Figure 5

α-globin gene expression is de-repressed in murine embryonic fibroblasts lacking KLF3. α-globin mRNA expression levels were determined by real time qRT-PCR analysis of (A) TER119⁺ erythrocytes purified from embryonic day E14.5 fetal liver (Klf3^+/+n = 3, Klf3^+/− n = 5, Klf3^−/− n = 6), (B) primary MEFs (Klf3^+/+n = 2, Klf3^−/− n = 2 or 3), (C) immortalized MEFs (Klf3^+/+n = 2, Klf3^−/− n = 2), and (D) immortalized Klf3^−/− MEFs rescued with KLF3-V5 or empty vector (n = 2 for each). (A-D) In each case, relative expression of α-globin mRNA was normalized to 18S rRNA levels, and the expression levels of Klf3^+/+(A-C) or Klf3^−/−(D) were set to 1.0. In (B), mRNA levels of Klf3 and two known KLF3-repressed targets, Klf8 and Fam132a[7, 25], have also been analyzed together with a negative control, Gapdh. In (A-D), error bars shown represent standard error of the mean, *P < 0.05 (one-tailed t-test relative to Klf3^+/+), **P < 0.002 (two-tailed t-test relative to Klf3^+/+), ***P < 0.05 (two-tailed t-test relative to Klf3^−/−). (E) KLF3 ChIP-Seq track across the murine α-globin locus in MEFs from [24]. The positions of the HS-12 and HS-26 regulatory regions are indicated. (F) EMSA showing the binding of KLF3 to two sites within the HS-26 region. Nuclear extracts were obtained from COS-7 cells that were mock-transfected (lanes 1 and 6) or transfected with pMT3-Klf3 (lanes 2, 3, 7 and 8). Nuclear extracts from MEFs are shown in lanes 4, 5, 9 and 10. Identification of KLF3:DNA complexes was achieved by addition of an antibody specific for KLF3 (αKLF3, lanes 3, 5, 8 and 10).