- Library Home /
- Search Collections /
- Open Collections /
- Browse Collections /
- UBC Faculty Research and Publications /
- XIST-induced silencing of flanking genes is achieved...
Open Collections
UBC Faculty Research and Publications
XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells Minks, Jakub; Baldry, Sarah E; Yang, Christine; Cotton, Allison M; Brown, Carolyn J
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.
Item Metadata
Title |
XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells
|
Creator | |
Publisher |
BioMed Central
|
Date Issued |
2013-08-01
|
Description |
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.
|
Subject | |
Genre | |
Type | |
Language |
eng
|
Date Available |
2016-01-12
|
Provider |
Vancouver : University of British Columbia Library
|
Rights |
Attribution 4.0 International (CC BY 4.0)
|
DOI |
10.14288/1.0223296
|
URI | |
Affiliation | |
Citation |
Epigenetics & Chromatin. 2013 Aug 01;6(1):23
|
Publisher DOI |
10.1186/1756-8935-6-23
|
Peer Review Status |
Reviewed
|
Scholarly Level |
Faculty
|
Copyright Holder |
Minks et al.; licensee BioMed Central Ltd.
|
Rights URI | |
Aggregated Source Repository |
DSpace
|
Item Media
Item Citations and Data
Rights
Attribution 4.0 International (CC BY 4.0)