Development and use of multiomics tools to dissect drivers of neuronal identity

Diwakar Shunmugapriya, Jeisimhan (2025): Development and use of multiomics tools to dissect drivers of neuronal identity. Dissertation, LMU München: Medizinische Fakultät

Abstract

Cell-type identities and development are guided by epigenetic mechanisms that modify chromatin to regulate gene expression without altering the DNA sequence. This process depends on transcription factors (TFs) to regulate RNA expression programs by affecting cis-regulatory elements and the chromatin landscape, all within the context of 3D nuclear architecture. Until recently, studies focused mainly on a few epigenetic layers, often neglecting the 3D genome architecture. Yet, high-throughput technologies like Hi-C have provided valuable insights into how 3D chromatin folding influences development. Therefore, in this PhD, we apply and develop multiomics assays that include chromosome conformation technologies to mechanistically understand how lineage-specifying factors dynamically rewire multiple epigenetic layers. We initiated our study by examining the epigenome remodelling involved in the direct neuronal reprogramming of astrocytes into induced neurons, facilitated by the overexpression of the proneural transcription factor Neurogenin2 (Ngn2) or its phosphorylation-resistant variant (PmutNgn2). Through the integration of single-cell multiomics and Methyl-HiC, we revealed that Ngn2 drives extensive multilayered epigenetic rewiring. Induction with PmutNgn2 resulted in the faster generation of more mature neurons, accompanied by enhanced chromatin remodelling. Interestingly, this effect was not due to superior pioneering activity but rather to the activation of downstream genes that act as co-factors. Among these, we identified Yy1 as a critical Ngn2-recruited co-factor, whose depletion impaired reprogramming efficiency. To explore how cellular and epigenetic contexts influence TF mediated rewiring, we overexpressed Ngn2 in mouse embryonic stem cells and neural progenitor cells. By integrating 3DRAM-seq with ChIP-mass spectrometry, we found that whilst Ngn2's direct activity at bound sites was largely consistent across both cell types, there were pronounced cell-type-specific indirect effects on the global epigenome, most notably in embryonic stem cells. These distinct effects appear to be modulated by Ngn2 interactors, which included subunits of the SWI/SNF and NuRD chromatin remodelling complexes. Recognising the intricate interplay between multiple epigenetic layers, we initially aimed to create a method for simultaneously profiling the 3D genome and transcription at single-cell resolution with high throughput. However, given the emergence of several similar methods in recent studies, we expanded upon existing techniques to develop sc-3DRAM-seq, capable of additionally measuring DNA methylation and chromatin accessibility. Bulk quality control experiments have shown promising results, and we are now conducting single-cell tests. The primary goal of this work is to uncover epigenetic variation within the heterogeneous tissues of mouse embryonic brains and human fetal brains.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 24. Juni 2025
• 1. Bericht­erstatter:in: Bonev, Boyan