation of chromatin modifiers plays an essential role in the pathogenesis of medulloblastoma, the most common paediatric malignant brain tumour. Here, we identify a BMI1-dependent sensitivity to deregulation of inositol metabolism in a proportion of medulloblastoma. We demonstrate mTOR pathway activation and metabolic adaptation specifically in medulloblastoma of the molecular subgroup G4 characterised by a BMI1 High ;CHD7 Low signature and show this can be counteracted by IP6 treatment. Finally, we demonstrate that IP6 synergises with cisplatin to enhance its cytotoxicity in vitro and extends survival in a pre-clinical BMI1 High ;CHD7 Low xenograft model. BMI1 and CHD7 are chromatin remodelling genes with a role in medulloblastoma pathogenesis. Here, the authors demonstrate that the BMI1 High /CHD7 Low signature mediates metabolic adaptation in G4 MB and predicts response to inositol treatment either alone or in combination with chemotherapy.

Neutrophils are vital innate immune cells shown to infiltrate glioblastomas, however we currently lack the molecular understanding of their functional states within the tumour niche. Given that neutrophils are known to display a prominent discordance between mRNA and protein abundance, we developed ultra-sensitive mini-bulk and single cell proteomic (SCP) workflows to study the heterogeneity of peripheral blood and tumour associated neutrophils (TAN) from patients with glioblastoma. Mini-bulk analysis enabled a deeper protein coverage of circulating immature, mature and TAN populations, defining signatures of maturity and demonstrating that TANs resemble mature circulating neutrophils. Analysis of the SCP data results in the detection of >1100 proteins from a single TAN providing a detailed characterization of neutrophil subsets in glioblastoma. Our approach shows evidence of pathogenic and anti-tumorigenic clusters and discovers cell states invisible to scRNAseq, opening new opportunities to selectively target pro-tumoural neutrophil states. Neutrophils infiltrate glioblastomas with the capacity to engage pro/anti tumoural responses. Here the authors developed proteomic workflows to stratify neutrophil heterogeneity by function. This work provides a platform to study neutrophil proteomes with single cell resolution in glioblastoma.

Brain organoids are becoming increasingly relevant to dissect the molecular mechanisms underlying psychiatric and neurological conditions. The in vitro recapitulation of key features of human brain development affords the unique opportunity of investigating the developmental antecedents of neuropsychiatric conditions in the context of the actual patients’ genetic backgrounds. Specifically, multiple strategies of brain organoid (BO) differentiation have enabled the investigation of human cerebral corticogenesis in vitro with increasing accuracy. However, the field lacks a systematic investigation of how closely the gene co-expression patterns seen in cultured BO from different protocols match those observed in fetal cortex, a paramount information for ensuring the sensitivity and accuracy of modeling disease trajectories. Here we benchmark BO against fetal corticogenesis by integrating transcriptomes from in-house differentiated cortical BO (CBO), other BO systems, human fetal brain samples processed in-house, and prenatal cortices from the BrainSpan Atlas. We identified co-expression patterns and prioritized hubs of human corticogenesis and CBO differentiation, highlighting both well-preserved and discordant trends across BO protocols. We evaluated the relevance of identified gene modules for neurodevelopmental disorders and psychiatric conditions finding significant enrichment of disease risk genes especially in modules related to neuronal maturation and synapsis development. The longitudinal transcriptomic analysis of CBO revealed a two-step differentiation composed of a fast-evolving phase, corresponding to the appearance of the main cell populations of the cortex, followed by a slow-evolving one characterized by milder transcriptional changes. Finally, we observed heterochronicity of differentiation across BO models compared to fetal cortex. Our approach provides a framework to directly compare the extent of in vivo/in vitro alignment of neurodevelopmentally relevant processes and their attending temporalities, structured as a resource to query for modeling human corticogenesis and the neuropsychiatric outcomes of its alterations.

Glioblastoma multiforme (GBM) is an aggressive incurable brain cancer. The cells that fuel the growth of tumours resemble neural stem cells found in the developing and adult mammalian forebrain. These are referred to as glioma stem cells (GSCs). Similar to neural stem cells, GSCs exhibit a variety of phenotypic states: dormant, quiescent, proliferative and differentiating. How environmental cues within the brain influence these distinct states is not well understood. Laboratory models of GBM can be generated using either genetically engineered mouse models, or via intracranial transplantation of cultured tumour initiating cells (mouse or human). Unfortunately, these approaches are expensive, time-consuming, low-throughput and ill-suited for monitoring live cell behaviours. Here, we explored whole adult brain coronal organotypic slices as an alternative model. Mouse adult brain slices remain viable in a serum-free basal medium for several weeks. GSCs can be easily microinjected into specific anatomical sites ex vivo, and we demonstrate distinct responses of engrafted GSCs to diverse microenvironments in the brain tissue. Within the subependymal zone – one of the adult neural stem cell niches – injected tumour cells could effectively engraft and respond to endothelial niche signals. Tumour-transplanted slices were treated with the antimitotic drug temozolomide as proof of principle of the utility in modelling responses to existing treatments. Engraftment of mouse or human GSCs onto whole brain coronal organotypic brain slices therefore provides a simplified, yet flexible, experimental model. This will help to increase the precision and throughput of modelling GSC-host brain interactions and complements ongoing in vivo studies. This article has an associated First Person interview with the first author of the paper.

Single‐cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA‐seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent‐like state in neuroepithelial‐derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non‐dividing neural progenitors. Putative glioblastoma stem‐like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down‐regulation of quiescence‐associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent‐like state found in neuroepithelial‐derived cells and gliomas. SYNOPSIS scRNA‐seq and functional genomics are applied to human neural stem cells to characterize cell cycle phases and factors increasing proliferative rate. A new cell cycle state “Neural G0” is discovered, offering insights into glioma patient tumors and patient survival. Unsupervised cell clustering reveals eight distinct cell cycle phases in human neural stem cells. A novel Neural G0 cell cycle phase is characterized by up‐regulation of genes with key roles in neural development. Increased expression of a GBM‐specific Neural G0 gene signature is associated with better prognosis for glioma patients, independent of tumor grade and IDH1/2 mutation. A functional‐genomics screen identifies genes that alter the time spent in G0/G1‐like states thereby increasing the proliferative rate of neural stem cells. Graphical Abstract scRNA‐seq and functional genomics are applied to human neural stem cells to characterize cell cycle phases and factors increasing proliferative rate. A new cell cycle state “Neural G0” is discovered, offering insights into glioma patient tumors and patient survival.