Hypoxia-induced Pontin methylation is mediated by G9a and GLP. (A) Protein expression levels of G9a, GLP, and Suv39h1 were examined by immunoblotting in HeLa and MCF7 cells exposed to normoxic and hypoxic conditions as indicated. (B and C) Pontin methylation was examined by immunoprecipitation of cell lysates from MCF7 cells exposed to hypoxia with anti-methyl-lysine antibody followed by immunoblotting with anti-Pontin, HIF-1α, G9a, and GLP antibodies for the indicated times (B) or in the presence and absence of an inhibitor of G9a and GLP, BIX-01294 (C). (D) Requirement of G9a for hypoxia-induced Pontin methylation was examined in WT and G9a-deficient MEFs exposed to normoxic and hypoxic condition. (E) HEK293 cells were cotransfected with plasmids encoding each GFP-tagged Pontin deletion constructs spanning the indicated amino acid residues exposed to normoxia or hypoxia for 6 h. Whole cell extracts were immunoprecipitated with antimethyl-lysine antibody followed by immunoblot assay using anti-GFP antibody to detect methylated Pontin. (F) Pontin methylation was examined as in (E) using constructs encoding Pontin WT or KA mutant exposed to normoxia or hypoxia for 6 h. (G) Dynamics of Pontin methylation was examined in 293HEK cells exposed to either normoxia, hypoxia, or hypoxia and normoxia and Pontin methylation was examined as in (E). (H) Endogenous interaction between Pontin and HIF-1α in MCF7 cells exposed to hypoxia using either anti-HIF-1α or normal IgG.

Pontin methylation potentiates HIF-1α transcriptional activity by enhancing interaction with p300. (A and B) Quantitative RT-PCR analysis of Pontin-dependent (A) and Pontin-independent (B) hypoxia target gene expressions identified (both 6 and 9 h). Results are expressed as relative mRNA levels compared to shNS under normoxic condition. Values are expressed as mean ± SEM of three independent experiments. (C and D) ChIP assays on the Ets1 and BNIP3L promoters in MCF7 cells with Pontin knockdown by shPontin (C) and reconstituted with exogenous shRNA-resistant Pontin WT^R and KA^R mutant (D) upon hypoxia. Promoter occupancy of proteins indicated was analyzed. (E) ChIP analysis performed in WT and HIF-1α-deficient (HIF-1α^-/-) MEFs examining the recruitment of various proteins to Ets1 promoter as indicated. (F) Endogenous interaction between Pontin and p300 in MCF7 cells exposed to either normoxia or hypoxia using anti-p300 antibody. (G and H) MCF7 cells were transfected with either Pontin WT or KA mutant and immunoprecipitation assay was performed using anti-p300 antibody. (I) Pontin WT or KA mutant was immunoprecipitated with anti-p300 antibody in WT or HIF-1α^-/- MEFs followed by immunoblotting using antibodies indicated.

G9a and GLP methylate Pontin. (A) Histone lysine methyltransferases were prepared as GST-fusion proteins and incubated with GST-Pontin in the presence of S-adenosyl-L-[methyl-³H] methionine (SAM). Reaction products were analyzed by autoradiogram. (B and C) In vitro methylation assay was performed using GST-G9a SET domain (B) or GST-GLP SET domain (C) with Coomassie brilliant blue staining (Left). Asterisks (*) indicate nonspecific bands, and the hash mark (#) indicates automethylation. (D) HEK293 cells were transfected with empty vector or with expression vector encoding G9a or GLP. Cell lysates were immunoprecipitated with antibody against antimethyl-lysine antibody followed by immunoblot analysis with anti-Pontin antibody to detect methylated Pontin. (E and F) Coimmunoprecipitation assay of endogenous Pontin with G9a (E) or GLP (F).

Pontin-dependent target identification by microarray analysis. (A and B) Three × HRE-luciferase reporter assay with knockdown of Pontin (A) or overexpression with Pontin WT and KA mutant (B) under normoxia or hypoxia for 6 h. Values are expressed as mean ± SEM of three independent experiments. (C) Flow chart showing the strategy of cDNA microarray analysis and Pontin-dependent gene selection process. (D) Pontin-dependent genes represent about 23.5% of all differentially expressed genes by hypoxia. (E and F) Identification of Pontin-independent genes (E) and Pontin-dependent up-regulated (cluster 3, red) and down-regulated (cluster 4, green) genes (F) by hierarchical clustering and comparing fold change of hypoxia-induced genes from cells expressing shNS and shPontin. (G) Known target genes for HIF-1, NFκB and TCF/LEF were collected and the enrichment of these genes was analyzed for each cluster is shown as a Z score within each cluster. (H) Heatmap diagram of Pontin-dependent genes that are activated by hypoxia (6 h) showing gene names with known HIF-1α target genes marked with an asterisk. Also see Table S1 for Pontin-dependent target genes at 9 h of hypoxia.

Pontin methylation increases tumorigenic properties via Ets1. (A and B) Proliferation was monitored over six days in hypoxic condition for MCF7 cells expressing shNS or shPontin (A) and expression of either Pontin WT or KA mutant (B). (C and D) Photomicrographs (40×) from Matrigel-coated transwell invasion assay of MCF7 cells expressing shNS and shPontin (C) and Pontin WT or Pontin KA (D) in normoxic and hypoxic conditions for 24 h. Bar graph shows mean number of cells per filter and p value is shown from student’s t-test analysis. Error bars represent SEM; n = 5 (Right). (E and F) Photomicrographs from a scratch-mobility assay of MCF7 cells expressing either shNS or shPontin and reconstituted with shRNA-resistant Pontin WT^R or KA^R mutant (E) or Pontin WT^R or KA^R mutant either transfected with control siRNA or Ets1 siRNA (F) in the hypoxia for the indicated times (Left). Gap distance between the two migrating fronts is measured and shown as a graph (Right). Asterisk (*) is indicated for p < 0.001 by two-tailed students t-test. (G) Proposed model of transcriptional activation role for methylated Pontin during hypoxia. Pontin is methylated by G9a/GLP by hypoxia and enhances the recruitment of p300 to a subset of hypoxia target promoters, thereby potentiating the transcriptional activity of HIF-1α leading to increase in hypoxia-mediated cellular migration, proliferation, and invasion.