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Autosomal chromosome epigenetic silencing to cure Down syndrome
XIST Mediated Autosomal Silencing

Dosage Compensation
Gene dosage compensation is ubiquitous across the animal kingdom, hinting at its critical importance. Different organisms have independently evolved distinct strategies of dosage compensation. These compensations are necessary to counterbalance gene dosage differences along chromosomes. Indeed, the use of sex chromosome for example, produce a imbalance in the copy number of genes, needing thus a compensation. Different organisms use distinct mechanisms to ensure that each sex has roughly the same level of expression of genes on its sex chromosomes. Caenorhabditis elegans represses both of its hermaphroditic X chromosomes by half; Drosophila melanogaster males transcribe their lone X chromosome at twice the rate of female flies; and female mammals routinely silence and compact one X chromosome into a Barr body in early embryonic development (Meyer et al., 2005).

Role of XIST in X-Inactivation
Though the mechanisms underlying mammalian X chromosome inactivation have been studied for several decades, much remains to be uncovered. The necessity and sufficiency of XIST, a long non-coding RNA, however, is well established (Wutz et al., 2000). XIST is a transcript of the X inactivation center (XIC) and the anti-sense strand of XIST produce the long non coding RNA Tsix.

A compelling model for X inactivation initiation proposes that both X chromosomes maintain an epigenetically identical profile and euchromatic formation prior to X chromosome inactivation (XCI). Prior to differentiation, both X chromosomes produce TSIX (the antisense partner to XIST), but not XIST. TSIX expression has been shown to inhibit expression of XIST. Before XIST is upregulated, the two X chromosomes will transiently pair at the X inactivation center (XIC) within the cell’s nucleus—this transient pairing allows for a stochastic redistribution of TSIX activating proteins from both X chromosomes to only one (Froberg et al., 2013). This drives expression of TSIX on the now active X (Xa) and triggers the expression of XIST from the soon to be inactive X (Xi).

Epigenetic Marks Associated with X Inactivation
The expression of the XIST gene product at Xi results in the recruitment of additional chromatin modifying factors—in particular, polycomb repressive complex 2 (PRC2) (Froberg et al., 2013). One particular subunit of PRC2, EZH2, directly binds the Repeat A (RepA) motif. RepA is both part of Xist and part of its own transcript and, in turn, leads to the spread of XIST and silencing of the X chromosome through, initially, tri-methylation of histone H3 at lysine 27 (H3K27me3) (Froberg et al., 2013). This is followed by H3K9 di-methylation (Okamoto et al., 2004). In addition to the tri-methylation of H3K27 and di-methylation of H3K9, other histone marks associated with maintaining the inactive X include: mono-methylation of H4K20, ubiquitination of H2A (Heard E., 2005), and association of macroH2A. Hypermethylation of CpG island promoters along Xi is the final epigenetic mark known to occur. It is thought to help stabilize the inactivation status of Xi (Csankovszki et al., 2001).

Autosomal Silencing with XIST
Recently, researchers have introduced human XIST cDNA into the trisomic 21st autosome in iPS cells derived from an adult male patient with Down syndrome (Jiang et al., 2013). The question underscoring this research is intriguing—Can the X-inactivation gene, XIST, inserted autosomally, effect chromosome wide changes that result in the silencing of a trisomic chromosome? And does epigenetic silencing of that chromosome functionally correct Down syndrome phenotype and genomic expression levels, and thus, lead to a therapy for this syndrome These are particularly pressing questions given the paucity of therapeutic options for individuals with Down syndrome.

Epigenetic Marks Associated with XIST-Mediated Autosomal Silencing
The results from this single-study hold exceptional promise. Following the successful integration of XIST cDNA, the formation of a “chromosome 21 Barr body” appeared. The trisomic 21st chromosome expressing XIST was also enriched for H3K27me3, UbH2A, H4K20me, and macroH2A, histone marks known to be associated with the inactive X chromosome. qRT-PCR and transcriptome profiling with a microarray confirmed both allele-specific silencing and genome-wide silencing and methylation. Finally, autosomal XIST expression resulted in larger, more numerous cell colonies, in which neural rosettes abounded.

The first version of this document was written as part of an epigenetics class at The University of Texas at Austin.

Reference
1. Meyer, B. J. (2005). X-Chromosome dosage compensation. WormBook, 1–14. DOI: http://dx.doi.org/10.1895/wormbook.1.8.1

2. Wutz, A., & Jaenisch, R. (2000). A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation. Molecular Cell, 5(4), 695–705. DOI: http://dx.doi.org/10.1016/S1097-2765(00)80248-8

3. Froberg, J. E., Yang, L., & Lee, J. T. (2013). Guided by RNAs: X-inactivation as a model for lncRNA function. Journal of Molecular Biology, 425(19), 3698–3706. DOI: http://dx.doi.org/10.1016/j.jmb.2013.06.031

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5. Heard, E. (2005). Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome. Current Opinion in Genetics & Development, 15(5), 482–489. DOI: http://dx.doi.org/10.1016/j.gde.2005.08.009

6. Csankovszki, G., Nagy, A., & Jaenisch, R. (2001). Synergism of Xist RNA, DNA methylation, and histone hypoacetylation in maintaining X chromosome inactivation. The Journal of Cell Biology, 153(4), 773–784. DOI: http://dx.doi.org/10.1083/jcb.153.4.773

7. Jiang, J., Jing, Y., Cost, G. J., Chiang, J.-C., Kolpa, H. J., Cotton, A. M., et al. (2013). Translating dosage compensation to trisomy 21. Nature, 500(7462), 296–300. DOI: http://dx.doi.org/10.1038/nature12394