MLL5 protein levels in primary cervical adenocarcinomas correlated with expression of OGT and USP7.

<p><b>(A)</b> Representative images of haematoxylin and eosin along with immuno-histochemical staining with antibodies against OGT, USP7 and MLL5 on paraffin-embedded sections of human primary cervical adenocarcinomas. Scale bars, 200 μm. Adenocarcinomas tissues were indicated with black arrows, and adjacent normal adenoid tissues were indicated with white arrows. <b>(B)</b> Model for OGT and USP7 cooperatively regulate the stability of MLL5 protein by inhibiting its ubiquitylation and degradation.</p

USP7 deubiquitinates MLL5 protein.

<p><b>(A)</b> Knockdown of USP7 in HeLa cells leads to down-regulation of MLL5 protein. Two different lentivirus-based shRNAs target to an mRNA sequence starting 1324 and 2426 bp downstream of the translation start site were used to knockdown USP7 expression in HeLa cells. Whole cell lysates were harvested at day 6 post-infection. The expression of endogenous USP7 and MLL5 proteins were detected by anti-USP7 and anti-MLL5 antibodies. Actin was used as a loading control. <b>(B)</b> Control or OGT knockdown HeLa cells were treated with CHX (100μg/ml) for the indicated times before harvesting. The expression of endogenous MLL5 proteins were detected by anti-MLL5 antibody. Actin was used as a loading control. <b>(C)</b> HEK293T cells were co-transfected with plasmids encoding FLAG-tagged MLL5 (2μg) along with increasing amount of Myc-tagged USP7 or its inactive C223S mutant (0, 0.1, 0.5, 2μg). The presence of MLL5 and USP7 or inactive C223S mutant proteins were examined by western blotting using anti-FLAG or anti-Myc antibodies. <b>(D)</b> HEK293T cells were transiently co-transfected with plasmids encoding His-tagged ubiquitin, FLAG-tagged MLL5, Myc-tagged USP7 or its inactive C223S mutant. The cells were treated with MG132 (5μM) for 12h before harvesting, and the ubiquitylated proteins were pulled down with Ni-NTA beads and the MLL5 was visualized by immunoblotting with by anti-FLAG antibody.</p

Characterization of post-translational modifications of MLL5 protein.

<p><b>(A)</b> The phosphorylation sites were highlighted in blue, ubiquitylation sites in red, O-GlcNAcylation sites in green. <b>(B)</b> A representiative MS data of the N-GlcNAcylation at Ser435 and Thr440 were shown.</p

OGT interacts with and stabilizes MLL5 protein.

<p><b>(A)</b> HEK293T cells were transiently co-transfected with expression vectors for HA-tagged MLL5 and FLAG-tagged OGT, and extracts prepared and immunoprecipitated with anti-HA or anti-FLAG antibodies. The presence of FLAG-tagged OGT or HA-tagged MLL5 protein were examined by western blotting using anti-FLAG or anti-HA antibodies, respectively. <b>(B)</b> Mapping the MLL5 interacting domains in the OGT protein. <i>Upper panel</i>, a schematic representation of the domain structure of the full length OGT and its truncated <i>Δ</i>TPR mutant. <i>Lower panel</i>, HEK293T cells were co-transfected with expression vectors for FLAG-tagged OGT or its truncated ΔTPR mutant and HA-tagged MLL5 and truncated mutants. 48h after transfection, cell lysates were harvested and FLAG-tagged OGT or its truncated <i>Δ</i>TPR mutant proteins were immunoprecipitated (IP) with anti-FLAG antibody, and the presence of MLL5 protein were examined by western blotting using anti-HA antibody. The asterisk indicate a nonspecific band. <b>(C)</b> Mapping the OGT interacting domains in the MLL5 protein. <i>Upper panel</i>, a schematic representation of the domain structure of the full length MLL5 and its truncated mutants. <i>Lower panel</i>, HEK293T cells were co-transfected with expression vector encoding MLL5-HA and OGT-FLAG or truncated mutants. Cells were lysed for co-immunoprecipitation as indicated. <b>(D)</b> Knockdown of OGT in HeLa cells leads to down-regulation of MLL5 protein. Two different lentivirus-based shRNAs target to an mRNA sequence located 1275 and 1412 bp downstream of the translation start site were used to knockdown OGT expression in HeLa cells. Whole cell lysates were harvested at day 6 post-infection. The expression of endogenous MLL5 and the global levels of O-GlcNAcylation were detected by anti-MLL5 and anti-GlcNAc antibodies. Actin was used as a loading control. <b>(E)</b> The mRNA levels of MLL5 remain unchanged by OGT knockdown. The mRNA levels of OGT and MLL5 in OGT knockdown cells were analyzed by quantitative RT-PCR. Two-tailed unpaired Student’s <i>t</i> tests were performed, p < 0.001. <b>(F-H)</b> Control or OGT knockdown cells were treated with CHX (100μg/ml), MG132 (30μM) or NH₄Cl (5mM) for the indicated times before harvesting. The expression of endogenous MLL5 proteins were detected by anti-MLL5 antibody. β-actin was used as a loading control. Quantification of relative MLL5 levels is shown in the bottom panel. Numbers below lanes indicate densitometry of the protein presented relative to β-actin.</p

