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. 2007 Jul 15;405(2):369-78.
doi: 10.1042/BJ20061873.

Polycomb protein Cbx4 promotes SUMO modification of de novo DNA methyltransferase Dnmt3a

Bing Li  1 , Jing Zhou, Peng Liu, Jialei Hu, Hong Jin, Yohei Shimono, Masahide Takahashi, Guoliang Xu
Affiliations

Polycomb protein Cbx4 promotes SUMO modification of de novo DNA methyltransferase Dnmt3a

Bing Li et al. Biochem J. .

Abstract

The 'de novo methyltransferase' Dnmt3a (DNA methyltransferase 3a) has been shown to mediate transcriptional repression. Post-translational modification of Dnmt3a by SUMOylation affects its ability to transcriptionally repress. However, very little is known about how the SUMOylation process is regulated. In the present study, we identified a PcG (Polycomb group) protein, Cbx4 (chromobox 4), as a specific interaction partner of Dnmt3a. Co-expression of Cbx4 and SUMO-1 (small ubiquitin-related modifier-1) along with Dnmt3a in transfected cells results in enhanced modification of Dnmt3a with SUMO-1. Purified Cbx4 also promotes SUMOylation of Dnmt3a in vitro. The modification occurs in the N-terminal regulatory region, including the PWWP (Pro-Trp-Trp-Pro) domain. Our results suggest that Cbx4 functions as a SUMO E3 ligase for Dnmt3a and it might be involved in the functional regulation of DNA methyltransferases by promoting their SUMO modification.

