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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.

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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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Cellular localization and dynamics of Uhrf2 depend on histone H3K9 methylation. A: Confocal mid sections of fixed wt J1, TKO and Suv39h dn ESCs transiently expressing Uhrf2-GFP and RFP-PCNA and counterstained with DAPI, which preferentially highlights PH. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. B: Confocal mid sections of fixed wt MEFs and Suv39h dn MEFs transiently expressing Uhrf2-GFP or Uhrf2 Y214A Y217A-GFP were immunostained for H3K9me3 and counterstained with DAPI. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. C: Dynamics of Uhrf2-GFP and Uhrf2 Y214A Y217A-GFP in living MEFs determined by half nucleus FRAP analysis. GFP is shown as reference. Curves represent means ± SEM from at least 8 nuclei.
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fig03: Cellular localization and dynamics of Uhrf2 depend on histone H3K9 methylation. A: Confocal mid sections of fixed wt J1, TKO and Suv39h dn ESCs transiently expressing Uhrf2-GFP and RFP-PCNA and counterstained with DAPI, which preferentially highlights PH. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. B: Confocal mid sections of fixed wt MEFs and Suv39h dn MEFs transiently expressing Uhrf2-GFP or Uhrf2 Y214A Y217A-GFP were immunostained for H3K9me3 and counterstained with DAPI. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. C: Dynamics of Uhrf2-GFP and Uhrf2 Y214A Y217A-GFP in living MEFs determined by half nucleus FRAP analysis. GFP is shown as reference. Curves represent means ± SEM from at least 8 nuclei.

Mentions: To monitor the subcellular localization of Uhrf2, we expressed Uhrf2-GFP constructs in cells with different genetic backgrounds. In wild type (wt) ESCs, Uhrf2 is localized in the nucleus and is enriched at pericentric heterochromatin (PH) (Fig. 3A,B and Supplementary Fig. S4A–C). To investigate which epigenetic marks at PH are recognized by Uhrf2 we determined the localization of Uhrf2 in genetically modified ESCs either lacking all three major DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) [Tsumura et al., 2006] or ESCs lacking the two major H3K9 methyltransferases Suv39H1/H2 (Suv39h dn) [Lehnertz et al., 2003]. TKO cells are practically devoid of genomic DNA methylation and Suv39h dn ESCs show substantially reduced H3K9me3 levels. We found Uhrf2 localized at PH in TKO but not in Suv39h dn ESCs, indicating that localization of Uhrf2 is dependent on H3K9 but not on DNA methylation (Fig. 3A). Consistently, immunostaining of wt mouse embryonic fibroblasts (MEFs) showed co-localization of Uhrf2 and H3K9me3 marks at PH, which was not observed in Suv39h dn MEFs [Peters et al., 2001] (Fig. 3B). Also, mutations in the TTD (Uhrf2 Y214A Y217A) that abolished binding to H3K9me3 peptides in vitro disrupted enrichment at PH in wt MEFs (Fig. 3B). The dependence of Uhrf2 localization on H3K9me3 was also confirmed by quantitative correlation analysis (Supplementary Fig. S4D,E).


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)

Cellular localization and dynamics of Uhrf2 depend on histone H3K9 methylation. A: Confocal mid sections of fixed wt J1, TKO and Suv39h dn ESCs transiently expressing Uhrf2-GFP and RFP-PCNA and counterstained with DAPI, which preferentially highlights PH. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. B: Confocal mid sections of fixed wt MEFs and Suv39h dn MEFs transiently expressing Uhrf2-GFP or Uhrf2 Y214A Y217A-GFP were immunostained for H3K9me3 and counterstained with DAPI. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. C: Dynamics of Uhrf2-GFP and Uhrf2 Y214A Y217A-GFP in living MEFs determined by half nucleus FRAP analysis. GFP is shown as reference. Curves represent means ± SEM from at least 8 nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: Cellular localization and dynamics of Uhrf2 depend on histone H3K9 methylation. A: Confocal mid sections of fixed wt J1, TKO and Suv39h dn ESCs transiently expressing Uhrf2-GFP and RFP-PCNA and counterstained with DAPI, which preferentially highlights PH. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. B: Confocal mid sections of fixed wt MEFs and Suv39h dn MEFs transiently expressing Uhrf2-GFP or Uhrf2 Y214A Y217A-GFP were immunostained for H3K9me3 and counterstained with DAPI. Merged images are displayed on the right side (GFP: green; DAPI: red). Scale bar 5 µm. C: Dynamics of Uhrf2-GFP and Uhrf2 Y214A Y217A-GFP in living MEFs determined by half nucleus FRAP analysis. GFP is shown as reference. Curves represent means ± SEM from at least 8 nuclei.
Mentions: To monitor the subcellular localization of Uhrf2, we expressed Uhrf2-GFP constructs in cells with different genetic backgrounds. In wild type (wt) ESCs, Uhrf2 is localized in the nucleus and is enriched at pericentric heterochromatin (PH) (Fig. 3A,B and Supplementary Fig. S4A–C). To investigate which epigenetic marks at PH are recognized by Uhrf2 we determined the localization of Uhrf2 in genetically modified ESCs either lacking all three major DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) [Tsumura et al., 2006] or ESCs lacking the two major H3K9 methyltransferases Suv39H1/H2 (Suv39h dn) [Lehnertz et al., 2003]. TKO cells are practically devoid of genomic DNA methylation and Suv39h dn ESCs show substantially reduced H3K9me3 levels. We found Uhrf2 localized at PH in TKO but not in Suv39h dn ESCs, indicating that localization of Uhrf2 is dependent on H3K9 but not on DNA methylation (Fig. 3A). Consistently, immunostaining of wt mouse embryonic fibroblasts (MEFs) showed co-localization of Uhrf2 and H3K9me3 marks at PH, which was not observed in Suv39h dn MEFs [Peters et al., 2001] (Fig. 3B). Also, mutations in the TTD (Uhrf2 Y214A Y217A) that abolished binding to H3K9me3 peptides in vitro disrupted enrichment at PH in wt MEFs (Fig. 3B). The dependence of Uhrf2 localization on H3K9me3 was also confirmed by quantitative correlation analysis (Supplementary Fig. S4D,E).

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