NFE2 regulates transcription of multiple enzymes in the heme biosynthesis pathway.

During erythroid differentiation, cooperative activities of erythroid transcription factors coordinate transcript levels of genes required for correct maturation. 1 These genes include the eight heme biosynthetic enzymes that are sequentially up-regulated to ensure augmented heme synthesis essential for the formation of hemoglobin. The transcription factor nuclear factor erythroid 2 (NFE2) plays a pivotal role in erythroid maturation and, together with other erythroid transcription factors such as EKLF and GATA1, 1 controls the transcription of erythroid-specific genes, including β-globin. 2 Furthermore, NFE2 is necessary for the transcriptional activation of two heme biosynthetic enzymes: porphobilinogen deaminase (PBGD) 3 and ferrochelatase (FECH). 4 This led us to investigate the role of NFE2 in the regulation of the remaining heme biosynthetic enzymes. In silico analysis revealed potential NFE2 consensus sites within haematologica 2014; 99:e208 LETTERS TO THE EDITOR Figure 1. (A) Predicted NFE2 consensus sites in the promoters of heme biosynthetic enzymes. (B) Chromatin immunoprecipitation of K562 cells was performed with either an NFE2 antibody or an unrelated IgG control, as indicated. Precipitated DNA was amplified using primers covering the predicted binding sites shown in A. In the lane marked " Input " , a 1:25 dilution of the input DNA was used. As an additional control, a genomic region of myogenin known not to interact with NFE2 was amplified (bottom panel). (C-E) HEL cells were treated with a lentiviral construct containing either shRNA against NFE2 (shNFE2) or scrambled RNA as negative control. (C) Western blot analysis of NFE2 protein expression. Equal protein loading was assessed by reprobing with an antibody against GAPDH. (D) mRNA expression of ALAD, PBGD, UROS, UROD, CPOX and the housekeeping gene β-2-microglobulin (β-2-M) was quantified by qRT-PCR. Expression levels of the heme biosynthetic enzymes were normalized to β-2-M expression and the expression in cells treated with scrambled RNA set to 1. Values are reported as fold change shRNA versus scrambled RNA-treated cells and represent the means and standard error of the mean of four individual experiments measured in duplicates. *P<0.05; **P<0.01; ***P<0.001 one sample t-test. (E) Heme content of the cells was determined by measuring the fluores-cence at an excitation wavelength of 400 nm and an emission wavelength of 662 nm. Values are shown relative to heme content in cells treated with scrambled RNA, which was set to one. The results represent the means and standard error of the mean of two independent transfec-tions with five independent measurements each. …