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 regulation during ES cell differentiation and find it to be more stable than previously anticipated. Reduction of the X:autosome ratio reveals semi-stable states which might correspond to distinct higher order chromatin conformations of the Xic. These studies also indicate that X-encoded factors are involved in Tsix activation. In the second part we present evidence for a special regulatory phase during early embryonic development in which dedicated chromatin modulators act to confer stable gene repression. A reporter construct containing an inducible antisense promoter showed that antisense transcription mediated repression in ES cells was reversible and dependent on properly assembled chromatin. Transcriptional interference did not seem to have a major repressive effect in our system. Repression was locked in by DNA methylation as soon as antisense transcription through the promoter of the reporter was induced during differentiation. Since the promoter of the reporter acquired H3K36me3 upon induction of antisense transcription, we propose that H3K36me3 might be a crucial component in the cascade of stably silencing antisense transcribed genes.