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. 2015 Mar;116(3):476-85.
doi: 10.1002/jcb.25001.

MBD4 interacts with and recruits USP7 to heterochromatic foci

Huan Meng  1 , David J Harrison, Richard R Meehan
Affiliations

MBD4 interacts with and recruits USP7 to heterochromatic foci

Huan Meng et al. J Cell Biochem. 2015 Mar.

Abstract

MBD4 is the only methyl-CpG binding protein that possesses a C-terminal glycosylase domain. It has been associated with a number of nuclear pathways including DNA repair, DNA damage response, the initiation of apoptosis, transcriptional repression, and DNA demethylation. However, the precise contribution of MBD4 to these processes in development and relevant diseases remains elusive. We identified UHRF1 and USP7 as two new interaction partners for MBD4. Both UHRF1, a E3 ubiquitin ligase, and USP7, a de-ubiquinating enzyme, regulate the stability of the DNA maintenance methyltransferase, Dnmt1. The ability of MBD4 to directly interact with and recruit USP7 to chromocenters implicates it as an additional factor that can potentially regulate Dnmt1 activity during cell proliferation.

Keywords: HETEROCHROMATIN REPLICATION AND FORMATION; MBD4; UHRF1; USP7.

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Figures

Figure 1
Figure 1
MBD4 complexes with UHRF1 and USP7. (A) UHRF1 and USP7 are identified as new interaction partners of MBD4. MBD4 was immunoprecipitated from nuclear extracts with MNase treatment and subjected to SDS–PAGE and Coomassie blue staining. Protein bands of UHRF1 and USP7 specific for the FLAG‐MBD4‐IP were identified by MS analysis (ctrl: Empty vector). Precipitated MBD4 and the molecular weight marker (M) are indicated. (B) Immunoprecipitation of ectopic proteins with FLAG epitope‐tagged (MNase treated nuclear extract) or GFP epitope‐tagged (no MNase treated nuclear extract) with anti‐FLAG or anti‐GFP antibodies. Co‐precipitated proteins were detected by Western Blot analysis. 3% of the input (Lane 1) and the antibody coupled IP (Lane 2) were loaded. The migration of the molecular weight is indicated on the left.
Figure 2
Figure 2
MBD4 interacts with UHRF1 via its intervening region. (A) Schematic representation of the domains of human MBD4 (MBD4 domains are to scale) and the representative scheme of generating the four MBD4 recombinant proteins. A black arrow indicates the natural occurring MBD4 protein truncation site 313 in human MMR‐deficiency carcinomas. (B) The schematic representation of the fusion proteins of MBD4 mutants, and coomaissie staining of the purified MBD4 proteins fused with trigger factor and ubiquitin. Respective total lysates of transformed E.coli cells of MBD4 recombinants 1–4 and their protein elutions are shown as indicated. The dye marker lanes are indicated on the left of lysate lanes with their molecular weight. The empty vector control (labeled as 0. TFUB control) containing TFUB only was from pre‐purified storage, and loaded on the left. The three elutions of individual MBD4 mutants were pooled together and 500 µg of each MBD4 protein mutant was measured for the subsequent reciprocal co‐immunoprecipitation (co‐IP) assays. (C) Flow chart of the reciprocal co‐IP procedure. (D & E) Co‐IP assays to determine the interaction of recombinant MBD4 mutants with Mlh1 (D. control) and UHRF1 (E). Co‐precipitated proteins were detected via western blot. Input (3% of 500 µg GFP‐Mlh1 (D. 6HIS IP) or FLAG‐UHRF1 (E. 6HIS IP), and 3% of 500 µg MBD4 protein mutants in D and E) and the antibody coupled IP are indicated with antibodies used for immunodetection. The migration of the molecular weight is indicated on the left of each blot. The regions labelled by a square on the schematic representation of MBD4 are identified the interacting domains responsible for the associations with Mlh1 (D. control) and UHRF1 (E).
Figure 3
Figure 3
MBD4 tightly co‐localizes with UHRF1 at chromocenters. Asynchronously growing mouse CMT93 cells were grown on coverslips and transfected by GFP‐MBD4 (A), FLAG‐UHRF1 (B), or co‐transfected with GFP‐MBD4 and FLAG‐UHRF1 (C). The cells were fixed 48 h later and analyzed directly by immunofluorescence (IF) (A), or immunostained with anti‐FLAG rabbit primary and goat anti‐rabbit IgG‐Alexafluor Red conjugate secondary antibodies and analyzed (B & C). Nuclear counterstaining was visualized with DAPI. Scale bars, 10 µm. (A) Distribution of GFP‐MBD4 in CMT93 cells. (B) Distribution of FLAG‐UHRF1 in CMT93 cells. (C) GFP‐MBD4 and FLAG‐UHRF1 exclusively colocalized with each other at chromocenters in CMT93 cells. In the immunostaining images in (C. i & ii), the cells exhibit an increase in cell size and marked large‐scale reorganization of heterochromatin, which may be indicative of heterochromatin reformation and replication in interphase. In (C. iii), the cells appear to be undergoing orderly division into two daughter cells. The insets on the right (A & B) or below (C. i, ii, iii) correspond to magnifications of the areas indicated by the two parallel white line. Scale bars, 10 µm.
Figure 4
Figure 4
MBD4 directly interacts with and recruits USP7 to chromocenters. (A) The representative scheme for alignment and mapping of a USP7 cDNA fragment that iinteracts with MBD4 in a Y2H screen, (i) USP7 coding sequence, (ii) corresponding peptide sequence, and (iii) schematic representation of domains of USP7. Protein domains are identified by searching Blast integrated SMART domain database (USP7 domains are to scale). The vertical gray shadow indicates the domain region of USP7 overlapping with interacting cDNA fragment. (B & C) Asynchronously growing mouse CMT93 cells were grown on coverslips and transfected by MCherry‐USP7 (B), or co‐transfected by GFP‐MBD4 and MCherry‐USP7 (C). The cells were fixed 48 h later and analyzed directly by IF. Nuclear counterstaining was visualized with DAPI. (B) Distribution of MCherry‐USP7 in CMT93 cells. (C) GFP‐MBD4 recruits MCherry‐USP7 exclusively to chromocenters in all the CMT93 cells co‐transfected. In all the immunostaining images in C, the cells exhibit marked heterochromatin remodeling. The insets on the right (B) or below (C. i, ii, iii) correspond to magnifications of the areas indicated by the two parallel white lines. White arrows indicate triple co‐localization of MBD4, USP7 and DAPI bright spots, while black arrows indicate colocalization of MBD4 and USP7 with diminishing or disappearing DAPI bright spots. Scale bars, 10 µm.
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