Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1 - PubMed (original) (raw)
Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1
Carina Frauer et al. PLoS One. 2011.
Abstract
Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1⁻/⁻ embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1⁻/⁻ ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Sequence and predicted structural homology of CXXC domains.
(A) Schematic representation of the domain structure in Dnmt1 and Tet1. The catalytic domain and the N-terminal region of Dnmt1 are connected by seven lysine-glycine repeats [(KG)7]. PBD: PCNA binding domain; TS: targeting sequence; CXXC: CXXC-type zinc finger domain; BAH1 and 2: bromo-adjacent homology domain; NLS: nuclear localization signal; Cys-rich: cysteine rich region. (B) Alignment of mammalian CXXC domains. Numbers on the right side indicate the position of the last amino acid in the corresponding protein. The Mbd1a isoform contains three CXXC motifs (Mbd1_1-3). Absolutely conserved residues, including the eight cysteines involved in zinc ion coordination are highlighted in red and the conserved KFGG motif is in red bold face. Positions with residues in red face share 70% similarity as calculated with the Risler algorithm . At the top residues of MLL1 involved in β sheets β1 and β2 (black arrows), α helices α1 and α2 and strict α turns (TTT) are indicated. All sequences are from M. musculus. Accession numbers (for GenBank unless otherwise stated): Dnmt1, NP_034196; Mll1, NP_001074518; Mll4, O08550 (SwissProt); CGBP, NP_083144; Kdm2a, NP_001001984; Kdm2b, NP_001003953; Fbxl19, NP_766336; Mbd1, NP_038622; CXXC4/Idax, NP_001004367; CXXC5, NP_598448; CXXC10 (see Materials and Methods). (C) A homology tree was generated from the alignment in (B). The three subgroups of CXXC domains identified are in different colors. Average distances between the sequences are indicated. (D–E) Homology models of the mouse Dnmt1 (D; red) and Tet1 (E; blue) CXXC domains superimposed to the CXXC domain of MLL1 (green; [35]). MLL1 residues that were described to contact DNA according to chemical shift measurements are cyan in (E), while cysteines involved in coordination of the two zinc ions are yellow. Arrows point to the KFGG motif in MLL1 and Dnmt1. The locations of α helices and β sheets are indicated as in (B).
Figure 2. Properties of isolated Dnmt1 and Tet1 CXXC domains.
(A–B) Subcellular localization (A) and binding kinetics (B) of GFP-CXXCDnmt1, GFP-CXXCDnmt1KF/AA, GFP-CXXCTet1 and GFP in mouse C2C12 myoblasts. Localization and binding kinetics were independent from the cell cycle stage (Figures S2 and S5 in File S1). Arrowheads in (A) point to nucleoli. Scale bar: 5 µm. Binding kinetics were analyzed by FRAP. (C) DNA binding specificity of the Dnmt1 and Tet1 CXXC domains. GFP, GFP-CXXCDnmt1, GFP-CXXCDnmt1KF/AA and GFP-CXXCTet1 were pulled down from extracts of transiently transfected HEK293T cells and incubated with fluorescent DNA substrates containing no CpG site or one central un-, hemi- or fully methylated CpG site in direct competition (noCGB, UMB, HMB, FMB, respectively). Shown are the mean DNA/protein ratios and corresponding standard errors from 5 (GFP), 4 (GFP-CXXCDnmt1 and GFP-CXXCDnmt1KF/AA) and 2 (GFP-CXXCTet1) independent experiments. * P = 0.01; ** P = 0.007; ***P = 0.001.
Figure 3. Cellular localization and in vivo catalytic activity of GFP-Tet1 and GFP-Tet1ΔCXXC.
(A) Live images of C2C12 myoblasts expressing GFP-Tet1. Scale bar: 5 µm. (B) Genomic hmC content in HEK293T cells overexpressing GFP, GFP-Tet1 and GFP-Tet1ΔCXXC. Shown are mean values and standard deviation of hmC percentage over total cytosine for three measurements from one transfection.
Figure 4. DNA binding specificity, intramolecular interaction and trapping of wild-type Dnmt1 and CXXC deletion constructs in vitro.
(A) Schematic representation of Dnmt1 expression constructs. (B) DNA binding specificity of GFP-Dnmt1 and GFP-Dnmt1ΔCXXC were assayed as described in Figure 2C. (C) Co-immunoprecipitation of the C-terminal domain of Dnmt1 (Ch-CTD-His) and the N-terminal region with and without deletion of the CXXC domain (GFP-NTR and GFP-NTRΔCXXC, respectively). GFP fusions were detected using an anti-GFP antibody, while the C-terminal domain construct was detected using an anti-His antibody. GFP was used as negative control. I = input, B = bound. (D) Comparison of binding and trapping activities for GFP-Dnmt1 and GFP-Dnmt1ΔCXXC to monitor irreversible covalent complex formation with hemimethylated substrates. (E) Relative covalent complex formation rate of GFP-Dnmt1 and GFP-Dnmt1ΔCXXC on substrates containing one un- (UMT) or hemi-methylated CpG site (HMT) in direct competition. The trapping ratio for GFP-Dnmt1 on unmethylated substrate was set to 1. In (D) and (E) the means and corresponding standard deviations of triplicate samples from three independent experiments are shown. GFP was used as negative control.
Figure 5. Cell cycle dependant cellular localization, protein mobility and trapping of wild-type Dnmt1 and CXXC deletion constructs in mouse C2C12 myoblasts.
(A) Cell cycle dependent localization of GFP-Dnmt1 and GFP-Dnmt1ΔCXXC constructs. Scale bar: 5 µm. (B) Analysis of binding kinetics of GFP-Dnmt1 and GFP-Dnmt1ΔCXXC in early and late S-phase cells by FRAP. The recovery curve for GFP is shown for comparison. (C) In vivo trapping by FRAP analysis in cells treated with 5-aza-dC. The trapped enzyme fraction is plotted over time for early and late S-phase cells. For each construct three to six cells in early-mid and late S phase were analysed per time point. Shown are mean values ± SEM. In (A–C) RFP-PCNA was cotransfected to identify cell cycle stages in living cells.
Figure 6. The CXXC deletion construct of Dnmt1 restores methylation in dnmt1 null cells.
Mouse dnmt1−/− ESCs transiently expressing GFP-Dnmt1 or GFP-Dnmt1ΔCXXC were isolated by FACS-sorting 48 h after transfection and CpG methylation levels within the indicated sequences were analyzed by bisulfite treatment, PCR amplification and direct pyrosequencing. Methylation levels of untransfected wild type and dnmt1−/− ESCs are shown for comparison.
References
- Suzuki MM, Bird A. DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet. 2008;9:465–476. - PubMed
- Mohn F, Weber M, Rebhan M, Roloff TC, Richter J, et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol Cell. 2008;30:755–766. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources