1 Nature 2013 Vol: 500(7462):296-300. DOI: 10.1038/nature12394

Translating dosage compensation to trisomy 21

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’.

Mentions
Figures
Figure 1: Genome editing integrates XIST into chromosome 21 in trisomic iPS cells.a, Concept for translating dosage compensation to trisomy 21. b, XIST construct (19 kb): two homologous arms and 14-kb XIST cDNA with inducible pTRE3G promoter. c, DNA/RNA FISH in interphase Down’s syndrome iPS cells shows that XIST overlaps one of three DYRK1A genes (left panels and insets) in a non-expressing cell (top, arrows), and a cell induced to express a large XIST RNA territory over the DYRK1A locus after 3 days in doxycycline (bottom, arrows). Right panels show green channel (DYRK1A) alone. Nuclear DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 2 μm. d, OCT4 immunostaining and XIST RNA FISH in a transgenic colony: highly consistent XIST expression throughout the colony. Scale bar, 100 μm. e, Metaphase DNA FISH shows one targeted chromosome 21. XIST gene (asterisk and close-up) overlaps one of three DYRK1A genes (arrows). Figure 2: XIST induces heterochromatin modifications and condensed chromosome 21 Barr body.a, XIST RNA recruits heterochromatic epigenetic marks (for example, UbH2A). Channels are separated for cell indicated with an arrow. Scale bar, 5 μm. b, Percentage of XIST territories with heterochromatin marks. Mean ± standard error, 100 nuclei in ~5 colonies. c, XIST RNA induces chromosome 21 Barr body visible by DAPI stain (arrow). Scale bar, 2 μm. Figure 3: XIST induces long-range silencing in targeted iPS cells.a, RNA FISH. APP RNA transcribes from three loci in uninduced cells (day 0), and is progressively silenced after induction (targeted chromosome 21, arrows). Scale bar, 2 μm. b, Quantification of APP silencing. Mean ± standard error, 100 nuclei. c, Silencing for four more chromosome-21-linked genes by RNA FISH. Mean ± standard error from 100 nuclei. d, Long-range silencing of chromosome 21 genes by XIST RNA. USP25 is ~21 Mb from the XIST integration site (black arrow). Figure 4: Genomic expression and methylation reveal widespread silencing of chromosome 21.a, Microarray: expression difference for three transgenic clones in doxycycline (dox) versus no doxycycline, compared to disomic line versus trisomic parental line. Total change in gene expression (n = 3) per chromosome shows chromosome 21 ‘correction’ near disomic levels, with only limited changes on other chromosomes. The right y axis is scaled for per cent gene expression change. Mean ± standard deviation, in triplicate. b, Distribution of individual gene repression across chromosome 21. c, Methylation of CpG island promoters. In treated clones, 97% of chromosome 21 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’ affects 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 ± s.e. (n = 4–6). c, Corrected cultures formed neural rosettes by day 14; trisomic (parental and non-induced) cultures took longer (17 days). Scale bar, 100 μm. d, Number of rosettes formed on day 14 and day 17. Mean ± standard error, 10–12 random fields in triplicate. P, parental; C1, clone 1; C3, clone 3.
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References
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    • . . . An inducible system for such ‘trisomy silencing’ would have immediate translational relevance as a resource to investigate the cellular pathology and gene pathways affected in Down’s syndrome, in a setting free from pervasive genetic or epigenetic variation that exists between individuals, sub-clones, or even isogenic cell isolates3, 7, 8. . . .
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    • . . . An inducible system for such ‘trisomy silencing’ would have immediate translational relevance as a resource to investigate the cellular pathology and gene pathways affected in Down’s syndrome, in a setting free from pervasive genetic or epigenetic variation that exists between individuals, sub-clones, or even isogenic cell isolates3, 7, 8. . . .
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    • . . . This is driven by a large (~17 kilobases (kb) in human), non-coding RNA, XIST, which is produced exclusively from the inactive X chromosome9, and ‘paints’ (accumulates across) the interphase chromosome structure10 . . .
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    • . . . This is driven by a large (~17 kilobases (kb) in human), non-coding RNA, XIST, which is produced exclusively from the inactive X chromosome9, and ‘paints’ (accumulates across) the interphase chromosome structure10 . . .
    • . . . This mirrored the unique behaviour of endogenous XIST RNA which ‘paints’ the inactive X chromosome nuclear territory10. . . .
