Precise regulation of neural progenitor states and neuronal positioning is essential for cortical development. Here, we identified Sox2 regulatory region 42 (SRR42), a regulatory region located 42 kb downstream of the Sox2 transcription start site that exhibits open chromatin, active histone modifications, and bidirectional transcription of enhancer RNAs (eRNAs). Using transgenic reporter mice, we found that SRR42 is active in neural progenitor cells in the developing cortex. CRISPR-Cas9-mediated deletion of SRR42 combined with single-cell multiome analysis revealed alterations in progenitor-state dynamics, neuronal migration, and cortical lamination without detectable changes in Sox2 expression. Furthermore, in vivo knockdown of SRR42-derived eRNAs impaired neuronal migration. These findings identify SRR42-derived RNAs as regulators of neuronal migration during cortical development and highlight enhancer-associated transcription as a functional component of neurodevelopmental gene regulation.

Neural progenitor cells (NPCs) must preserve lineage identity while remaining responsive to developmental cues. Here, we discuss the hypothesis that ATRX condensates help organize enhancer-centered nuclear microenvironments in NPCs. ATRX has long been studied in heterochromatin maintenance, histone variant deposition, and chromatin remodeling; earlier work has also shown that ATRX can occupy euchromatic and active regulatory regions and contribute to transcriptional regulation. Recent evidence in human NPCs indicates that ATRX forms nuclear puncta with condensate-like properties, associates with neurogenic enhancer-rich regions, and incorporates regulatory factors such as CHD7 and p300. Perturbation of ATRX condensate formation is associated with changes in enhancer-associated ATRX occupancy, neural gene-expression programs, and neuroepithelial organization, suggesting a regulatory mode that may complement canonical heterochromatin-associated functions. We propose a dual-mode model in which folded domains contribute to chromatin anchoring at repressive regions, whereas intrinsically disordered regions support condensate-associated organization at active developmental enhancers. We emphasize that whether ATRX condensates activate enhancers de novo, stabilize pre-existing enhancer states, buffer transcriptional variability, or primarily organize cofactor localization remains unresolved. We also discuss limitations of the current evidence and outline acute, locus-specific experiments needed to test the model.

ATRX is a chromatin-remodelling protein associated with neurodevelopmental disorders and gliomas, forming distinct nuclear foci associated with chromatin organization and phase-separated nuclear compartments. To visualize ATRX dynamics in live cells, we generated a human iPS cell (hiPSC) line with EGFP knocked in at the ATRX N-terminus using CRISPR/Cas12a-mediated genome editing. The modified hiPSCs retained pluripotency, normal karyotype, and trilineage differentiation potential. EGFP-ATRX localized to discrete nuclear foci, consistent with the phase-separated condensates described for ATRX, enabling real-time visualization of its chromatin association. This reporter line offers a valuable tool for studying ATRX function during differentiation and chromatin architecture development.

Mutations in the ATRX genes cause alpha-thalassemia X-linked intellectual disability (ATR-X) syndrome. Here, we show that ATRX influences the fate of human neural progenitor cells (hNPCs) by forming condensates through liquid-liquid phase separation (LLPS). The intrinsically disordered region (IDR) of ATRX is essential for LLPS and enables ATRX to form dynamic condensates that recruit co-activators. These condensates are necessary for ATRX localization at super-enhancers (SEs) in hNPCs, linking its compartmentalization to transcriptional regulation. Disruption of ATRX condensates alters gene expression and impairs neuronal differentiation. Our findings support a model in which ATRX phase separation regulates gene networks required for hNPC identity. These findings extend current understanding of ATRX function beyond its roles in chromatin structure and suggest that LLPS is a key regulatory mechanism by which ATRX supports neurodevelopment. This study opens avenues for further investigation into how dysregulation of ATRX and its phase-separation ability may contribute to the pathogenesis of ATR-X syndrome and related neurodevelopmental disorders.

Although neural stem/progenitor cells derived from human induced pluripotent stem cells (hiPSC-NS/PCs) are expected to be a cell source for cell-based therapy, tumorigenesis of hiPSC-NS/PCs is a potential problem for clinical applications. Therefore, to understand the mechanisms of tumorigenicity in NS/PCs, we clarified the cell populations of NS/PCs. We established single cell-derived NS/PC clones (scNS/PCs) from hiPSC-NS/PCs that generated undesired grafts. Additionally, we performed bioassays on scNS/PCs, which classified cell types within parental hiPSC-NS/PCs. Interestingly, we found unique subsets of scNS/PCs, which exhibited the transcriptome signature of mesenchymal lineages. Furthermore, these scNS/PCs expressed both neural (PSA-NCAM) and mesenchymal (CD73 and CD105) markers, and had an osteogenic differentiation capacity. Notably, eliminating CD73+ CD105+ cells from among parental hiPSC-NS/PCs ensured the quality of hiPSC-NS/PCs. Taken together, the existence of unexpected cell populations among NS/PCs may explain their tumorigenicity leading to potential safety issues of hiPSC-NS/PCs for future regenerative medicine.