SOX2-Dependent Regulatory Networks Shaping Human Brain Development and Disease

This thesis investigates the molecular and functional architecture underlying early human brain development, with a particular focus on the transcription factor SOX2, a central regulator of neural progenitor identity and cortical patterning. By integrating cutting-edge genomic, epigenomic, and functional approaches, the work aims to elucidate how SOX2 shapes chromatin organization and transcriptional programs that drive neurodevelopmental trajectories, and how its perturbation contributes to human disease. Chapter I describes the development and optimization of a high-resolution CRISPR-LICHT (CRISPR–lineage tracing at cellular resolution in heterogeneous tissue) screening platform, specifically adapted for three-dimensional human cortical organoids. This methodological advancement enables systematic, spatially resolved functional genomics in a physiologically relevant model of the developing brain. The chapter details the validation of the approach using SOX2 and other well-characterized neurodevelopmental regulators, followed by its large-scale application to a set of poorly annotated Tdark genes under SOX2 regulation. The results uncover previously unrecognized components of the molecular circuitry governing neural progenitor proliferation, differentiation, and survival. Chapter II investigates the impact of SOX2 haploinsufficiency using patient-derived pluripotent stem cell lines. Through a combination of transcriptomic, epigenetic, and phenotypic analyses, this section delineates the cellular and molecular alterations that underlie SOX2-related neurodevelopmental disorders. The findings provide mechanistic insights into how subtle dosage imbalances in a key transcription factor can lead to profound developmental outcomes. Chapter III examines SOX2-mediated chromatin regulation in greater depth, focusing on long-range enhancer–promoter interactions that orchestrate gene expression programs during human corticogenesis. In this chapter, I characterize three evolutionarily conserved SOX2-bound enhancers that establish long-range chromatin contacts with the OLIG1/OLIG2 locus, key regulators of neural lineage specification. Through complementary genomic mapping and perturbation approaches, these studies reveal how SOX2 coordinates the spatial organization of chromatin to control the precise temporal activation of OLIG genes. Given the implication of OLIG1 and OLIG2 in Down syndrome and other neurodevelopmental disorders, the findings provide mechanistic insights into how enhancer dynamics contribute to both normal and pathological brain development. Chapters IV and V broaden the scope of the thesis through collaborative and complementary studies. The first investigates the role of noncoding genomic regions in human brain evolution and susceptibility to neurological disease, emphasizing the contribution of regulatory sequence divergence to species-specific cognitive traits. The second maps three-dimensional chromatin contacts in neural cells at high resolution, identifying new candidate genes and regulatory elements implicated in neurodevelopmental disorders. Together, these studies provide a comprehensive view of how transcriptional and chromatin-based mechanisms interact to shape the developing human brain. By combining methodological innovation with mechanistic investigation, the thesis advances our understanding of SOX2 function and establishes a framework for exploring the molecular underpinnings of human neurodevelopment and its disorders.