Regulation of erythroid differentiation by trnscription factors and heme

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1) We showed that dimethyl sulfoxide increased the eryThroid transcription factor NF-E2' DNA complex formation in the wild -type MEL cells, in part by the induction of p45 gene expression, and that both DNA binding and transactivation activity of NF-E2 are regulated by Ser/Thr phosphorylation suchas Ras siganling cascade. An Mafk homodimer may suppress transcription not only by competition for the DNA binding site, but also by directly inhibiting transcription.2) Using a yeast two-hybrid screen with a GAL4-MafK fusion protein, we identified two novel bZip transcription factors, Bach1 and Bach2, as heterodimerization partners of MafK, In addition to a CNC-type bZip domain, these Bach proteins possess another protein interaction motif, BTB domain. Expression of Bach1 appears ubiquitous, but that of Bach2 is restricted to monocytes and neuronal cells. Bach proteins bind in vitro to NF-E2 binding sites by forming heterodimers with MafK.Bach1 and Bach2 function as transcription repressors i … More n transfection assays using fibroblast cells, but they function as a transcription activator and repressor, respectively, in cultured erythroid cells.3) A short amino acid sequence, the heme regulatory motif (HRM), has been shown to be involved in the hemin inhibition of protein transport into mitochondria in vitro. To elucidate the role of HRM in the home regulation of 5-aminolevulinate synthase (ALAS) transport in vivo, we constructed a series of mutants of the non-specific isoform of ALAS (ALAS-N) in which the specific cysteine residues within the HRMs were converted to serines. Wild-type and mutant enzymes were expressed in QT6 fibroblasts through transient transfection, followed by analyses of the mitochondrial import of the enzymes. The home inhibition, which was observed in the wild-type ALAS-N, abolished completely when all the three HRMs in the enzyme were mutated, indicating that the FIRM is actually required for the heme regulation of ALAS-N transport within the cells. In contrast, exogenous hemin did not affect the import of the erythroid ALAS (ALAS-E) under the comparable experimental conditions.4) We analyzed ALAS-E gene (ALAS2) of Japanese males with pyridoxine-refractory X-l inked sideroblastic anemia (XLSA) and identified an ALAS2 mutation (D19OV). The mutation did not affect the enzyme activity in vitro. However, the amount of the enzyme in bone marrow cells were found to be reduced to approximately 5% of the normal control. Additionally, accumulation of aberrantly processed proteins, the sizes of which were larger than that of mature ALAS-E, was found in mitochondria in transient transfection analyses. These larger products were reproducibly detected in assays combining in vitro transcription/translation and precursor import into isolated mitochondria. These results indicate that the mutation causing pyridoxine-refractory XLSA may affect the processing of ALAS-E precursor, thus provoking instability of the ALAS-E protein. Less