USP7 interacts with MLL5 protein.

<p><b>(A)</b> The reciprocal immunoprecipitation of USP7 and MLL5. HEK293T cells were transiently co-transfected with plasmids encoding HA-tagged USP7 and FLAG-tagged MLL5. 48h after transfection, cells lysates were harvested and FLAG-tagged MLL5 or HA-tagged USP7 proteins were immunoprecipitated (IP) with anti-FLAG or anti-HA antibodies, and the presence of USP7 or MLL5 protein were examined by western blotting using anti-HA or anti-FLAG antibodies, respectively. <b>(B)</b> Mapping the MLL5 interacting domain in USP7 protein. <i>Left panel</i>, a schematic representation of the domain structure of the full length USP7 and its truncated mutants. <i>Right panel</i>, HEK293T cells were co-transfected with expression vectors encoding FLAG-tagged MLL5 and Myc-tagged USP7 or truncated mutants. FLAG-tagged MLL5 proteins were co-immunoprecipitated with anti-FLAG antibody, and the presence of USP7 protein and truncated mutants were examined by western blotting using anti-Myc antibody. <b>(C)</b> Mapping the USP7 interacting domain in MLL5 protein. <i>Left panel</i>, a schematic representation of the domain structure of the full length MLL5 and its truncated mutants. <i>Right panel</i>, HEK293T cells were co-transfected with expression vectors encoding HA-tagged USP7 and FLAG-tagged MLL5 or truncated mutants. HA-tagged USP7 or truncated mutants were co-immunoprecipitated with anti-HA antibody, and the presence of MLL5 protein was examined by western blotting using anti-FLAG antibody.</p

MLL5 forms a complex with OGT and USP7.

<p><b>(A)</b> The reciprocal immunoprecipitation of MLL5, OGT and USP7. HEK293T cells were transiently co-transfected with plasmids encoding FLAG-tagged MLL5, V5-tagged OGT and HA-tagged USP7. 48h after transfection, cell lysates were harvested, and co-immunoprecipitation assays were performed as indicated. <b>(B)</b> Mapping the USP7 interacting domain in the OGT protein. <i>Upper panel</i>, a schematic representation of the domain structure of the full length OGT and its <i>Δ</i>TPR truncated mutant. <i>Lower panel</i>, HEK293T cells were transiently co-transfected with expression vectors for HA-tagged USP7 and FLAG-tagged OGT or its truncated <i>Δ</i>TPR mutant. 48h after transfection, the cells lysates were harvested and HA-tagged USP7 proteins were immunoprecipitated (IP) with anti-HA antibody, and the presence of OGT or its truncated <i>Δ</i>TPR mutant were examined by western blotting using anti-FLAG antibody. <b>(C)</b> Mapping the OGT interacting domain in the USP7 protein. <i>Upper panel</i>, a schematic representation of the domain structure of the full length USP7 and its truncated mutants. <i>Lower panel</i>, HEK293T cells were transiently co-transfected with expression vectors for FLAG-tagged OGT and Myc-tagged USP7 or its truncated mutants. 48h after transfection, cell lysates were harvested and FLAG-tagged OGT proteins were immunoprecipitated (IP) with anti-FLAG antibody, and the presence of USP7 or its truncated mutants were examined by western blotting using anti-Myc antibody. <b>(D)</b> Western blot profiles of selected fractions are shown. The bulk of MLL5 protein was found in the fraction C1, MLL5 protein were also presented in fraction B5, where OGT and USP72 were also present. <b>(E)</b> HeLa cells were transiently co-transfected with plasmids encoding FLAG-tagged MLL5, V5-tagged OGT and HA-tagged USP7, and then fixed and subjected to immunofluorescence using anti-FLAG monoclonal antibody to detect MLL5, anti-OGT polyclonal antibody to detect OGT, and anti-HA monoclonal antibody to detect USP7. Cells were stained with DAPI to highlight the nuclei.</p