    • . . . We next used multi-colour RNA FISH to determine the presence of transcription foci at each allele for six specific chromosome 21 genes, an established approach that we earlier showed10, 15 discriminates active versus silenced genes on inactive X chromosome . . .
    • . . . DNA and RNA FISH were carried out as previously described10, 15, 16, 33 . . .
  11. Heard, E. Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome Curr. Opin. Genet. Dev. 15, 482-489 (2005) .
    • . . . During early development, the XIST RNA induces numerous heterochromatin modifications and architectural changes which transcriptionally silence the inactive X chromosome and manifest cytologically as a condensed Barr body (reviewed in refs 11, 12) . . .
    • . . . The natural inactivated X chromosome forms a condensed Barr body which carries repressive histone marks11 . . .
  12. Hall, L. L.; Lawrence, J. B. XIST RNA and architecture of the inactive X chromosome: implications for the repeat genome Cold Spring Harb. Symp. Quant. Biol. 75, 345-356 (2010) .
    • . . . During early development, the XIST RNA induces numerous heterochromatin modifications and architectural changes which transcriptionally silence the inactive X chromosome and manifest cytologically as a condensed Barr body (reviewed in refs 11, 12) . . .
    • . . . Thus, XIST RNA evolved for the X chromosome uses epigenome-wide mechanisms12 . . .
  13. Carrel, L.; Willard, H. F. X-inactivation profile reveals extensive variability in X-linked gene expression in females Nature 434, 400-404 (2005) .
    • . . . There is evidence for some DNA sequence specificity to XIST function, as certain human genes escape X-inactivation13; however, autosomal chromatin has substantial capacity to be silenced14, 15, 16 . . .
  14. Lee, J. T.; Strauss, W. M.; Dausman, J. A.; Jaenisch, R. A 450 kb transgene displays properties of the mammalian X-inactivation center Cell 86, 83-94 (1996) .
    • . . . There is evidence for some DNA sequence specificity to XIST function, as certain human genes escape X-inactivation13; however, autosomal chromatin has substantial capacity to be silenced14, 15, 16 . . .
  15. Hall, L. L.; Clemson, C. M.; Byron, M.; Wydner, K.; Lawrence, J. B. Unbalanced X;autosome translocations provide evidence for sequence specificity in the association of XIST RNA with chromatin Hum. Mol. Genet. 11, 3157-3165 (2002) .
    • . . . There is evidence for some DNA sequence specificity to XIST function, as certain human genes escape X-inactivation13; however, autosomal chromatin has substantial capacity to be silenced14, 15, 16 . . .
    • . . . We next used multi-colour RNA FISH to determine the presence of transcription foci at each allele for six specific chromosome 21 genes, an established approach that we earlier showed10, 15 discriminates active versus silenced genes on inactive X chromosome . . .
    • . . . DNA and RNA FISH were carried out as previously described10, 15, 16, 33 . . .
  16. Hall, L. L. An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells Proc. Natl Acad. Sci. USA 99, 8677-8682 (2002) .
    • . . . There is evidence for some DNA sequence specificity to XIST function, as certain human genes escape X-inactivation13; however, autosomal chromatin has substantial capacity to be silenced14, 15, 16 . . .
    • . . . To measure overall transcription across the XIST-targeted chromosome 21, we used an approach that we developed to broadly assay heterogeneous nuclear RNA (hnRNA) expression and to distinguish inactive from active X chromosome16, on the basis of in situ hybridization to CoT-1 repeat RNA . . .
    • . . . DNA and RNA FISH were carried out as previously described10, 15, 16, 33 . . .
  17. Moehle, E. A. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases Proc. Natl Acad. Sci. USA 104, 3055-3060 (2007) .
    • . . . We pursued zinc finger nuclease (ZFN)-driven targeted addition17 of an inducible XIST transgene to the gene-rich core of chromosome 21 in induced pluripotent stem (iPS) cells derived from a Down’s syndrome patient . . .
    • . . . Southern blotting for targeted gene addition was performed exactly as described17, 32 on SphI-digested genomic DNA probed with a fragment corresponding to positions Chr21:38825803+38826056 (hg19). . . .
  18. Urnov, F. D.; Rebar, E. J.; Holmes, M. C.; Zhang, H. S.; Gregory, P. D. Genome editing with engineered zinc finger nucleases Nature Rev. Genet. 11, 636-646 (2010) .
    • . . . Thus, our first goal was to demonstrate that ZFNs could accurately insert the largest transgene to date, substantially larger than sequences commonly used for genome editing18 . . .
