1. Klinik für Innere Medizin mit SP Hämatologie, Onkologie und Immunologie, PhilippsUniversität Marburg, Baldinger Strasse, 35043 Marburg, Germany 2. Institut für Medizinische Biometrie und Epidemiologie; Philipps-Universität, Bunsenstraße 3, 35037 Marburg, Germany 3. Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-MannkopffStr. 2, 35037 Marburg, Germany 4. Medizinische Klinik und Poliklinik I, Universitätsklinikum Carl Gustav Carus, Fetscherstr. 74, 01307 Dresden, Germany 5. Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794-5222, USA

Acute myeloid leukemia (AML) is a heterogeneous disease with multiple different cytogenetic and molecular aberrations contributing to leukemic transformation. We compared gene expression profiles of 4608 genes using cDNA-arrays from 20 AML patients (nine with −7/del7q and 11 with normal karyotype) with 23 CD34+ preparations from healthy bone marrow donors. SKI , a nuclear oncogene, was highly up regulated. In a second set of 183 AML patients analyzed with real-time PCR, the highest expression level of SKI in AML with −7/del7q could be confirmed. As previously described, Ski associates with the retinoic acid receptor (RAR) complex and can repress transcription. We wanted to investigate the interference of Ski with RAR α signaling in AML. Ski was co-immunoprecipitated and colocalized with RAR α . We also found that overexpression of wild-type Ski inhibited the prodifferentiating effects of retinoic acid in U937 leukemia cells. Mutant Ski, lacking the N-CoR binding, was no more capable of repressing RAR α signaling. The inhibition by wild-type Ski could partially be reverted by the histone deacetylase blocking agent valproic acid. In conclusion, Ski seems to be involved in the blocking of differentiation in AML via inhibition of RAR α signaling.

We have cloned and sequenced the gene encoding the chicken A2B adenosine receptor, a member of the family of G-protein-coupled seven-transmembrane-domain receptors which is activated by extracellular adenosine. The gene occupies about 10 kb of genomic sequences and contains two exons. The promoter of the gene is associated with a CpG-rich island and lacks a canonical TATA-box. Previous work has shown that the chicken A2B adenosine receptor is a direct target gene of the transcription factors v-Myb and c-Myb and that its expression in the hematopoietic system is specific for myelomonocytic as well as erythroid cells. To understand the molecular basis of this cell type specificity we have searched for cis-regulatory DNA elements that direct the expression of the gene to specific hematopoietic lineages. As a first step towards this aim we have mapped DNase I-hypersensitive sites in the chromatin of the A2B adenosine receptor gene. Our studies reveal multiple nuclease-sensitive sites which are located in the intron of the gene and in the upstream region. A number of these sites are cell-type-specific suggesting that they correspond to cell-type-specific cis-regulatory DNA elements. To characterize the possible function of these elements we have performed reporter gene studies. Our results show that several of the nuclease-sensitive regions act as myelomonocytic-specific cis-acting stimulatory elements. Taken together, our data suggest that the expression of the A2B adenosine receptor gene in myelomonocytic cells is controlled by multiple cell type-specific cis-acting sequences located upstream and within the intron of the gene.

Numerous studies have shown that the retroviral oncogene v-myb encodes a transcription factor (v-Myb) which interferes with the differentiation program of myelomonocytic cells. It is generally thought that v-Myb deregulates the expression of specific target genes and thereby causes transformation of these cells. By using an estrogen-inducible version of v-Myb we have previously identified the gene for the chicken A2B adenosine receptor (A2B-AR), a member of the seven-pass transmembrane receptor superfamily, as a bona fide target gene for v-Myb. The chicken A2B-AR gene is expressed in v-myb transformed myeloblasts as well as in c-myb expressing erythroblasts, offering the opportunity to study how Myb transcription factors activate a target gene in two different hematopoietic lineages. Here, we report the characterization of the promoter of the A2B-AR gene. We show that the A2B-AR promoter region contains an exceptionally large number of Myb binding sites, many of which contribute to the Myb-inducibility of the promoter. The same sites were required for promoter activity in myelomonocytic and erythroid cells. In contrast to the promoters of other Myb target genes the A2B-AR promoter was not activated synergistically by Myb and other lineage-specific transcription factors that have been identified as Myb cooperation partners before. Taken together, our data suggest that the activation of the A2B-AR promoter by Myb depends on the simultaneous binding of a large number of Myb molecules.

The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which disrupts the myelomonocytic differentiation program and transforms myelomonocytic cells in vivo and in vitro. It is thought that v-Myb exerts its biological effects by deregulating the expression of specific target genes, most of which are still unknown. c-myb, the cellular progenitor of v-myb, is expressed in all immature hematopoietic cells and is presumed to regulate the expression of genes that are essential for the development of the hematopoietic system. Recently, we have identified the chicken Pdcd4 gene as a novel v-myb target gene. Pdcd4 has originally been identified in a screen for genes upregulated in apoptotic cells and, more recently, has been implicated in tumor progression. As a myb-regulated gene Pdcd4 is of interest because unlike most other myb target genes it is expressed in a broad spectrum of hematopoietic cells. As a first step to study the regulation of Pdcd4 expression in more detail, we here report the identification and preliminary characterization of the myb-inducible promoter of the Pdcd4 gene.