Dynamics of X Chromosome Inactivation

__Abstract__ Dosage compensation evolved to account for the difference in expression of sex chromosome-linked genes. In mammals dosage compensation is achieved by inactivation of one X chromosome during early female embryogenesis in a process called X chromosome inactivation (XCI). Central players in this process are two overlapping antisense transcribed noncoding genes, Xist and Tsix. The nature of this XCI process places this field of research at the interface between stem cell biology, epigenetics and gene regulation. The aim of this thesis is to shed further light onto the different levels of regulation that ensure faithful initiation and maintenance of XCI in a developmental context. These different levels include transcription factors, noncoding RNAs, antisense transcription, and epigenetic processes including DNA methylation, histone modifications and chromosomal conformation. In the first part we analyze trans- and cis-acting networks that regulate the initiation of XCI. Classical transcription factors and their regulators, most prominently the ubiquitin ligase RNF12 and its primary target in ES cells, REX1, have an essential function during initiation and early maintenance of XCI. Moreover, our deletion and transgene studies argue that many of the noncoding RNAs located in the Xic predominantly function in cis, and that X chromosome pairing events are not necessary for XCI to occur. We also utilize Xist and Tsix reporter lines to study the dynamics of Xist and Tsix re

FGF treatment of host embryos injected with ES cells increases rates of chimaerism

In spite of the emergence of genome editing tools, ES cell mediated transgenesis remains the most controllable way of creating genetically modified animals. Although tetraploid (4N) complementation of 4N host embryos and ES cells, is the only method guaranteeing that offspring are entirely ES cell derived, this technique is challenging, not always successful and difficult to implement in some laboratory settings. The current study shows that pretreatment of host blastocysts with FGF4 prior to ES cell injection can provide an alternative method for the generation of animals displaying high rates of chimaerism. Chimaerism assessment in E11 fetuses and born pups shows that a large percentage of resulting conceptuses show a high ES cell contribution from implantation onwards and that developing pups do not necessitate c-section for delivery

Xist and Tsix transcription dynamics is regulated by the X-to-autosome ratio and semistable transcriptional states

In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between Xlinked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist. Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation. Our results revealed mutually antagonistic roles for Tsix on Xist and vice versa and indicate the presence of semistable transcriptional states of the Xic locus predicting the outcome of XCI. These transcriptional states are instructed by the X-t

Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation

Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene

A Conserved Noncoding Locus Regulates Random Monoallelic Xist Expression across a Topological Boundary

cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.Galupa et al. uncover elements important for Xist regulation in its neighboring TAD and reveal that these elements can influence gene regulation both within and between topological domains. These findings, in a context where dynamic, developmental expression is necessary, challenge current models for TAD-based gene-regulatory landscapes

Chromatinmediated reversible silencing of sense-antisense gene pairs in embryonic stem cells is consolidated upon differentiatio

Genome-wide gene expression studies have indicated that the eukaryotic genome contains many gene pairs showing overlapping sense and antisense transcription. Regulation of these coding and/or noncoding gene pairs involves intricate regulatory mechanisms. In the present study, we utilized an enhanced green fluorescent protein (EGFP)-tagged reporter plasmid cis linked to a doxycycline-inducible antisense promoter, generating antisense transcription that fully overlaps EGFP, to study the mechanism and dynamics of gene silencing after induction of noncoding antisense transcription in undifferentiated and differentiating mouse embryonic stem cells (ESCs). We found that EGFP silencing is reversible in ESCs but is locked into a stable state upon ESC differentiation. Reversible silencing in ESCs is chromatin dependent and is associated with accumulation of trimethylated lysine 36 on histone H3 (H3K36me3) at the EGFP promoter region. In differentiating ESCs, antisense transcription-induced accumulation of H3K36me3 was associated with an increase in CpG methylation at the EGFP promoter. Repression of the sense promoter was affected by small-molecule inhibitors which interfere with DNA methylation and histone demethylation pathways. Our results indicate a general mechanism for silencing of fully overlapping sense-antisense gene pairs involving antisense transcription-induced accumulation of H3K36me3 at the sense promoter, resulting in reversible silencing of the sense partner, which is stabilized during ESC differentiation by CpG methylation

Epigenetic characterization of the FMR1 promoter in induced pluripotent stem cells from human fibroblasts carrying an unmethylated full mutation

Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene