Introduction: Due to the discrepancy in the number of X chromosomes between the sexes in many animal species, dosage compensation mechanisms have evolved to equalize X-linked gene dosage between males and females. In female mammals, dosage compensation is achieved by a remarkable process known as X-chromosome inactivation (XCI), that leads to the epigenetic silencing of an entire X chromosome. XCI is master-regulated by a long non-coding RNA (lncRNA) called X-inactive specific transcript (Xist in mouse/XIST in human), which is exclusively expressed from the future inactive X-chromosome (Xi). Xist gene is peculiar, since it encodes for a 17 kb capped, spliced and polyadenylated RNA that is never translated. This lncRNA is monoallelically upregulated randomly from only one of the X chromosomes chosen for inactivation, coating the entire chromosome in cis and inducing transcriptional silencing and heterochromatin formation that is stably inherited through cell divisions. Xist lncRNA is poorly conserved at sequence level, with the exception of six tandem-repeated regions, known as the A to F repeats. These regions are believed to act as regulatory modules that exert their function through the interaction with specific RNA-binding proteins. However, the relative contribution of each of these RNA modules to XCI is not fully understood. Methods: Using a well-described system whereby Xist is expressed from a tetracycline-inducible promoter in its endogenous position in J1 male mouse embryonic stem cells, we performed deletions for Xist D and E tandem-repeated regions by CRISPR/Cas9. Then, we validated and characterized these mutants alongside another mutant type previously generated in the lab (Xist ΔF). Three independent clones per mutant type were selected and their potential impact on XCI was addressed. We specifically explored several features related to XCI, namely: Xist coating of the X chromosome, through RNA Fluorescence In Situ Hybridization (FISH) for Xist; Xist capacity to silence the genes on the X chromosome, by RT-qPCR and RNA-FISH for X-linked genes; recruitment of typical heterochromatin marks of the Xi, using RNA FISH for Xist combined with Immunofluorescence for the histone marks H3K27me3, H2AK119ub and H4K20me1. Results: Xist-TetOP mutants for D- and E-repeats were generated, and, jointly with the previous deletion for the F-repeat, successfully validated. All the characterized mutants (Xist ΔF, ΔD and ΔE) were able to form Xist domains (or clouds) around the Xi, as observed by RNA-FISH for Xist. Xist ΔF clouds visually seemed smaller, but this was not quantified. By measuring Xist expression levels by RT-qPCR analysis, we suspected a possible instability in Xist ΔD transcripts that should be further explored. The X-linked gene silencing of some candidate genes seemed not to be affected in all the Xist mutants, but further characterization at X chromosome global level would give a definitive answer. Finally, typical heterochromatin marks of the Xi were explored for two clones of Xist ΔF mutants and no major defects were observed, although the two clones (F2 and F3) exhibited some variability. Conclusion: As a result of this work, a new series of inducible Xist mutants was generated, which will be a valuable set of experimental tools for further investigation in the field of XCI. Our initial characterization gave the first hints on the functional role of some unexplored conserved repeats of Xist during XCI. In the future, these Xist-inducible mutants can be used in high-throughput approaches to enquiry about gene silencing (RNA-seq), chromatin status (ChIP-seq) or to fish protein interactors of the different repeats (ChIRP-MS), to get mechanistic insights in this puzzled process of XCI.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019