Translating Dosage Compensation to Trisomy 21

Down syndrome is a common disorder with enormous medical and social costs, caused by trisomy for Chr21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected in DS patient stem cells by manipulating a single gene, XIST. Using 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. In this study, we used microarrays to understand the genome-wide impacts of inducible XIST expression on Chr21 in trisomy 21 human iPS cell lines, and to evaluate the extent of Chr21 silencing trisomic samples versus a disomic male iPS cell line. Three independently targeted subclones plus the parental Chr21 trisomic (non-targeted) iPS cell line were grown ± doxycycline (2 µg/ml) for 22 d. Normal male iPS line was also cultured for 22 d and total RNA was extracted with a High Pure RNA extraction kit (Roche) in triplicate for each, processed with a Gene Chip 3’ IVT Express Kit (Affymetrix), and hybridized to Affymetrix human gene expression PrimeView arrays. Array normalization was performed with Affymetrix Expression Console Software with Robust Multichip Analysis (RMA). Probesets with the top 60% of signal values were considered present and “expressed” and were used for all further analysis. In total 9 RNA samples were prepared in triplicate for a total of 27 arrays.