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Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways.

Pichler G, Wolf P, Schmidt CS, Meilinger D, Schneider K, Frauer C, Fellinger K, Rottach A, Leonhardt H - J. Cell. Biochem. (2011)

Bottom Line: Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation.Ectopic expression of Uhrf2 in uhrf1(-/-) embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences.We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany.

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Uhrf1 and Uhrf2 are not functionally redundant in ESCs. DNA methylation analysis of wt E14 ESCs, uhrf1−/− ESCs and of uhrf1−/− ESCs ectopically expressing Uhrf1-GFP or Uhrf2-GFP. ESCs transiently expressing Uhrf1-GFP and Uhrf2-GFP were isolated by FACS sorting 48 h after transfection and CpG methylation levels of major satellites repeats were analysed by bisulfite treatment, PCR amplification and direct pyrosequencing. Statistical significance of differences in DNA methylation levels between uhrf1−/− ESCs and uhrf1−/− ESCs with ectopically expressed Uhrf1-GFP or Uhrf2-GFP are indicated; *P < 0.05. Shown are means ± SD from three independent experiments.
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fig05: Uhrf1 and Uhrf2 are not functionally redundant in ESCs. DNA methylation analysis of wt E14 ESCs, uhrf1−/− ESCs and of uhrf1−/− ESCs ectopically expressing Uhrf1-GFP or Uhrf2-GFP. ESCs transiently expressing Uhrf1-GFP and Uhrf2-GFP were isolated by FACS sorting 48 h after transfection and CpG methylation levels of major satellites repeats were analysed by bisulfite treatment, PCR amplification and direct pyrosequencing. Statistical significance of differences in DNA methylation levels between uhrf1−/− ESCs and uhrf1−/− ESCs with ectopically expressed Uhrf1-GFP or Uhrf2-GFP are indicated; *P < 0.05. Shown are means ± SD from three independent experiments.

Mentions: To investigate whether Uhrf1 and Uhrf2 are functionally redundant we performed interaction and rescue assays. Like Uhrf1, also Uhrf2 interacts with Dnmts (Supplementary Fig. S6) suggesting a similar function in DNA methylation. To test for such a functional role, we ectopically expressed Uhrf2-GFP or Uhrf1-GFP in uhrf1−/− ESCs and determined DNA methylation levels at major satellites by pyrosequencing. While ectopic expression of Uhrf1-GFP led to significant increase of DNA methylation levels at CpG sites of major satellite DNA in uhrf1−/− ESCs, Uhrf2-GFP did not restore DNA methylation at these sites (Fig. 5). These results point to functional differences between Uhrf1 and Uhrf2 in vivo.


Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways.

Pichler G, Wolf P, Schmidt CS, Meilinger D, Schneider K, Frauer C, Fellinger K, Rottach A, Leonhardt H - J. Cell. Biochem. (2011)

Uhrf1 and Uhrf2 are not functionally redundant in ESCs. DNA methylation analysis of wt E14 ESCs, uhrf1−/− ESCs and of uhrf1−/− ESCs ectopically expressing Uhrf1-GFP or Uhrf2-GFP. ESCs transiently expressing Uhrf1-GFP and Uhrf2-GFP were isolated by FACS sorting 48 h after transfection and CpG methylation levels of major satellites repeats were analysed by bisulfite treatment, PCR amplification and direct pyrosequencing. Statistical significance of differences in DNA methylation levels between uhrf1−/− ESCs and uhrf1−/− ESCs with ectopically expressed Uhrf1-GFP or Uhrf2-GFP are indicated; *P < 0.05. Shown are means ± SD from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569875&req=5

fig05: Uhrf1 and Uhrf2 are not functionally redundant in ESCs. DNA methylation analysis of wt E14 ESCs, uhrf1−/− ESCs and of uhrf1−/− ESCs ectopically expressing Uhrf1-GFP or Uhrf2-GFP. ESCs transiently expressing Uhrf1-GFP and Uhrf2-GFP were isolated by FACS sorting 48 h after transfection and CpG methylation levels of major satellites repeats were analysed by bisulfite treatment, PCR amplification and direct pyrosequencing. Statistical significance of differences in DNA methylation levels between uhrf1−/− ESCs and uhrf1−/− ESCs with ectopically expressed Uhrf1-GFP or Uhrf2-GFP are indicated; *P < 0.05. Shown are means ± SD from three independent experiments.
Mentions: To investigate whether Uhrf1 and Uhrf2 are functionally redundant we performed interaction and rescue assays. Like Uhrf1, also Uhrf2 interacts with Dnmts (Supplementary Fig. S6) suggesting a similar function in DNA methylation. To test for such a functional role, we ectopically expressed Uhrf2-GFP or Uhrf1-GFP in uhrf1−/− ESCs and determined DNA methylation levels at major satellites by pyrosequencing. While ectopic expression of Uhrf1-GFP led to significant increase of DNA methylation levels at CpG sites of major satellite DNA in uhrf1−/− ESCs, Uhrf2-GFP did not restore DNA methylation at these sites (Fig. 5). These results point to functional differences between Uhrf1 and Uhrf2 in vivo.

Bottom Line: Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation.Ectopic expression of Uhrf2 in uhrf1(-/-) embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences.We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany.

Show MeSH
Related in: MedlinePlus