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Telomere length homeostasis and telomere position effect on a linear human artificial chromosome are dictated by the genetic background.

Weuts A, Voet T, Verbeeck J, Lambrechts N, Wirix E, Schoonjans L, Danloy S, Marynen P, Froyen G - Nucleic Acids Res. (2012)

Bottom Line: Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression.We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice.We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium.

ABSTRACT
Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression. Previous studies suggested that TPE depends on genetic background. As these analyses were performed on different chromosomes, cell types and species, it remains unclear whether TPE represents a chromosome-rather than genetic background-specific regulation. We describe the development of a Linear Human Artificial Chromosome (L-HAC) as a new tool for telomere studies. The L-HAC was generated through the Cre-loxP-mediated addition of telomere ends to an existing circular HAC (C-HAC). As it can be transferred to genetically distinct cell lines and animal models the L-HAC enables the study of TPE in an unprecedented manner. The HAC was relocated to four telomerase-positive cell lines via microcell-mediated chromosome transfer and subsequently to mice via blastocyst injection of L-HAC(+)-ES-cells. We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice. Expression of the subtelomeric neomycin gene was inversely correlated with telomere length and subtelomeric methylation. We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent.

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Related in: MedlinePlus

In vivo bisulfite sequencing data for the C- and L-HAC. Analogous to the in vitro analysis, a region of 60 bp surrounding EcoRI(2) was analysed. All the CpGs are marked by horizontal yellow bars. They are termed 5–12 and correspond to CpGs 5–12 in Figure 5. All C nucleotides that precede a G nucleotide are highlighted in red. Genomic DNA of the mice containing the C-HAC was extracted from mouse tail fibroblast derived from mice backcrossed to the NMRI background for 11 generations. For the L-HAC, solely mice from generation 1 (F1) were available. The graphs represent screenshots from the chromatogram obtained from the 3130x Genetic Analyzer (Life Technologies). The scale on the left (0–1300 and 0–1500) reflects the peak intensity for each nucleotide readout. The CpG′s in the subtelomeric region of the L-HAC are clearly hypermethylated compared to their counterparts on the circular chromosome. Untreated seq: the untreated wild-type sequence at that specific locus; bisulfite seq: the sequence that was obtained upon bisulfite treatment and sequencing PCR; F11: the 11th generation of offspring; F1: the 1st generation of offspring.
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gks926-F8: In vivo bisulfite sequencing data for the C- and L-HAC. Analogous to the in vitro analysis, a region of 60 bp surrounding EcoRI(2) was analysed. All the CpGs are marked by horizontal yellow bars. They are termed 5–12 and correspond to CpGs 5–12 in Figure 5. All C nucleotides that precede a G nucleotide are highlighted in red. Genomic DNA of the mice containing the C-HAC was extracted from mouse tail fibroblast derived from mice backcrossed to the NMRI background for 11 generations. For the L-HAC, solely mice from generation 1 (F1) were available. The graphs represent screenshots from the chromatogram obtained from the 3130x Genetic Analyzer (Life Technologies). The scale on the left (0–1300 and 0–1500) reflects the peak intensity for each nucleotide readout. The CpG′s in the subtelomeric region of the L-HAC are clearly hypermethylated compared to their counterparts on the circular chromosome. Untreated seq: the untreated wild-type sequence at that specific locus; bisulfite seq: the sequence that was obtained upon bisulfite treatment and sequencing PCR; F11: the 11th generation of offspring; F1: the 1st generation of offspring.

Mentions: To study the artificial telomeres in vivo we performed TRF analysis on genomic DNA isolated from mouse lung tissue using PstI and the BLAS probe. Four littermates displayed telomeres of similar length between 30 kb and 70 kb (Supplementary Figure S4). Bisulfite sequencing of tail fibroblast DNA derived from two F1-L-HAC+-mice as well as from three mice carrying the C-HAC (backcrossed to the NMRI and 129/Sv backgrounds for 15 and 11 generations, respectively) was performed to check the methylation status of the HAC-subtelomere. All analysed CpG sites were fully methylated in mice carrying the L-HAC whereas in mice carrying the C-HAC the methylation status at each CpG was between 25% and 50% at most (Figure 8).Figure 8.


Telomere length homeostasis and telomere position effect on a linear human artificial chromosome are dictated by the genetic background.