    • . . . ZFNs against the DYRK1A locus were designed using an archive of pre-validated zinc finger modules18, 29, and validated for genome editing activity by transfection into K562 cells and Surveyor endonuclease-based measurement of endogenous locus disruption (‘Cel1’30, 31) exactly as described29 . . .
  19. DeKelver, R. C. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome Genome Res. 20, 1133-1142 (2010) .
    • . . . We first attempted this with a ~16-kb XIST transgene in a transformed cell line (HT1080), using established ZFNs to the AAVS1 locus on chromosome 19 (ref. 19) . . .
    • . . . In one step, we integrated both the doxycycline-controlled XIST transgene into chromosome 21 (Fig. 1b and Supplementary Fig. 3a) and a transgene carrying the doxycycline control component (rtTA) into the AAVS1 chromosome 19 safe harbour, disruption of which creates no known adverse effects19 (Supplementary Fig. 3b). . . .
    • . . . ZFNs against the human AAVS1 locus on chromosome 19 have been previously described19 . . .
    • . . . The pEF1α-3G rtTA-pA cassette from pEF1α-Tet3G vector (Clontech) was subcloned into a plasmid for targeted gene addition to the PPP1R12C/AAVS1 locus19, which contains a unique HindIII site flanked by two 800-bp stretches of homology to the ZFN-specified position in the genome. . . .
  20. Park, I. H. Disease-specific induced pluripotent stem cells Cell 134, 877-886 (2008) .
    • . . . We used a male Down’s syndrome iPS cell line20 which we confirmed maintains pluripotency markers and trisomy 21 . . .
    • . . . Daley (Children’s Hospital Boston)20 and maintained on irradiated mouse embryonic fibroblasts (iMEFs) (R&D Systems, PSC001) in hiPSC medium containing DMEM/F12 supplemented with 20% knockout serum replacement (Invitrogen), 1 mM glutamine (Invitrogen), 100 μM non-essential amino acids (Invitrogen), 100 μM β-mercaptoethanol (Sigma) and 10 ng ml−1 FGF-β (Invitrogen, PHG0024) . . .
  21. Aït Yahya-Graison, E. Classification of human chromosome 21 gene-expression variations in Down syndrome: impact on disease phenotypes Am. J. Hum. Genet. 81, 475-491 (2007) .
    • . . . Because evidence suggests that many chromosome 21 genes are not increased the theoretical 1.5-fold in trisomy21, 22, we also directly compared trisomic to disomic cells . . .
    • . . . Trisomy 21 may have an impact on genome-wide expression pathways, but differences attributable to trisomy 21 are confounded by genetic and epigenetic variability21 . . .
  22. Biancotti, J. C. Human embryonic stem cells as models for aneuploid chromosomal syndromes Stem Cells 28, 1530-1540 (2010) .
    • . . . Because evidence suggests that many chromosome 21 genes are not increased the theoretical 1.5-fold in trisomy21, 22, we also directly compared trisomic to disomic cells . . .
    • . . . The fraction of chromosome 21 genes detected as overexpressed varies with the study and tissue, but generally is in the 19–36% range3, 22, with individual gene increases often in the ~1.2–1.4 range (less than the theoretical 1.5) . . .
    • . . . For example, one study of Down’s syndrome embryoid bodies showed that only 6–15% of genes appeared significantly upregulated, but this was comparing non-isogenic samples of different ES cell isolates22. . . .
  23. Csankovszki, G.; Nagy, A.; Jaenisch, R. Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation J. Cell Biol. 153, 773-784 (2001) .
    • . . . In addition to transcriptional silencing, X-inactivation is stabilized by hypermethylation of promoter CpG islands23, 24, which occurs late in the silencing process . . .
    • . . . First, we show that heterochromatic silencing is stably maintained, even upon removal of doxycycline and XIST expression (Supplementary Fig. 9a, b), consistent with previous studies23 . . .
  24. Cotton, A. M. Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation Hum. Genet. 130, 187-201 (2011) .
    • . . . In addition to transcriptional silencing, X-inactivation is stabilized by hypermethylation of promoter CpG islands23, 24, which occurs late in the silencing process . . .
    • . . . Here, 97% of CpG-island-containing genes exhibited a robust increase in promoter DNA methylation, within the range of that seen for the inactive X chromosome24 (adjusted for active/inactive chromosomes; see Methods) . . .
