Down syndrome (DS) is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21 (Chr21). We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST. Using genome editing with zinc finger nucleases, we targeted a large, inducible XIST transgene into the Chr21 DYRK1A locus, in DS pluripotent stem cells. XIST RNA coats Chr21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a “Chr21 Barr Body.” This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Remarkably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one Chr21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of “chromosome therapy”.

SUMMARY Recent studies recognize a vast diversity of non-coding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using CoT-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (“CoT-1 RNA”), including LINE-1. CoT-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis, and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The CoT-1 RNA territory resists mechanical disruption and fractionates with the non-chromatin scaffold, but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. CoT-1 RNA has several properties similar to XIST chromosomal RNA, but is excluded from chromatin condensed by XIST. These findings impact two “black boxes” of genome science: the poorly understood diversity of non-coding RNA and the unexplained abundance of repetitive elements.

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SUMMARYThe discovery of small non-coding microRNAs has revealed novel mechanisms of post-translational regulation of gene expression, the implications of which are still incompletely understood. We focused on microRNA 21 (miR-21), which is expressed in cardiac valve endothelium during development, in order to better understand its mechanistic role in cardiac valve development. Using a combination of in vivo gene knockdown in zebrafish and in vitro assays in human cells, we show that miR-21 is necessary for proper development of the atrioventricular valve (AV). We identify pdcd4b as a relevant in vivo target of miR-21 and show that protection of pdcd4b from miR-21 binding results in failure of AV development. In vitro experiments using human pulmonic valve endothelial cells demonstrate that miR-21 overexpression augments endothelial cell migration. PDCD4 knockdown alone was sufficient to enhance endothelial cell migration. These results demonstrate that miR-21 plays a necessary role in cardiac valvulogenesis, in large part due to an obligatory downregulation of PDCD4.