CONTROL OF POLYCOMB BY CIS-REPRESSIVE LONG NON-CODING RNAS

Cis-repressive long non-coding RNAs (lncRNAs) spread Polycomb Repressive Complexes (PRCs) within specific genomic regions to achieve chromatin compaction and stable gene silencing. Xist is the best characterized lncRNA; it is required to spread PRCs and silence genes across the entire 165 Mb X chromosome. Despite decades of research using the Xist lncRNA as a model, the relationship between lncRNAs and PRCs remains unclear. Airn and Kcnq1ot1 lncRNAs function similarly to Xist, but in smaller genomic regions. In this dissertation, we gained novel insights into lncRNA and PRC mechanism by comparing and contrasting lncRNA features and the genomic environments of Xist, Airn, and Kcnq1ot1. First, we found that Airn and Kcnq1ot1 spread PRCs and silence genes across multi megabase domains in mouse trophoblast stem cells (TSCs). Similar to the X chromosome, Airn and Kcnq1ot1 targeted regions contained non-uniform patterns of PRCs. We showed that PRC density in the 13 Mb Airn target region correlated with Airn abundance and was dependent on multiple aspects of genome architecture: linear distance to the Airn locus, pre-existing structure, TAD boundaries, and high-affinity chromatin sites of Airn. In Airn overexpression TSCs, eight PRC-bound CpG islands (CGIs) appeared to nucleate the spread of Polycomb. Deletion of one 2kb CGI caused loss of Polycomb across 4.5 Mb. Xist and Kcnq1ot1 targeted regions showed similar patterns of Polycomb at PRC-bound CGIs. This suggests a common mechanism where lncRNAs depend on pre-bound CGIs to specifically target and spread Polycomb in cis.Doctor of Philosoph

A Survey of Imprinted Gene Expression in Mouse Trophoblast Stem Cells

Several hundred mammalian genes are expressed preferentially from one parental allele as the result of a process called genomic imprinting. Genomic imprinting is prevalent in extra-embryonic tissue, where it plays an essential role during development. Here, we profiled imprinted gene expression via RNA-Seq in a panel of six mouse trophoblast stem lines, which are ex vivo derivatives of a progenitor population that gives rise to the placental tissue of the mouse. We found evidence of imprinted expression for 48 genes, 31 of which had been described previously as imprinted and 17 of which we suggest as candidate imprinted genes. An equal number of maternally and paternally biased genes were detected. On average, candidate imprinted genes were more lowly expressed and had weaker parent-of-origin biases than known imprinted genes. Several known and candidate imprinted genes showed variability in parent-of-origin expression bias between the six trophoblast stem cell lines. Sixteen of the 48 known and candidate imprinted genes were previously or newly annotated noncoding RNAs and six encoded for a total of 60 annotated microRNAs. Pyrosequencing across our panel of trophoblast stem cell lines returned levels of imprinted expression that were concordant with RNA-Seq measurements for all eight genes examined. Our results solidify trophoblast stem cells as a cell culture-based experimental model to study genomic imprinting, and provide a quantitative foundation upon which to delineate mechanisms by which the process is maintained in the mouse

Proximity-dependent recruitment of Polycomb repressive complexes by the lncRNA Airn

Summary: During mouse embryogenesis, expression of the long non-coding RNA (lncRNA) Airn leads to gene repression and recruitment of Polycomb repressive complexes (PRCs) to varying extents over a 15-Mb domain. The mechanisms remain unclear. Using high-resolution approaches, we show in mouse trophoblast stem cells that Airn expression induces long-range changes to chromatin architecture that coincide with PRC-directed modifications and center around CpG island promoters that contact the Airn locus even in the absence of Airn expression. Intensity of contact between the Airn lncRNA and chromatin correlated with underlying intensity of PRC recruitment and PRC-directed modifications. Deletion of CpG islands that contact the Airn locus altered long-distance repression and PRC activity in a manner that correlated with changes in chromatin architecture. Our data imply that the extent to which Airn expression recruits PRCs to chromatin is controlled by DNA regulatory elements that modulate proximity of the Airn lncRNA product to its target DNA