Supplementary Figure S3 from Crucial Functions of the JMJD1/KDM3 Epigenetic Regulators in Cancer

Somatic mutations across JMJD1 proteins.</p

Supplementary Figure S4 from Crucial Functions of the JMJD1/KDM3 Epigenetic Regulators in Cancer

Reaction rate of JMJD1A in dependence of the oxygen concentration.</p

Supplementary Figure S1 from Crucial Functions of the JMJD1/KDM3 Epigenetic Regulators in Cancer

Relationship between HR and the three JMJD1 proteins.</p

Supplementary Figure S2 from Crucial Functions of the JMJD1/KDM3 Epigenetic Regulators in Cancer

JMJD1 gene alteration frequencies.</p

Supplementary Table S1 from Crucial Functions of the JMJD1/KDM3 Epigenetic Regulators in Cancer

Correlation between JMJD1 mRNA levels in tumors.</p

Activation of the p21 promoter by JMJD2D in HEK293T cells.

<p>(<b>A</b>) Activity of a p21 luciferase reporter construct upon cotransfection of control vector pEV3S or wild-type or H192A JMJD2D is depicted. As indicated, empty vector pcDNA3 or pcDNA3-p53 was also transfected. (<b>B</b>) Analogous, response of a CMV or MMP-1 luciferase reporter construct to cotransfection of p53 and/or JMJD2D. (<b>C</b>) Chromatin immunoprecipitation assay in HCT116 cells treated without and with adriamycin.</p

Mapping of interaction domains.

<p>(<b>A</b>) Indicated Flag-tagged amino acids of JMJD2D were coexpressed with p53 in HEK293T cells and complex formation assessed in coimmunoprecipitation assays as in Fig. 1B. A sketch of human JMJD2D highlighting its JmjC domain is presented at the bottom. (<b>B</b>) Comparable amounts of GST or indicated GST-p53 fusion proteins were bound to glutathione agarose. After incubation with Flag-tagged JMJD2D, bound JMJD2D was revealed by anti-Flag Western blotting. The location of the DNA binding domain within p53 is indicated in the sketch at the bottom.</p

Binding of JMJD2D to p53.

<p>(<b>A</b>) Flag-tagged JMJD2D or HSPBAP1 were coexpressed with p53 in HEK293T cells. After anti-p53 immunoprecipitation (IP), coprecipitated proteins were revealed by anti-Flag immunoblotting (top panel). The bottom two panels show input levels of Flag-tagged proteins or p53. IgH, immunoglobulin heavy chain. (<b>B</b>) Indicated Flag-tagged JMJD proteins were coexpressed with p53 in HEK293T cells. After anti-Flag immunoprecipitation, coprecipitated p53 was detected by anti-p53 Western blotting (top panel). The middle and bottom panels show p53 and Flag-JMJD protein input levels, respectively. (<b>C</b>) HCT116 cell extracts were challenged with no, control or anti-JMJD2D antibodies and coprecipitated p53 detected by immunoblotting (top panel). The bottom panel shows that respective JMJD2D input levels were equal.</p

JMJD2D protects from apoptosis.

<p>(<b>A</b>) The level of sub-G1 HCT116 cells was determined in cells that expressed control shRNA or JMJD2D shRNA#3. Cells were treated for 72 h with 1 µM adriamycin or DMSO as indicated. (<b>B</b>) The same in case of p53^−/− HCT116 cells. (<b>C</b>) Model of JMJD2D action.</p

Sumoylation of p68 and p72 RNA Helicases Affects Protein Stability and Transactivation Potential

The p68 (DDX5) and p72 (DDX17) proteins are members of the DEAD-box (DDX) family of RNA helicases. We show that both p68 and p72 are overexpressed in breast tumors. Bioinformatical analysis revealed that the SUMO pathway is upregulated in breast tumors and that both p68 and p72 contain one consensus sumoylation site, implicating that sumoylation of p68 and p72 increases during breast tumorigenesis and potentially contributes to their overexpression. We determined that p68 and p72 are indeed sumoylated at a single, homologous site. Importantly, sumoylation significantly increased the stability of p68 and p72. In contrast to p72 and consistent with an ∼3-fold lesser half-life, p68 was found to be polyubiquitylated, and mutation of the sumoylation site increased polyubiquitylation, suggesting that sumoylation increases p68 half-life by reducing proteasomal degradation. Moreover, whereas p68 robustly coactivated transcription from an estrogen response element, its sumoylation mutant showed a drastically reduced coactivation potential. In contrast, the p68 sumoylation status did not affect the ability to enhance p53-mediated MDM2 transcription. On the contrary, preventing sumoylation of p72 caused an increase in its ability to transactivate both estrogen receptor and p53. Furthermore, sumoylation promoted the interaction of p68 and p72 with histone deacetylase 1 but had no effect on binding to histone deacetylases 2 and 3, the coactivator p300, or estrogen receptor and also did not affect homo/heterodimerization of p68/p72. In conclusion, sumoylation exerts pleiotropic effects on p68/p72, which may have important implications in breast cancer by modulating estrogen receptor and p53 activity