Transgenic Expression of Bach1 Transcription Factor Results in Down-Regulation of the p45 Target Genes in Megakaryocytic Lineage Cells.

Both p45 and BACH transcription factors can form dimers with one of the small Maf proteins, and these heterodimers bind to Maf recognition elements (MARE). MARE is known to act as a critical cis-regulatory element of erythroid and megakaryocytic genes. While detailed analyses of p45 -null mutant mice and small maf compound mutant mice revealed that these factors are both critical for platelet production, the functional contributions of BACH1 and the relationship or redundancy between BACH1 and p45 in megakaryocytes remain to be clarified. To address these issues, we generated transgenic lines of mice bearing human BACH1 cDNA under the control of the GATA-1 locus hematopoietic regulatory domain. The transgenic mouse lines showed significant thrombocytopenia associated with impaired maturation of the megakaryocytes, and they developed myelofibrosis. The megakaryocytes overexpressing the BACH1 transgene exhibited reduced proplatelet formation. Since the phenotype of the BACH1 transgenic mice resembled that of the p45 -deficient mice, we examined the expression of the p45 NF-E2 target genes in the primary megakaryocytes from fetal liver cells of the BACH1 transgenic mice. RT-PCR analyses showed that expression of the hematopoietic-specific ß1-tubulin, thromboxane synthase ( TXAS), and of the 3ß-hydroxy-steroid dehydrogenase genes was significantly downregulated in the megakaryocytes from BACH1 transgenic mice. The TXAS gene is a well-known MARE-dependent gene containing functional MAREs in its promoter and in the second intron. To ask whether BACH1 actually binds to MARE in the megakaryocytic genes, we then performed chromatin immunoprecipitation (ChIP) analysis with a BACH1-specific antibody. A ChIP assay with a human megakaryocytic cell line, UT-7/TPO, demonstrated that BACH1 bound to the promoter and enhancers region in vivo. As expected, co-transfection with BACH1 or Bach1-MafK fusion protein (B1K) expression plasmids repressed the reporter gene activity driven by the TXAS promoter. These findings thus provide evidence that BACH1 acts as a transcriptional repressor in the regulation of MARE-dependent genes in megakaryocytes.