<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Exploring the mechanisms of escape from X chromosome inactivation in neural progenitor cells"^^ . "During female mammalian development one of the two X chromosomes becomes silenced\r\nthrough X chromosome inactivation (XCI). XCI ensures proper X-linked gene dosage between\r\nfemales (XX) and males (XY), and is essential for female development. Intriguingly, some\r\nX-linked genes escape the chromosome-wide gene silencing during XCI (i.e., the “escapees”)\r\nand are expressed from both the active (Xa) and inactive X chromosome (Xi) within the same\r\nnucleus. The relevance of escape from XCI for female development and sexual dimorphism is\r\nbecoming increasingly recognized, but the mechanisms remain elusive. Escapees can be\r\nclassified into constitutive (3 - 5% of X-linked genes in mice), which escape in all tissues and\r\nindividuals and tissue- and/or cell-specific facultative escapees (> 20%). Constitutive escapees\r\nhave Y chromosome homologs and thus expression from the Xi is presumably required for\r\nhigher CTCF binding on the Xi in close proximity to escapees in NPCs proper female\r\ndevelopment. Expression from the Xi of a subset of facultative escapees on the other hand,\r\nmay be due to inefficient maintenance of XCI silencing, and this in some cases could underlie\r\nsex-biased disorders.\r\nClusters of facultative escapees have been found to reside in 3D chromatin domains that\r\nresemble Topologically Associating Domains (TADs) on the Xi. The Xi is largely depleted for\r\nTADs. However, a few TAD-like domains can be detected on the Xi that correlate with\r\nexpressed clusters of escapees, suggesting that there might be an interplay between 3D\r\norganization and transcriptional activity of facultative escapees. The main goal of my PhD\r\nproject was to explore the relationship between gene activity and 3D chromosome topology on\r\nthe Xi. To this end, I first established a fast and straightforward Capture Hi-C protocol, which\r\nallows for the allele-specific interrogation of the local 3D genome topology of megabase-sized\r\nregions of interest. The allele-specific analysis of this protocol relies on the mapping of known\r\nsingle nucleotide polymorphisms (SNPs) in F1 hybrid mouse embryonic stem cells (mESCs),\r\ngenerated from a cross between two highly polymorphic mouse strains. Compared to genomewide\r\nHi-C this new easy-to-follow and time-effective Capture Hi-C protocol with its\r\ncommercial capture strategy enables increased resolution at a reduced cost, thereby making it\r\naccessible for a wide variety of applications.\r\nI used this high-resolution Capture Hi-C protocol to investigate the high-resolution 3D genome\r\ntopology of facultative escape clusters in several clonal neural progenitor cell (NPC) lines,\r\nwhere different subsets of facultative escapees are found, showing clone-specific,\r\ntranscription-correlated local 3D genome topology at these clusters. I further characterized\r\nthese facultative escape clusters by performing CUT&RUN for different chromatin marks and\r\nstructural factors to investigate differences in these features at escape clusters compared to the\r\nsilenced chromatin of the Xi. I observed NPC clone-specific escape from XCI as well as\r\nstructural features, with active loci in the different NPC clones being exclusively engaged in\r\nTAD-like structures and long-range loops. To test the role of structural factors such as the\r\nCCCTC-binding factor (CTCF) in these TAD-like structures and in the maintenance of escapee\r\nexpression, I established clonal NPC cell lines with a degron-based system that allowed acute\r\nand reversible depletion of CTCF. Surprisingly, depletion of CTCF for several days did not\r\nresult in a major effect on 3D chromatin structure nor in the expression of escapees on the Xi,\r\ndespite higher CTCF binding on the Xi in close proximity to escapees in NPCs. This implies\r\nthat escapee expression patterns and 3D structure on the Xi are largely independent of CTCF\r\nin NPCs. This argues for a transcription-induced local 3D genome topology at the facultative\r\nescape clusters of the Xi, rather than a CTCF-loop extrusion-based model. Furthermore, I\r\nfound that depletion of CTCF did not lead to a spread of escape status into nearby regions of\r\nthe Xi. Thus, contrary to previous hypotheses, CTCF does not play a role as a boundary factor\r\nthat prevents spread from facultative or constitutive escapee regions into neighbouring silent\r\ngenes.\r\nI also explored the novel and unexpected role that the XCI master regulatory RNA, Xist, plays\r\nin modulating the expression of escapees in NPCs. The prevailing view has been that Xist\r\nRNA together with XCI initiation partners including SPEN, only plays a major role in\r\nestablishing XCI during early embryogenesis, and that X-linked genes then remain repressed\r\nthroughout life due to epigenetic mechanisms, mainly at the chromatin level. However, recent\r\nevidence has revealed that reduced Xist RNA levels can in fact increase escapee expression in\r\nsomatic cells, and that even a slight change in the dosage of some escapee-encoded proteins,\r\ncan in turn influence cell states that impact tissue homeostasis and disease susceptibility. I\r\nexamined NPCs with an inducible Xist gene on the Xi, which allowed me to increase Xist\r\nlevels in NPCs and assess the impact on facultative and constitutive escapees. Remarkably,\r\nincreased Xist RNA levels resulted in efficient silencing of escapee genes. Furthermore, I\r\nshowed that this silencing is SPEN-dependent implying that a similar mechanism to the\r\ninitiation of XCI is used in NPCs. Although both constitutive and facultative escapees are\r\ndownregulated, constitutive escapees seem to be more resistant to complete silencing via Xist\r\neven after prolonged exposure. On the other hand, facultative escapees can be irreversibly\r\nsilenced. Using Capture Hi-C, I further show that higher levels of Xist in NPCs lead to loss of\r\nlocal 3D genome topology at facultative escape clusters. This reinforces the hypothesis from\r\nthe first part of my PhD that TAD-like structures on the Xi are transcription-induced structures.\r\nTaken together, I have been able to show during my PhD that facultative escape from XCI is\r\nhighly variable between different clonal NPC lines and that although this correlates with CTCF\r\nbinding on the Xi and occurs in the context of local TAD-like structures on the Xi, both escape\r\nand associated 3D structures seem to be maintained largely independently of CTCF. I have\r\nalso shown that increased Xist RNA levels in NPCs can impose silencing of both facultative\r\nand constitutive escapees. Xist RNA together with its partner SPEN, thus play a significant\r\nrole in initiating the silencing of escapees, even in a somatic context. This discovery has\r\nimplications for physiology and disease, as natural variations in Xist levels during development\r\nor in adult tissues will likely influence the extent to which genes escape XCI, thereby\r\nprofoundly impacting global gene expression, differentiation potential, and tissue homeostasis."^^ . "2026" . . . . . . . "Antonia Raphaela"^^ . "Hauth"^^ . "Antonia Raphaela Hauth"^^ . . . . . . "Exploring the mechanisms of escape from X chromosome inactivation in neural progenitor cells (PDF)"^^ . . . "Exploring the mechanisms of escape from X chromosome inactivation in neural progenitor cells (Other)"^^ . . . . . . "Exploring the mechanisms of escape from X chromosome inactivation in neural progenitor cells (Other)"^^ . . . . . . "Exploring the mechanisms of escape from X chromosome inactivation in neural progenitor cells (Other)"^^ . . . . . . "Exploring the mechanisms of escape from X chromosome inactivation in neural progenitor cells (Other)"^^ . . . . . . 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