title: XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells.
abstract: BACKGROUND
  The establishment of facultative heterochromatin by X-chromosome inactivation requires the long non-coding RNA XIST/Xist. However, the molecular mechanism by which the RNA achieves chromosome-wide gene silencing remains unknown. Mouse Xist has been shown to have redundant domains for cis-localization, and requires a series of well-conserved tandem 'A' repeats for silencing. We previously described a human inducible XIST transgene that is capable of cis-localization and suppressing a downstream reporter gene in somatic cells, and have now leveraged these cells to dissect the sequences critical for XIST-dependent gene silencing in humans.
  
  
  RESULTS
  We demonstrated that expression of the inducible full-length XIST cDNA was able to suppress expression of two nearby reporter genes as well as endogenous genes up to 3 MB from the integration site. An inducible construct containing the repeat A region of XIST alone could silence the flanking reporter genes but not the more distal endogenous genes. Reporter gene silencing could also be accomplished by a synthetic construct consisting of nine copies of a consensus repeat A sequence, consistent with previous studies in mice. Progressively shorter constructs showed a linear relationship between the repeat number and the silencing capacity of the RNA. Constructs containing only two repeat A units were still able to partially silence the reporter genes and could thus be used for site-directed mutagenesis to demonstrate that sequences within the two palindromic cores of the repeat are essential for silencing, and that it is likely the first palindrome sequence folds to form a hairpin, consistent with compensatory mutations observed in eutherian sequences.
  
  
  CONCLUSIONS
  Silencing of adjacent reporter genes can be effected by as little as 94 bp of XIST, including two 'monomers' of the A repeat. This region includes a pair of essential palindromic sequences that are evolutionarily well-conserved and the first of these is likely to form an intra-repeat hairpin structure. Additional sequences are required for the spread of silencing to endogenous genes on the chromosome.
authors.affiliation: Department of Medical Genetics, Molecular Epigenetics Group, University of British Columbia, Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. carolyn.brown@ubc.ca.
authors.forename: Jakub
authors.initials: J
authors.lastname: Minks
authors.forename: Sarah El
authors.initials: SE
authors.lastname: Baldry
authors.forename: Christine
authors.initials: C
authors.lastname: Yang
authors.forename: Allison M
authors.initials: AM
authors.lastname: Cotton
authors.forename: Carolyn J
authors.initials: CJ
authors.lastname: Brown
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