Identification of Novel Heme-Regulated Genes during Erythroid Differentiation.

(Introduction) During erythroid differentiation, a large amount of heme is synthesized for hemoglobin formation. Besides its fundamental role as an oxygen carrier, heme is known to play a key role in transcriptional regulation of certain genes and in translational control of protein synthesis. Since it is known that both the level of erythroid-specific genes and heme content increase together during erythroid differentiation, heme may likely regulate the expression of erythroid-specific genes. It is also possible that heme might regulate even a wider variety of genes than hemoglobin synthesis in cells undergoing erythroid differentiation. With this view in mind, we have searched for novel genes that are under the control of heme. For this purpose, we used the wild-type and heme-deficient erythroblasts generated from the wild-type and ALAS2 (-) ES cells in vitro and compared their gene expression profiles. By this approach, we have identified and reported four novel erythroid-specific genes previously (ASH meeting 2003). In the present study, we have further investigated the mechanisms of heme-mediated regulation of these genes.

(Methods) cDNA sequences of these genes were determined by 5′RACE and data-base search. In order to generate erythroblasts containing various amounts of heme, ALAS2 (-) ES subclones, which had been partially rescued by human ALAS2 cDNA driven under the erythroid-specific promoter, were established and induced to undergo erythroid differentiation. Correlation between the level of expression of these genes and intracellular heme content was examined. In addition, the promoter region of one of these genes, NuSAP, was cloned, and its cis-element, through which heme regulates the expression, was determined by promoter analysis and EMSA.

(Results) The level of expression of all these genes was closely correlated with intracellular heme content in the partially rescued ALAS2 (-) erythroblasts, indicating that expression of these genes is clearly under the control of heme. The results of 5′-RACE and database search have allowed us to identify that these genes consist of uncoupling protein2 (UCP2), nucleolar spindle-associated protein (NuSAP) and two as yet uncharacterized genes (EST1 and 2). While EST1 consists of 110 a.a. with a GK motif which is characteristic of acetyltransferase, EST2 consists of 972 a.a, with nucleotide sequence indicative of a serine/threonine kinase with a putative transmembrane domain. In order to determine the heme regulatory motif, we also performed the promoter analysis of the NuSAP gene. The results showed that CCAAT box, located 34 nucleotides upstream from the transcription initiation site, was essential for the promoter activity, and the binding activity of a protein complex to this element was enhanced in DMSO-treated MEL cells, suggesting that heme regulates the expression of the NuSAP gene through this motif.

(Conclusion) These results demonstrate that expression of a wide variety of genes, which may have quite different functions from hemoglobin formation, may also be regulated by heme in erythroid cells undergoing cell differentiation.