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. 2011 Sep;112(9):2585-93.
doi: 10.1002/jcb.23185.

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

Garwin Pichler  1 , Patricia Wolf, Christine S Schmidt, Daniela Meilinger, Katrin Schneider, Carina Frauer, Karin Fellinger, Andrea Rottach, Heinrich Leonhardt
Affiliations

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

Garwin Pichler et al. J Cell Biochem. 2011 Sep.

Abstract

Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. 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. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. 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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Figures

Figure 1
Figure 1
Opposite expression pattern of uhrf1 and uhrf2 during differentiation. A: Schematic outline of the multi-domain architecture of Uhrf1 in comparison to Uhrf2. An N-terminal ubiquitin-like domain (Ubl) is followed by a tandem Tudor domain (TTD), a plant homeodomain (PHD), a SET and RING associated (SRA) domain and a C-terminal really interesting new gene (RING) domain. Numbers indicate primary sequence similarities of single domains determined by BlastP search [Altschul, 1991]. Expression analysis of uhrf1 and uhrf2 by Real-time PCR in ESCs and somatic cells (B), during differentiation of wt J1 ESCs (C) and in various adult mouse tissues in comparison to the expression data in ESCs (D). Expression levels are relative to uhrf1 in wtJM8A (B), day 0 of differentiation (C) and to kidney (D) (uhrf1 set to 1). Shown are means ± SD of at least two independent experiments.
Figure 2
Figure 2
Cooperative binding of repressive epigenetic marks by Uhrf2. In vitro binding ratios of fluorescently labeled substrate over bound GFP fusion proteins were determined. A: Histone H3- and H4-tail binding specificities of Uhrf2. Shown are means ± SD of biological duplicates. B: Histone H3 tail binding specificity of Uhrf2, its tandem Tudor domain (TTD), its PHD domain and its TTD mutant (Y214A Y217A). Shown are means ± SEM of at least three independent experiments. C: DNA binding properties of Uhrf1, Uhrf2 and of single (SRA, TTD) and combined Uhrf2 domains (TTD–PHD–SRA). Shown are means ± SEM of three independent experiments. D: DNA binding properties of Uhrf1, Uhrf2 and Uhrf2 Y214A Y217A in combination with histone-tail peptide binding. Shown are means ± SD of three independent experiments (Uhrf1, Uhrf2) and of two independent experiments (Uhrf2 Y214A Y217A). Values were normalized to the binding ratio of each GFP fusion for unmethylated DNA without histone-tail peptide. Statistical significance of differences between the binding ratios with un- and hemimethylated DNA is indicated; *P < 0.05. E + F: H3K9me3 peptide binding by Uhrf1, Uhrf2, and Uhrf1ΔSRA with increasing concentrations of DNA substrate containing either one central hemimethylated (E) or noCpG site (F). Shown are means ± SD of biological duplicates. Values were normalized to the binding ratio of Uhrf1ΔSRA without DNA.
Figure 3
Figure 3
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.
Figure 4
Figure 4
Cooperative binding of the combined tandem Tudor–PHD domain of Uhrf2. A: Histone H3 N-terminal tail binding specificity of the TTD of Uhrf2 and of the combined TTD and PHD domain (TTD–PHD) of Uhrf1 and Uhrf2. Shown are means ± SEM from at least six independent experiments. B: Histone H3K9me3 binding of the combined TTD–PHD domains of Uhrf1 and Uhrf2, hybrid proteins (L1 and L2 specify inserted linker sequences derived from Uhrf1 and Uhrf2, respectively) and a stretch deletion Uhrf2 construct. Shown are means ± SEM from at least three independent experiments.
Figure 5
Figure 5
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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