Translating dosage compensation to trisomy 21

Abstract

Down's syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a 'chromosome 21 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. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of 'chromosome therapy'.

Figure 1. Genome-editing integrates XIST into Chr21 in trisomic iPSCs

a. Concept for translating dosage compensation to trisomy 21. b. XIST construct (19kb): two homologous arms and 14kb XIST cDNA with inducible pTRE3G promoter. c. DNA/RNA FISH in interphase DS iPSCs shows XIST overlaps one of three DYRK1A genes in non-expressing cell (top, arrows), and a large XIST RNA territory with DYRK1A after 3 days in dox (bottom, arrows). Scale: 2 μm. d. OCT4 immunostaining & XIST RNA FISH in a transgenic colony: highly consistent XIST expression throughout the colony. Scale: 100 μm. e. Metaphase DNA FISH shows one targeted Chr21. XIST gene (asterisk and close-up) overlaps one of three DYRK1A genes (arrows).

Figure 2. XIST induces heterochromatin modifications and condensed Chr21 Barr Body

a. XIST RNA recruits heterochromatic epigenetic marks (e.g. UbH2A). Channels separated for indicated cell. b. Percentage of XIST territories with heterochromatin marks. Mean ± SE, 100 nuclei in ~5 colonies. c. XIST RNA induces “Chr21 Barr Body” visible by DAPI stain (arrow). Scale: 2 μm.

Figure 3. XIST induces long-range silencing in targeted iPSCs

a. RNA FISH. APP RNA transcribes from three loci in uninduced cells (Day 0), and is progressively silenced following induction (targeted Chr21, arrows). Scale: 2 μm. b. Quantification of APP silencing. Mean ± SE, 100 nuclei. c. Silencing for four more Chr21-linked genes by RNA FISH. Mean ± SE from 100 nuclei. d. Long range silencing of Chr21 genes by XIST RNA. USP25 is ~21 Mb from XIST integration site (black arrow).

Figure 4. Genomic expression and methylation reveal widespread silencing of Chr21

a. Microarray: expression difference for three transgenic clones in dox versus no dox, compared to disomic line versus trisomic parental line. Total change in gene expression (N=3) per chromosome shows Chr21 “correction” near disomic levels, with only limited changes on other chromosomes. Right Y-axis scaled for percent gene expression change. b. Distribution of individual gene repression across Chr21. c. Methylation of CpG island promoters. In treated clones, 97% of Chr21 genes increased by at least 5% (2 fold greater than average), compared to none in the parental line. P: parental line; 1: Clone 1; 2: Clone 2.

Figure 5. “Trisomy correction” effects cell proliferation and neurogenesis

a. One week of XIST expression resulted in larger, more numerous colonies (representative sample). b. Changes in cell number for parental line (PL), parental line subclone (PL-s), and six transgenic clones (C1–C6). Mean ±SE. (n=4–6). c. Corrected cultures formed neural rosettes by day 14; trisomic (parental and non-induced) cultures took longer (17days). Scale: 100μm. e. Number of rosettes formed on day 14 and day 17. Mean ±SE, 10–12 random fields in triplicate. P: parental; C1: Clone 1; C3: Clone 3.