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      Efficiency of Xist-mediated silencing on autosomes is linked to chromosomal domain organisation

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          Abstract

          Background

          X chromosome inactivation, the mechanism used by mammals to equalise dosage of X-linked genes in XX females relative to XY males, is triggered by chromosome-wide localisation of a cis-acting non-coding RNA, Xist. The mechanism of Xist RNA spreading and Xist-dependent silencing is poorly understood. A large body of evidence indicates that silencing is more efficient on the X chromosome than on autosomes, leading to the idea that the X chromosome has acquired sequences that facilitate propagation of silencing. LINE-1 (L1) repeats are relatively enriched on the X chromosome and have been proposed as candidates for these sequences. To determine the requirements for efficient silencing we have analysed the relationship of chromosome features, including L1 repeats, and the extent of silencing in cell lines carrying inducible Xist transgenes located on one of three different autosomes.

          Results

          Our results show that the organisation of the chromosome into large gene-rich and L1-rich domains is a key determinant of silencing efficiency. Specifically genes located in large gene-rich domains with low L1 density are relatively resistant to Xist-mediated silencing whereas genes located in gene-poor domains with high L1 density are silenced more efficiently. These effects are observed shortly after induction of Xist RNA expression, suggesting that chromosomal domain organisation influences establishment rather than long-term maintenance of silencing. The X chromosome and some autosomes have only small gene-rich L1-depleted domains and we suggest that this could confer the capacity for relatively efficient chromosome-wide silencing.

          Conclusions

          This study provides insight into the requirements for efficient Xist mediated silencing and specifically identifies organisation of the chromosome into gene-rich L1-depleted and gene-poor L1-dense domains as a major influence on the ability of Xist-mediated silencing to be propagated in a continuous manner in cis.

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          Most cited references30

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          Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes.

          LINE-1 (L1) elements are the most abundant autonomous retrotransposons in the human genome, accounting for about 17% of human DNA. The L1 retrotransposon encodes two proteins, open reading frame (ORF)1 and the ORF2 endonuclease/reverse transcriptase. L1 RNA and ORF2 protein are difficult to detect in mammalian cells, even in the context of overexpression systems. Here we show that inserting L1 sequences on a transcript significantly decreases RNA expression and therefore protein expression. This decreased RNA concentration does not result from major effects on the transcription initiation rate or RNA stability. Rather, the poor L1 expression is primarily due to inadequate transcriptional elongation. Because L1 is an abundant and broadly distributed mobile element, the inhibition of transcriptional elongation by L1 might profoundly affect expression of endogenous human genes. We propose a model in which L1 affects gene expression genome-wide by acting as a 'molecular rheostat' of target genes. Bioinformatic data are consistent with the hypothesis that L1 can serve as an evolutionary fine-tuner of the human transcriptome.
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            A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.

            During early mammalian female development, one of the two X chromosomes becomes inactivated. Although X-chromosome coating by Xist RNA is essential for the initiation of X inactivation, little is known about how this signal is transformed into transcriptional silencing. Here we show that exclusion of RNA Polymerase II and transcription factors from the Xist RNA-coated X chromosome represents the earliest event following Xist RNA accumulation described so far in differentiating embryonic stem (ES) cells. Paradoxically, exclusion of the transcription machinery occurs before gene silencing is complete. However, examination of the three-dimensional organization of X-linked genes reveals that, when transcribed, they are always located at the periphery of, or outside, the Xist RNA domain, in contact with the transcription machinery. Upon silencing, genes shift to a more internal location, within the Xist RNA compartment devoid of transcription factors. Surprisingly, the appearance of this compartment is not dependent on the A-repeats of the Xist transcript, which are essential for gene silencing. However, the A-repeats are required for the relocation of genes into the Xist RNA silent domain. We propose that Xist RNA has multiple functions: A-repeat-independent creation of a transcriptionally silent nuclear compartment; and A-repeat-dependent induction of gene repression, which is associated with their translocation into this silent domain.
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              Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.

              A general strategy for selecting insertion mutations in mice has been devised. Constructs lacking a promoter and including a beta-galactosidase gene, or a reporter gene encoding a protein with both beta-galactosidase and neomycin phosphotransferase activity, were designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Such insertion events create a mutation in the tagged gene and allow its expression to be followed by beta-galactosidase activity. Introduction of promoter trap constructs into embryonic stem (ES) cells by electroporation or retroviral infection has led to the derivation of transgenic lines that show a variety of beta-galactosidase expression patterns. Intercrossing of heterozygotes from 24 strains that express beta-galactosidase identified 9 strains in which homozygosity leads to an embryonic lethality. Because no overt phenotype was detected in the remaining strains, these results suggest that a substantial proportion of mammalian genes identified by this approach are not essential for development.
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                Author and article information

                Journal
                Epigenetics Chromatin
                Epigenetics & Chromatin
                BioMed Central
                1756-8935
                2010
                7 May 2010
                : 3
                : 10
                Affiliations
                [1 ]MRC Clinical Sciences Centre, Faculty of Medicine ICSTM, Hammersmith Hospital, London, UK
                [2 ]Morgan Building, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
                [3 ]Centre for Bioinformatics, Division of Molecular Biosciences, Imperial College London, London, UK
                [4 ]Department of Mathematics, Imperial College, London, UK
                [5 ]Department of Biochemistry, University of Oxford, Oxford, UK
                Article
                1756-8935-3-10
                10.1186/1756-8935-3-10
                2873326
                20459652
                46449931-d427-488b-9551-18726ab1a8b3
                Copyright ©2010 Tang et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 14 January 2010
                : 7 May 2010
                Categories
                Research

                Genetics
                Genetics

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