TY  - GEN
A1  - Yildiz, Umut
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/35099/
N2  - Gene regulation is central in orchestrating complex biological processes during development and in disease. The underlying networks are instrumental in defining cell states, fine-tuning transcriptional programs for lineage commitment, and conferring cellular plasticity to adapt to environmental cues. Transcription factors (TFs) and chromatin regulators (CRs) are de facto effectors of gene regulatory networks (GRNs) governing these processes, and their dysfunction is implicated in several diseases, including schizophrenia. Despite accumulating evidence linking genetic variants of schizophrenia risk to the impaired activity of CRs and TFs, the precise molecular mechanisms underlying the disease state remain elusive. This is partly due to limitations in suitable neuronal cell culture models for large-scale functional assays within the human context.
To address this, I established CRISPR interference (CRISPRi) and activation (CRISPRa) human induced pluripotent stem cell (hiPSC) models, enabling systematic investigation of risk genes and pathways in neural progenitor cells (NPCs) and induced glutamatergic neurons. Additionally, I utilized high-throughput single-cell gene expression assays and co-developed a similar approach to profile chromatin accessibility at the single-cell level, overcoming the cost-dependent limitations of large-scale screening experiments. A joint effort, that enables the co-profiling of chromatin accessibility and gene expression from the same cell in a scalable and high-throughput manner, is included in this thesis (Appendix A).
Combining the established CRISPRi cell culture and single-cell technology platforms, I characterized the function of 65 CRs and TFs implicated in schizophrenia risk throughout in vitro neurodevelopment. Screen deconvolution revealed several perturbations interfering with neuronal differentiation trajectories. For instance, knockdown of several CRs and TFs resulted in a transcriptionally delayed neurodevelopmental phenotype, indicating their potential regulatory function in promoting neurodevelopment. Recurrently correlated pathways, including Wnt signaling, NRSF/REST, and the PRC2 pathway, suggest that the function of these CRs and TFs potentially converge on the same molecular programs associated with cell type specification and repression of transcriptional programs related to alternative cell fates. Additionally, I identified a potential novel function for the Microspherule protein 1 (MCRS1) in restraining differentiating cells at the NPC state. Follow-up CRISPRi screens coupled to fluorescence-activated cell sorting (FACS) in combination with cell cycle and metabolic indicators confirmed the differentiation-promoting or restricting regulatory functions of these risk CRs and TFs. Notably, the individual knockdown of MCRS1 confirmed its role in cell cycle regulation, as evidenced by reduced growth and proliferation rates.
Gene regulatory network inference from single-cell transcriptomic readouts identified TCF4 and ZEB1 as critical modulators of neurodevelopmental processes, with inverse expression and activity patterns throughout in vitro neurodevelopment. Discrepancies in TCF4 and ZEB1 expression and activity correlated with altered differentiation trajectories, highlighting their critical role in regulating neurodevelopmental pacing. Integrating chromatin accessibility readouts following CRISPRi/a of TCF4 and ZEB1 with the scCRISPRi screen data suggested that the increased activity of ZEB1 at the NPC stage, as a consequence of reduced MCRS1 expression, induced an epithelial-to-mesenchymal-like transition, which facilitated neuronal commitment and accelerated differentiation. On the contrary, the impaired ability to shut down the TCF4-driven transcriptional program may have resulted in the delayed differentiation phenotypes in CRISPRi-perturbed induced neurons.
Collectively, the study demonstrates the importance of functional genetic screens in disease-relevant cell types, coupled with multimodal single-cell readouts, in understanding the neurodevelopmental origins of schizophrenia. The findings shed light on the convergent roles of risk CRs and TFs in regulating critical neurodevelopmental processes and underscored the significance of their regulation in shaping proper developmental trajectories.
CY  - Heidelberg
AV  - restricted
Y1  - 2025///
TI  - Functional dissection of schizophrenia risk genes using single-cell CRISPR screens
KW  - Single-Cell functional genomics
ID  - heidok35099
ER  -