Journal: Molecular and Cellular Biology

Article Title: EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300

doi: 10.1128/MCB.00718-13

Figure Lengend Snippet: EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the p2.1 reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.

Article Snippet: The p2.1 reporter was purchased from ATCC.

Techniques: Over Expression, Activity Assay, Knock-Out, Mutagenesis, Luciferase, Transfection

Journal: Molecular and Cellular Biology

Article Title: EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300

doi: 10.1128/MCB.00718-13

Figure Lengend Snippet: The repression of HIF-1α by EAF2 is independent of FIH-1. (A) The transcriptional activities of HIF-1α C-TAD (aa 786 to 826) and the N803A mutant were suppressed by EAF2 overexpression. (B) Coimmunoprecipitation assays showed that EAF2 could not interact with FIH-1 when the two proteins were overexpressed in HEK293T cells. (C) EAF2 suppressed the transcriptional activity of the p2.1 reporter (C1) and the BNIP3 promoter reporter (C2) induced by the HIF-1α PP mutant (P402A P564A) and PPN mutant (P402A P564A N803A) in HEK293T cells under normoxic conditions. (D) EAF2 suppressed the transcriptional activity of the p2.1 reporter (D1) and the BNIP3 promoter reporter (D2) induced by HIF-1α PP and PPN mutants in HepG2 cells under normoxic conditions.

Article Snippet: The p2.1 reporter was purchased from ATCC.

Techniques: Mutagenesis, Over Expression, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300

doi: 10.1128/MCB.00718-13

Figure Lengend Snippet: EAF2 protects against hypoxia-induced cell death and inhibits cellular uptake of glucose under hypoxic conditions. (A, left) Under hypoxic conditions (1% O2 for 24 h), EAF2+/+ MEFs had a significantly higher cell survival rate than EAF2−/− MEFs (P = 0.0005). (Right) Under hypoxic conditions (1% O2 for 24 h), overexpression of full-length EAF2 in EAF2−/− MEFs enhanced the cell survival rate (P = 0.0014), but overexpression of domains III to VI of EAF2 did not do so (P = 0.4095). Cells were counted using a Bio-Rad cell counter after trypan blue staining. (B1) The representative pictures of EAF2+/+ and EAF2−/− MEFs stained with Hoechst 33342 after treatment by normoxia (21% O2) or hypoxia (1% O2) for 24 h. The dying cells are marked by arrows. (B2) Quantitative analysis for Hoechst staining. EAF2−/− MEFs had a significantly higher dead cell ratio than EAF2+/+ MEFs (P = 0.0104). Data are means and SEM representing nine randomly selected fields of three wells in a six-well plate. (C1) EAF2−/− MEFs had significantly higher intracellular glucose levels than EAF2+/+ MEFs. The glucose level was measured by a glucose assay kit (BioVision). (C2) Knockdown of EAF2 in HepG2 cells caused an increase in cellular uptake of glucose under hypoxia (P = 0.0048 and P = 0.0139). (D) Inhibition of p300 activity abolished the suppressive role of EAF2 on p2.1 promoter luciferase activity in EAF2−/− MEFs (P = 0.9370 versus P = 0.0047). Anacardic acid (AA) was added to the culture medium (50 μM) for 18-h treatment. Data are means and SEM of three independent experiments performed in triplicate. (E) Anacardic acid treatment (50 μM) abrogated the effect of EAF2 on cellular glucose uptake. Without anacardic acid treatment, EAF2−/− MEFs had higher intracellular glucose levels than EAF2+/+ MEFs (green curve versus orange curve). With anacardic acid treatment, the intracellular glucose levels in both EAF2−/− MEFs and EAF2+/+ MEFs dropped to the same level as that of EAF2+/+ MEFs without anacardic acid (AA) treatment (purple, dark green, and orange curves versus green curve). Glucose uptake was measured by FACS, following 1.5 h exposure to 2-NBDG (100 μM).

Article Snippet: The p2.1 reporter was purchased from ATCC.

Techniques: Over Expression, Staining, Glucose Assay, Inhibition, Activity Assay, Luciferase