Identification of a Novel Acetyl-CoA Binding Protein (EST1) Which Epigenetically Regulates the Expression of Erythroid-Specific Genes

Abstract

(Introduction) Embryonic stem (ES) cells that lack 5-aminolevulinate synthase-2 (ALAS2) gene provide a valuable model in dissecting molecular events in which heme is required during erythroid differentiation. Recently, we identified a novel acetyltransferase-like gene (EST1) through differential expression analysis between wild-type and heme-deficient erythroblasts as a potential downstream target gene of heme (BBRC 2006; 340:105–110, ASH 2007). EST1 belongs to the GNAT (GCN5-related N-acetyltransferase) superfamily since it contains the highly conserved amino acid residues (motif A), known as acetyl-CoA binding domain. Since the GNAT superfamily contains a wide variety of acetyltransferases with different substrates or with unknown functions, probing how EST1 might be required for biological events in erythroid cells, could be informative. Here, we investigated the role of EST1 during erythroid differentiation.(Methods) EST1 was constitutively expressed using Flag/HA-tagged retroviral vector into mouse erythroleukemia (MEL) cell line. EST1 protein complex was purified by affinity chromatography from nuclear extract of EST1-expessed MEL cells. To obtain dominant-negative EST1-expressing cells, both Arg-62 and Gly-65 within EST1 were substituted to glutamic acid, and similarly transduced into MEL cells. These mutations have been widely applied for abolishment of acetyl-CoA binding activity. For depletion of endogenous EST1, siRNAs specific for EST1 were introduced into Hepa1c1c7 cells(Results) Although recombinant EST1 protein did not have acetylase activity for free histones in vitro (ASH 2007), EST1 protein forms a multimeric protein complex. Western blot analysis using FLAG-eluted polypeptides revealed the presence of GCN5, TRRAP, SPT3 and GATA-1, implying that this protein complex might participate in the transcriptional regulation of erythroid-specific genes. Following EST1 depletion in Hepa1c1c cells, a significant decrease in the acetylation of H3 and a mild decrease in that of H4, were observed by Western blot analyses. Similarly, a significant decrease in the acetylation of H3 and a mild decrease in that of H4 were also observed in dominant-negative than in wild-type EST1-expressing MEL cells. Furthermore, the level of bmajor and ALAS2 mRNA were significantly lower in dominant-negative than in wild-type EST1-expressing MEL cells upon treatment with 1.5% DMSO for both 48h and 72h. We are currently exploring mechanisms of how EST1 participates in the regulation of histone modification in erythroid cells.(Conclusion) EST1 may epigenetically regulate a subset of erythroid-specific genes under control of heme. Further elucidation of the function of the EST1 gene would enhance our understanding of the transcriptional network involving erythroid differentiation.