Project description:Although Down Syndrome (DS) is the leading genetic cause of intellectual disability in children, the developmental pathogenesis remains largely unknown, and better strategies are needed to investigate this. We previously showed that one copy of chromosome 21 can be epigenetically silenced in DS iPSCs by insertion of an XIST transgene, which produces a non-coding RNA that normally silences one X chromosome in female cells. XIST was shown to induce heterochromatin and silence transcription across chromosome 21 in pluripotent stem cells, the natural developmental context of XIST function. Prior literature indicated that initiation of chromosome silencing is only possible within 48 hours of mES cell differentiation, however it would be highly advantageous experimentally if trisomy silencing could be initiated in differentiated cells, and this is critical for any therapeutic potential of XIST. Here we use RNAseq and molecular cytology to investigate the effectiveness of XIST for trisomy silencing in cells undergoing in vitro neural differentiation and examine the potential cell phenotypic effects of chromosomal silencing. Induction of XIST from the onset of differentiation resulted in comprehensive silencing of chromosome 21 genes, providing a powerful approach to examine effects of trisomy on neurogenesis. To determine whether human neural stem cells can initiate XIST-mediated silencing, we induced XIST at several times during neural differentiation. We demonstrate for the first time that differentiated normal human cells can initiate chromosome silencing in response to XIST expression. While this process only takes a few days in pluripotent cells, we show that it takes 2-3 weeks in differentiated cells. Importantly, single-cell RNAseq revealed that cells which express XIST preferentially differentiate into neurons, providing evidence of phenotypic improvement with trisomy silencing, which is seen even when XIST is initiated weeks into differentiation. We are currently investigating whether silenced neurons show other non-chromosome 21 transcriptional changes suggestive of potentially improved function. These studies have important implications for the understanding of XIST biology and DS neurobiology, and also further open the possibility of a chromosomal therapy for DS using a single gene: XIST.

Dataset Information

Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation.

Publications

Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation.

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