Sp1-dependent increase in G6PD transcription in response to HDACi. (A) Schematic of the G6PD promoter drawn to scale. Vertical boxes represent Sp1 motifs; predicted motifs are shown in red; two previously validated Sp1 binding sites (Motifs 3 and 4) are shown in dark green. The Sp1 motifs that are included in the 562-bp promoter region, also boxed in Figure 4B, are numbered 1 through 7. R1 is the reverse primer and F1 through F6 are the forward primers used to generate promoter fragments F1 through F6 shown in (B). There are 2 transcription start sites (TSS). NEMO, a gene whose promoter overlaps with that of G6PD is not shown. (B) Transcriptional activity of G6PD promoter assessed by luciferase (Luc) reporter assays. Luciferase activity was measured at baseline and upon 3 mM NaBu treatment of 24 hours. Activity is normalized against promoterless pGL3 basic and is shown relative to the baseline levels of the longest promoter fragment, F6 (n = 3). (C) Luciferase reporter assays conducted as in (B), using G6PD promoter constructs containing mutated putative Sp1 binding sites as shown, at baseline and upon 3 mM NaBu treatment for 24 hours. Mean ± standard error of the mean (SEM); n = 3. One-way ANOVA was used to compare pGL3-F5 to the mutated construct activities. (D) Sp1 binding in the GPPP gene promoters assessed by ChIP-RQ-PCR in a WT B-cell line. DHFR was used as a positive control for Sp1 binding. Data are shown as mean ± SEM for 3 independent experiments. Paired Student t test was performed to compare untreated vs 5-hour and 24-hour treatment with NaBu (n = 3). (E) The effect of a dominant negative (DN) form of Sp1 on G6PD transcription. In all, 293 T cells were transfected either with a plasmid containing a complementary DNA (cDNA) encoding a DN form of Sp1 or with a no-insert control plasmid. G6PD mRNA levels were assessed 48 hours later by qRT-PCR (n = 3).