The Presence of a Chromatin Boundary Appears to Shield a Transgene in Tobacco from RNA Silencing

Mlynárová, Ľudmila; Hricová, Andrea; Loonen, Annelies E. H. M.; Nap, Jan-Peter

doi:10.1105/tpc.012070

Cited by 37 publications

(29 citation statements)

References 77 publications

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“…However, we have observed that cells with the same genotype can include both GFP negative and GFP positive cells and that the Rb7 MAR reduces the frequency of GFP negative, nonexpressing cells. These results are consistent with previous suggestions (Allen et al, 2000;Mlynarova et al, 2003) that MARs can reduce the severity of gene silencing. A large portion of the MAR effect can now be understood as an effect on the frequency of silencing events within a clone of transgenic cells.…”

Section: Discussionsupporting

confidence: 83%

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

2005

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Many studies in both plant and animal systems have shown that matrix attachment regions (MARs) can increase expression of transgenes in whole organisms or cells in culture. Because histochemical assays often indicate variegated transgene expression, a question arises: Do MARs increase transgene expression by increasing the percentage of cells expressing the transgene (likelihood), by increasing the level of expression in expressing cells (magnitude), or both? To address this question, we used flow cytometry to measure green fluorescent protein (GFP) expression in individual tobacco (Nicotiana tabacum) cells from lines transformed by Agrobacterium tumefaciens. We conclude that MAR-mediated overall increases in transgene expression involve both likelihood and magnitude. On average, cell lines transformed with the Rb7 MAR-containing vector expressed GFP at levels 2.0- to 3.7-fold higher than controls. MAR lines had fewer nonexpressing cells than control lines (10% versus 45%), and the magnitude of GFP expression in expressing cells was greater in MAR lines by 1.9- to 2.9-fold. We also show that flow cytometry measurements on cells from isogenic lines are consistent with those from populations of independently transformed cell lines. By obviating the need to establish isogenic lines, this use of flow cytometry could greatly simplify the evaluation of MARs or other sequence elements that affect transgene expression

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Section: Discussionsupporting

confidence: 83%

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

2005

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“…This has not been found for weak S/MARs, though the possibility remains that weak S/MARs are involved in dynamic folding of loops into subdomains (Avramova et al, 1995). S/MARs have been further demonstrated in a variety of functional tests to act as insulators (Mlynarova et al, 2003), according to the loop domain model, by protecting a loop from the effects of the neighboring chromatin or associated enhancer sequences. However, this is not a general rule since other S/MARs do not show such insulating activity in enhancer-blocking assays, and some known insulators do not show properties typical for S/MARs (Jarman and Higgs, 1988;Kellum and Schedl, 1992;Li and Stamatoyannopoulos, 1994;Antes et al, 2001;Razin, 2001).…”

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confidence: 99%

Genome-Wide in Silico Mapping of Scaffold/Matrix Attachment Regions in Arabidopsis Suggests Correlation of Intragenic Scaffold/Matrix Attachment Regions with Gene Expression

et al. 2004

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We carried out a genome-wide prediction of scaffold/matrix attachment regions (S/MARs) in Arabidopsis. Results indicate no uneven distribution on the chromosomal level but a clear underrepresentation of S/MARs inside genes. In cases where S/MARs were predicted within genes, these intragenic S/MARs were preferentially located within the 5#-half, most prominently within introns 1 and 2. Using Arabidopsis whole-genome expression data generated by the massively parallel signature sequencing methodology, we found a negative correlation between S/MAR-containing genes and transcriptional abundance. Expressed sequence tag data correlated the same way with S/MAR-containing genes. Thus, intragenic S/MARs show a negative correlation with transcription level. For various genes it has been shown experimentally that S/MARs can function as transcriptional regulators and that they have an implication in stabilizing expression levels within transgenic plants. On the basis of a genome-wide in silico S/MAR analysis, we found a significant correlation between the presence of intragenic S/MARs and transcriptional down-regulation.

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“…Remnants of retroelements have been identifi ed in the vicinity of unstably expressed transgenes [18], and methylation of retroelements has been suggested to affect adjacent sequences, leading to transcriptional repression [25,26]. Conversely, matrix attachment regions (MARs), dispersed throughout eukaryotic genomes [27] have in some cases been shown to confer position-and copy-numberindependent control of transgene expression [28][29][30]. Furthermore, abrupt changes in GC content between genomic DNA and a transgenic insert have been suggested to make the transgene conspicuous to silencing mechanisms [31,32].…”

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confidence: 99%

Transgene silencing may be mediated by aberrant sense promoter sequence transcripts generated from cryptic promoters

Eike

Mercy

Aalen

2005

Cell. Mol. Life Sci.

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To investigate whether silencing of a T-DNA-carried nptII gene in five single-copy transgenic Arabidopsis lines might be due to position effects, genomic DNA flanking the insertions was analysed for gene density, GC content, presence of short repeats and transposable elements, i.e. factors suggested to promote silencing. No single, common factor could explain the observed silencing. However, in two lines, a transcript covering the nos promoter driving the nptII gene was detected. In sibling sublines with approximately 100% silencing, the nos promoter was heavily methylated. In silico analysis suggested the presence of cryptic core promoters upstream of the nos promoter, in one case in the plant DNA and in the other in a short inverted TDNA region. These fragments were able to drive reporter gene expression in transgenic Arabidopsis plants. Our results indicate that methylation and silencing of transgenic promoters may be mediated by aberrant RNA transcribed from cryptic promoters at the transgene insertion site.

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The Presence of a Chromatin Boundary Appears to Shield a Transgene in Tobacco from RNA Silencing

Cited by 37 publications

References 77 publications

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

The Rb7 Matrix Attachment Region Increases the Likelihood and Magnitude of Transgene Expression in Tobacco Cells: A Flow Cytometric Study

Genome-Wide in Silico Mapping of Scaffold/Matrix Attachment Regions in Arabidopsis Suggests Correlation of Intragenic Scaffold/Matrix Attachment Regions with Gene Expression

Transgene silencing may be mediated by aberrant sense promoter sequence transcripts generated from cryptic promoters

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