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Abstract

Gene expression has to be regulated in a cell type-specific manner to ensure proper functionality of cell types and tissues. In a diploid organism, the two alleles of a gene can be regulated independently, causing differential contribution to mRNA levels and thus to cellular function. Allelic imbalance in gene expression has long been recognized as a contributor to cellular phenotypes, however, it is not well understood how and to which extent allelic imbalance is shaped by the regulatory environment of different cell types. Recent advances in single-cell technologies provide the opportunity to profile gene expression and its regulation in a cell type-specific manner at scale, extending our understanding of genome function. In this thesis, I performed a comprehensive analysis of allele-specific expression (ASE) at single-cell resolution in interspecific mouse hybrids. I first analysed the differentiation-dependence of allelic imbalance caused by strain-specific genetic effects during murine spermatogenesis. This analysis shows that across cell types, variation in ASE is extremely pervasive. Using an F1 trio design, I further separated cis- and trans-contributions to gene expression divergence and showed that cell type-specific action of regulatory variants is mainly driven by the interaction of cis-effects with the cellular environment. Finally, I investigated the contribution of dynamic genetic effects to cell type-specific transcriptional evolution. Next, I focussed on ASE caused by an epigenetic mechanism, namely X-chromosome inactivation (XCI). In female humans and mice, XCI causes mosaic haplotype-specific expression of X-linked genes, and escape from XCI can lead to increased gene dosage compared to males. Using single-cell genomics assays, I developed an analysis approach to distinguish active and inactive X-chromosomes in individual cells, which allowed me to identify cell type-specific escape. I further showed that T-cell expansion during ageing leads to globally impaired silencing of the inactive X which is associated with an exhaustion phenotype. These findings replicated on the level of chromatin accessibility, demonstrating that variation in escape is associated with an active chromatin state. Collectively, I showed that escape can vary at the cell type level and during organismal ageing. While these results show that escape from XCI is plastic, they do not address how it might be regulated in different cell types. In the final chapter, I therefore explored whether the Xist long non-coding RNA can regulate escapee expression. Using allele-specific RNA-Seq data, I showed that increased Xist-levels lead to almost complete silencing of escapees in neural progenitor cells. Modelling of silencing trajectories showed substantial variability among genes in both their resistance to silencing as well as their reversibility, suggesting that escape is genomically encoded. Finally, I demonstrated that over-expression of Xist leads to escapee silencing in early embryogenesis. These results provide a potential mechanism that might drive variability in expression from the inactive X. Taken together, this thesis delineates to which extent allelic imbalance is driven by cell typespecific regulatory environments and suggests analysis approaches for allele-resolved single-cell data. This provides the basis for a comprehensive survey of allelic usage in vivo and the molecular mechanisms causing its context-specificity.