Weuts A, Voet T, Verbeeck J, Lambrechts N, Wirix E, Schoonjans L, Danloy S, Marynen P, Froyen G - Nucleic Acids Res. (2012)

In vivo bisulfite sequencing data for the C- and L-HAC. Analogous to the in vitro analysis, a region of 60 bp surrounding EcoRI(2) was analysed. All the CpGs are marked by horizontal yellow bars. They are termed 5–12 and correspond to CpGs 5–12 in Figure 5. All C nucleotides that precede a G nucleotide are highlighted in red. Genomic DNA of the mice containing the C-HAC was extracted from mouse tail fibroblast derived from mice backcrossed to the NMRI background for 11 generations. For the L-HAC, solely mice from generation 1 (F1) were available. The graphs represent screenshots from the chromatogram obtained from the 3130x Genetic Analyzer (Life Technologies). The scale on the left (0–1300 and 0–1500) reflects the peak intensity for each nucleotide readout. The CpG′s in the subtelomeric region of the L-HAC are clearly hypermethylated compared to their counterparts on the circular chromosome. Untreated seq: the untreated wild-type sequence at that specific locus; bisulfite seq: the sequence that was obtained upon bisulfite treatment and sequencing PCR; F11: the 11th generation of offspring; F1: the 1st generation of offspring.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526267&req=5

gks926-F8: In vivo bisulfite sequencing data for the C- and L-HAC. Analogous to the in vitro analysis, a region of 60 bp surrounding EcoRI(2) was analysed. All the CpGs are marked by horizontal yellow bars. They are termed 5–12 and correspond to CpGs 5–12 in Figure 5. All C nucleotides that precede a G nucleotide are highlighted in red. Genomic DNA of the mice containing the C-HAC was extracted from mouse tail fibroblast derived from mice backcrossed to the NMRI background for 11 generations. For the L-HAC, solely mice from generation 1 (F1) were available. The graphs represent screenshots from the chromatogram obtained from the 3130x Genetic Analyzer (Life Technologies). The scale on the left (0–1300 and 0–1500) reflects the peak intensity for each nucleotide readout. The CpG′s in the subtelomeric region of the L-HAC are clearly hypermethylated compared to their counterparts on the circular chromosome. Untreated seq: the untreated wild-type sequence at that specific locus; bisulfite seq: the sequence that was obtained upon bisulfite treatment and sequencing PCR; F11: the 11th generation of offspring; F1: the 1st generation of offspring.
Mentions: To study the artificial telomeres in vivo we performed TRF analysis on genomic DNA isolated from mouse lung tissue using PstI and the BLAS probe. Four littermates displayed telomeres of similar length between 30 kb and 70 kb (Supplementary Figure S4). Bisulfite sequencing of tail fibroblast DNA derived from two F1-L-HAC+-mice as well as from three mice carrying the C-HAC (backcrossed to the NMRI and 129/Sv backgrounds for 15 and 11 generations, respectively) was performed to check the methylation status of the HAC-subtelomere. All analysed CpG sites were fully methylated in mice carrying the L-HAC whereas in mice carrying the C-HAC the methylation status at each CpG was between 25% and 50% at most (Figure 8).Figure 8.

Bottom Line: Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression.We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice.We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent.

View Article: PubMed Central - PubMed

Affiliation: Human Genome Laboratory, VIB Center for the Biology of Disease, Leuven, Belgium.

ABSTRACT
Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression. Previous studies suggested that TPE depends on genetic background. As these analyses were performed on different chromosomes, cell types and species, it remains unclear whether TPE represents a chromosome-rather than genetic background-specific regulation. We describe the development of a Linear Human Artificial Chromosome (L-HAC) as a new tool for telomere studies. The L-HAC was generated through the Cre-loxP-mediated addition of telomere ends to an existing circular HAC (C-HAC). As it can be transferred to genetically distinct cell lines and animal models the L-HAC enables the study of TPE in an unprecedented manner. The HAC was relocated to four telomerase-positive cell lines via microcell-mediated chromosome transfer and subsequently to mice via blastocyst injection of L-HAC(+)-ES-cells. We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice. Expression of the subtelomeric neomycin gene was inversely correlated with telomere length and subtelomeric methylation. We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent.

Show MeSH
Related in: MedlinePlus