FIG 6.

Activation of E2f1 transcription by PU.1. (A) Reduction of E2f1 mature nascent mRNA transcript levels by PU.1 induction. E2f1 mature mRNA transcript levels were determined using RT-qPCR of nascent mRNA prepared 72 h after induction with 1,000 ng/ml Dox. (B) Induction of immature E2f1 nascent mRNA transcript levels by PU.1 induction. cDNA was synthesized using strand-specific primers. Immature E2f1 mRNA transcripts levels were determined using a primer set specific for E2f1 exon 1. (C) Induction of E2f1 immature nascent mRNA transcript levels by PU.1 induction. Immature E2f1 mRNA transcripts levels were determined using a primer set specific for E2f1 intron 1. (D) Induction of H3K27 acetylation by PU.1 at the Cybb promoter. Chromatin was prepared from iBN cells 72 h after induction with 1,000 ng/ml Dox. ChIP was performed using anti-H3K27Ac antibody. qPCR analysis was performed using primers recognizing the E2f1 or Cybb promoter. (E) Reduction of H3K27 trimethylation by PU.1 at the E2f1 and Cybb promoters. Chromatin was prepared from iBN cells 72 h after induction with 1,000 ng/ml Dox. ChIP was performed using anti-H3K27Me3 antibody. qPCR analysis was performed using primers recognizing the E2f1 or Cybb promoters. (F) A regulatory region in intron 1 of E2f1 behaves as a transcriptional activator. Plasmid DNA for pGL3-promoter (control), pGL3-promoter-E2F1 intron 1, or pGL3-promoter-E2F1 intron 1 with a mutated PU.1 binding site (Mut) was transiently transfected into WEHI-3B cells. Normalized luciferase activity was determined 24 h after transfection and is expressed as fold change relative to the level of the control. *, P < 0.05; ***, P < 0.001.