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Chromosome silencing to study Down Syndrome

XIST transgenes can silence an autosome

The X-linked human XIST gene produces a large, non-coding RNA, which we showed is expressed exclusively from the inactive X chromosome (Xi) and accumulates to coat the X chromosome in cis. XIST RNA on the Xi induces a series of chromatin modifications that collectively silence the chromosome. Subsequent work from our lab and others indicates XIST RNA is also capable of silencing autosomal chromosomes.

Unbalanced X;autosome translocations provide evidence for sequence specificity in the association of XIST RNA with chromatin. Hum Mol Genet. 2002 Dec 1;11(25):3157-65.

An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells. Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8677-82.

Translating dosage compensation to trisomy 21

Using genome editing with zinc finger nucleases (and currently CRISPR/Cas9), we targeted a large, inducible XIST transgene into the one of three Chromosome 21s in induced pluripotent stem cells (iPSCs) derived from Down syndrome patient cells. XIST RNA coats the extra chromosome 21 and triggers chromosome-wide transcriptional silencing and stable heterochromatin modifications, to form a “Chromosome 21 Barr Body.”

Translating dosage compensation to trisomy 21. Nature. 2013 Aug 15;500(7462):296-300.

Trisomy silencing normalizes Down syndrome hematopoietic defects

Trisomy 21 confers approximately 500-fold greater incidence of acute megakaryocytic leukemia (AMKL) and a ~20-fold greater risk for acute lymphoblastic leukemia (ALL). In addition, less severe hematopoietic abnormalities are present in most individuals with DS, including immune system defects.
We have demonstrated that trisomy silencing by XIST-induced expression from one chromosome 21 can correct the regulatory mechanisms that underlie the hematopoietic cell pathologies of DS. Trisomy silencing reduced the overproduction of megakaryocyte and erythrocyte colonies in multiple transgenic clones. This also normalized an excess of CD43⁺ hematopoietic progenitors but did not impact earlier CD34⁺/CD43⁻ population. Overall, results support that XIST normalized the hematopoietic process by rebalancing dosage-sensitive chromosome 21 genes.

Trisomy 21 initially increases the production of hematopoietic progenitor cells as they emerge from the endothelial-to-hematopoietic transition. A likely enhanced EHT process is accompanied by increased IGF signaling. Trisomy 21 also increases the colony-forming potential of these hematopoietic progenitor cells.

Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro. Nat Commun. 2018 Dec 5;9(1):5180.

Silencing Trisomy 21 Promotes Neuronal Differentiation

We have demonstrated that XIST mediated chromosome silencing occurs in differentiated cells, contrary to the expectation that pluripotency factors are required.

The cell pathologies that underlie cognitive disabilities in Down syndrome are not well understood. We have investigated the effects of trisomy 21 on in vitro DS neurodevelopment, combining bulk and single-cell RNA sequencing (scRNA-seq) with molecular cytology of otherwise identical cells, with and without XIST-mediated dosage compensation induced from pluripotency or later in differentiation. This study demonstrated that over-expression of chr21 genes confers a developmental delay in terminal differentiation of neural stem cells (NSCs) to neurons.

Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation. Dev Cell. 2020 Feb 10;52(3):294-308.

Trisomy 21 causes deficits in angiogenesis and Notch signaling

To understand which cell types and pathways are more directly affected by trisomy 21 (T21), we used an inducible-XIST system to silence one chromosome 21 in vitro.
T21 caused the dysregulation of Notch signaling in iPSCs, potentially affecting cell-type programming. Further analyses identified dysregulation of pathways important for two cell types: neurogenesis and angiogenesis.
An in vitro assay for microvasculature formation revealed a cellular pathology involving delayed tube formation in response to angiogenic signals. Parallel transcriptomic analysis of endothelia further showed deficits in angiogenesis regulators.

Chromosome silencing in vitro reveals trisomy 21 causes cell-autonomous deficits in angiogenesis and early dysregulation in Notch signaling. Cell Rep. 2022 Aug 9;40(6):111174.