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Figures

Figure 1
Figure 1. Cbx4 interacts with Dnmt3a in vitro and in vivo
(A) Direct interaction of Cbx4 with Dnmt3a: Western-blotting analysis of the His-tagged Cbx4 in fractions obtained from GST pull-down assays using GST (lanes 2 and 5) or GST–Dnmt3a (lanes 3 and 6). About 10% of the input Cbx4 was loaded (lanes 1 and 4). To eliminate the possibility of false-positive interactions caused by contaminating nucleic acid, micrococcal nuclease (MNase) or ethidium bromide (EtBr) was used to treat the protein preparations during pull-down assays. (B) Co-immunoprecipitation (IP) of Dnmt3a with Cbx4 expressed from transfected cells. HEK-293T cells were transiently transfected with pcDNA3-HA-Dnmt3a and pFlag-CMV-Cbx4 alone or in combination as indicated at the top. Whole-cell extracts were immunoprecipitated with anti-Flag antibody. The precipitated proteins were analysed by Western blotting using anti-HA or anti-Flag antibody as indicated. (C) Co-immunoprecipitation of Cbx4 with Dnmt3a from transfected cells. Samples immunoprecipitated with anti-HA antibody were analysed by Western blotting using anti-Cbx4 or anti-HA antibody as indicated.
Figure 2
Figure 2. Mapping of interaction domains in Cbx4 and Dnmt3a
(A) Schematic representation of Cbx4 fragments with their Dnmt3a-interacting abilities (upper panel, shown on the right) based on the data from yeast two-hybrid assays (lower panel). The full-length Dnmt3a was fused to GAL4BD. The fusion construct was co-transformed into yeast strain AH109 with the indicated portions of Cbx4, which were fused to the GAL4AD. Growth of colonies on the SD minimal medium (SD-L-T-A-H) lacking leucine, tryptophan, adenine and histidine reflects positive interaction of Cbx4 fragments with Dnmt3a. Regions of conservation (the chromodomain and the COOH box) are depicted with black shading in the bars. (B) Schematic representation of Dnmt3a fragments with their Cbx4-interacting abilities (upper panel, shown on the right) based on the data from yeast two-hybrid assays (lower panel). Full-length Cbx4 was fused to GAL4AD and was co-transformed into yeast strain AH109 with the indicated portions of Dnmt3a, which are fused to the GAL4BD. Growth of colonies on the SD minimal medium (SD-L-T-A-H) reflects positive interactions.
Figure 3
Figure 3. Dnmt3a interacts with Cbx4 specifically
(A) Amino acid alignment of the COOH boxes and their flanking regions of the Cbx family members. Identical residues are indicated by black shading and highly conserved residues by grey shading. (B) Schematic representation of Cbx proteins with their Dnmt3a-interacting abilities (upper panel, shown on the right) based on the data from yeast two-hybrid assays (middle panel). Cbx proteins were fused to GAL4AD and co-transformed into yeast strain AH109 with GAL4BD–Dnmt3a. The interactions were accessed by growth of colonies on the SD minimal medium (SD-L-T-A-H). The GAL4AD-Cbx and GAL4BD-Dnmt3a fusion proteins also contain an HA or a myc epitope tag respectively, which were used to confirm the expression of Cbx proteins and Dnmt3a in transformed yeast by Western blotting (bottom panel).
Figure 4
Figure 4. Dnmt3a is SUMOylated in HEK-293T cells
(A) Western-blotting analysis of the SUMOylation of Dnmt3a. HEK-293T cells were transiently transfected with expression plasmids as indicated on the top. Cell lysates were analysed by Western blotting with anti-HA antibody (left panel) or with anti-Dnmt3a antibody (right panel). The positions of Dnmt3a and its SUMOylated forms are indicated. (B) Immunoprecipitation analysis of the SUMOylation of Dnmt3a. HEK-293T cells were transiently transfected with expression plasmids as indicated on the top. The cell extracts were immunoprecipitated with anti-HA antibody. The precipitated proteins were analysed by Western blotting with anti-Dnmt3a antibody (lanes 1 and 2), anti-GFP antibody (lanes 3 and 4) or anti-HA antibody (lanes 5 and 6). The positions of Dnmt3a and its SUMOylated forms are indicated.
Figure 5
Figure 5. Mapping of the Dnmt3a region necessary for SUMOylation
(A) Schematic overview of the Dnmt3a deletion mutants used. The NLS sequences are indicated by arrows. The ability to be SUMOylated was assessed on the basis of the data in (B) and is indicated on the right. (B) Western-blotting analysis of the SUMOylation of Dnmt3a deletion mutants. HEK-293T cells were transiently transfected with expression plasmids encoding different regions of Dnmt3a, as indicated on the top. Cell lysates were analysed by Western blotting with anti-HA antibody. The positions of SUMOylated forms of Dnmt3a fragments are marked by arrowheads. Arrows depict the additional bands which might represent an unknown form of post-translational modification of Dnmt3a.
Figure 6
Figure 6. Cbx4 enhances SUMOylation of Dnmt3a in vivo and in vitro
(A) Cbx4 promotes SUMO-1 conjugation to CtBP in transfected cells. HEK-293T cells were transiently transfected with expression plasmids encoding Flag–SUMO-1, Flag–Cbx4 and myc-CtBP as indicated. The cell lysates were analysed by Western blotting using anti-myc (upper panel) or anti-Cbx4 (lower panel) antibody. (B) Cbx4 promotes SUMO-1 conjugation to Dnmt3a in transfected cells. HEK-293T cells were transiently transfected with expression plasmids encoding GFP–SUMO-1, Flag–Cbx4 and HA–Dnmt3a as indicated. The cell lysates were analysed by Western blotting using anti-HA (upper panel) or anti-Cbx4 antibody (lower panel). (C) SUMOylation of Dnmt3a in vitro. Purified GST-tagged Dnmt3a (amino acids 1–482, GST–Dnmt3aN) was incubated in the absence or presence of 250 ng of recombinant E1 (Aos1/Uba2 heterodimer), 100 ng of recombinant E2 (Ubc9) and 3 μg of recombinant SUMO-1 as indicated. Reactions were terminated by the addition of SDS loading buffer. Proteins were analysed by Western blotting using anti-GST antibody. (D) Cbx4 enhances SUMOylation of Dnmt3a in vitro. Purified GST–Dnmt3aN was incubated with 250 ng of E1 (Aos1/Uba2 heterodimer), 20 ng of E2 (Ubc9) and 3 μg of SUMO-1. Lanes 1–4 contained 0, 25, 50 or 100 ng of purified His-Cbx4 respectively. Note that the amount of Ubc9 used here was 5-fold lower than that used in (C).
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