Analyses of Heme-Regulated Genes during Erythoid Differentiation.

(Introduction) During erythroid differentiation, the level of erythroid-specific genes increases synchronizing with the intracellular heme content. In addition, heme has been shown to play a role in transcription and protein synthesis. Based on these evidences, it is possible that heme widely regulates the expression of erythroid specific genes. With this hypothesis, we compared the gene expression profile between wild-type and heme-deficient erythroblasts generated from wild-type and ALAS2 (−) ES cells in in vitro, and identified 4 heme-regulated erythroid-specific genes (UCP2, CNBP, NuSAP and unknown EST1), (

(Methods) First, the expression and regulation of NuSAP gene during erythroid differentiation was examined. For the expression analysis, in vivo erythroblasts were fractionated according to the surface expression of TER119/CD71, and the level of expression of NuSAP mRNA was examined by quantitative RT-PCR. For the promoter analysis, the promoter region of mouse NuSAP gene was cloned, and the regulatory cis-element was determined by luciferase assay and EMSA. Next, for defining the properties of EST1, EST1 was constitutively expressed using Flag/HA tagged retroviral vector into mouse erythroleukemia (MEL) cell line, and nuclear extract of EST1-expessed MEL cells was purified by affinity chromatography, which was loaded on an SDS/PAGE gel and subjected to electrophoresis. In addition, for in vitro histone acetyltransferase (HAT) assay, free histone and purified EST1 protein were incubated with [³H]acetyl-CoA, and acetyltransferase activity was measured by scintigraphy.

(Results) (1) NuSAP mRNA was more significantly abundant in the subset corresponding to immature erythroblasts (TER119⁺CD71^high) than mature erythroblasts (TER119⁺CD71^low), and it was significantly increased in TER119⁺ cells from in vivo phlebotomized mice compared with control mice. Furthermore, during erythroid maturation of MEL cells by dimethylsulfoxide, NuSAP mRNA was increased at 24–72 hrs. Promoter analyses of NuSAP gene demonstrated that duplicated CCAAT boxes located at −81/−85 and −30/−34 were essential for promoter activity, which was trans-activated by NF-YA. These results suggested that NuSAP might contribute to the expansion of immature erythroblast pool under the control of NF-Y. (2) By the gel electrophoresis, it was revealed that EST1 protein forms a multimeric protein complex. Furthermore, whereas recombinant EST1 protein did not show HAT activity, EST1 complex could acetylate free histones in vitro, suggesting that EST1 might be a component of HAT complex.

(Conclusion) The novel functions of EST1 and NuSAP suggest that heme regulates erythroid differentiation by controlling the expression of variety of genes.