    • . . . Because any methylation increase on the transgenic chromosome would be diluted by the presence of three chromosome 21 copies, this suggests the range of 60% methylation on the one XIST-coated chromosome, which is within the range seen for the inactive X chromosome24. . . .
  25. Guidi, S.; Ciani, E.; Bonasoni, P.; Santini, D.; Bartesaghi, R. Widespread proliferation impairment and hypocellularity in the cerebellum of fetuses with down syndrome Brain Pathol. 21, 361-373 (2011) .
    • . . . There is some evidence of proliferative impairment in Down’s syndrome brains4, 25; however, we observed that this varied in vitro between our Down’s syndrome fibroblast samples, and this would be highly sensitive to culture history . . .
    • . . . XIST expression triggers not only chromosome 21 repression, but a defined effect on the genomic expression profile, and reverses deficits in cell proliferation and neural progenitors, which has implications for hypocellularity in the Down’s syndrome brain4, 25 . . .
  26. Shi, Y. A human stem cell model of early Alzheimer’s disease pathology in Down syndrome Sci. Transl. Med. 4, 124ra29 (2012) .
    • . . . Variability in the kinetics of neural differentiation between various iPS cell lines can obscure differences due to trisomy 21 (ref. 26) . . .
  27. Lavon, N. Derivation of euploid human embryonic stem cells from aneuploid embryos Stem Cells 26, 1874-1882 (2008) .
    • . . . Inducible trisomy silencing in vitro compares otherwise identical cultures, allowing greater discrimination of differences directly due to chromosome 21 overexpression distinct from genetic and epigenetic variation between transgenic sub-clones, or potentially even rare disomic sub-clones isolated from a trisomic population (refs 27, 28 and this study) . . .
  28. Li, L. B. Trisomy correction in down syndrome induced pluripotent stem cells Cell Stem Cell 11, 615-619 (2012) .
    • . . . Inducible trisomy silencing in vitro compares otherwise identical cultures, allowing greater discrimination of differences directly due to chromosome 21 overexpression distinct from genetic and epigenetic variation between transgenic sub-clones, or potentially even rare disomic sub-clones isolated from a trisomic population (refs 27, 28 and this study) . . .
  29. Doyon, J. B. Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells Nature Cell Biol. 13, 331-337 (2011) .
    • . . . ZFNs against the DYRK1A locus were designed using an archive of pre-validated zinc finger modules18, 29, and validated for genome editing activity by transfection into K562 cells and Surveyor endonuclease-based measurement of endogenous locus disruption (‘Cel1’30, 31) exactly as described29 . . .
  30. Miller, J. C. An improved zinc-finger nuclease architecture for highly specific genome editing Nature Biotechnol. 25, 778-785 (2007) .
    • . . . ZFNs against the DYRK1A locus were designed using an archive of pre-validated zinc finger modules18, 29, and validated for genome editing activity by transfection into K562 cells and Surveyor endonuclease-based measurement of endogenous locus disruption (‘Cel1’30, 31) exactly as described29 . . .
  31. Guschin, D. Y. A rapid and general assay for monitoring endogenous gene modification Methods Mol. Biol. 649, 247-256 (2010) .
    • . . . ZFNs against the DYRK1A locus were designed using an archive of pre-validated zinc finger modules18, 29, and validated for genome editing activity by transfection into K562 cells and Surveyor endonuclease-based measurement of endogenous locus disruption (‘Cel1’30, 31) exactly as described29 . . .
  32. Urnov, F. D. Highly efficient endogenous human gene correction using designed zinc-finger nucleases Nature 435, 646-651 (2005) .
    • . . . Southern blotting for targeted gene addition was performed exactly as described17, 32 on SphI-digested genomic DNA probed with a fragment corresponding to positions Chr21:38825803+38826056 (hg19). . . .
  33. Byron, M.; Hall, L. L.; Lawrence, J. B. A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures Curr. Protoc. Hum. Gen Chapter 4, (2013) .
    • . . . DNA and RNA FISH were carried out as previously described10, 15, 16, 33 . . .
  34. Irizarry, R. A. Summaries of Affymetrix GeneChip probe level data Nucleic Acids Res. 31, e15 (2003) .
    • . . . Array normalization was performed with Affymetrix Expression Console Software with Robust Multichip Analysis (RMA)34 . . .
  35. Weber, M. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome Nature Genet. 39, 457-466 (2007) .
    • . . . CpG islands were defined as high and intermediate CpG densities using the CpG density classifications based on those used previously35 . . .
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