FOXO3A directly activates PRDM1α in cHL cell lines. (A) PRDM1 and FOXO3 mRNA levels positively correlate in microdissected HRS cells. Data were mined from GEO (GSE39133). PRDM1, probe set 228964_at; FOXO3, probe set 224891_at. Statistical significance was analyzed by a 2-tailed Student t test. r, Pearson correlation coefficient. (B-F) The cHL cell lines KM-H2 and L428 expressing the constitutively active version of human FOXO3A (FOXO3(A3)ER [F3ER]) or EV were treated with 200 nM 4-OHT for 24 hours to induce nuclear translocation of the construct. (B) F3ER protein expression in stable cHL cell lines was assessed by immunoblot analysis. (C) Functional activity of F3ER in stably transfected cHL cell lines was demonstrated by transfection with a reporter vector containing the forkhead response element upstream of firefly luciferase and cotransfection with a Renilla luciferase reporter under the control of the ubiquitin promoter. F3ER was activated by 4-OHT immediately after transfection. Luminescence was measured as described in “Materials and methods.” Bars indicate average firefly luciferase activity normalized to Renilla luciferase luminescence. (D-E) FOXO3A increases PRDM1α mRNA (D) and protein (E) expression levels. cHL cells expressing F3ER were treated with 200 nM 4-OHT or vehicle. mRNA and protein was isolated and analyzed via RT-qPCR or immunoblot, respectively. (F) FOXO3A activates the PRDM1α promoter. L428 cells expressing F3ER were transiently transfected with the pGL4.20 vector containing the FOXO1 core promoter (CP) or a vector containing PRDM1 promoter regions, together with ubi-Renilla as reference vector. The FOXO1 CP, which does not contain FOXO-binding sites, served as negative control. After transfection, cells were treated with 4-OHT or vehicle and relative luciferase activity was measured 24 hours later. (G) FOXO3A directly binds to the PRDM1α promoter. L428 cells were transfected with bFOXO3 and BirA or with EV and BirA. Twenty-four hours later, cells were harvested and ChIP was performed. The position of targeted FOXO-binding sites at PRDM1α promoter regions is indicated counting from the start of transcription. A FOXP1 enhancer region served as positive control. The precipitated chromatin was amplified by specific primers and quantified with qPCR. Data are shown as fold enrichment compared with the gene desert region located on Chr12. All data are shown as mean ± SD of 3 independent experiments. For immunoblots, a representative of 3 independent experiments is depicted. TUBB served as loading control.