LETTER TO THE EDITOR CREB regulates Meis1 expression in normal and malignant hematopoietic cells

CREB is a 43 kDa basic leucine zipper nuclear protein that is ubiquitously expressed and conserved from Drosophila to humans. Transcriptional activation by CREB is mediated through its interaction with a consensus octanucleotide sequence (TGANNTCA) termed the cyclic AMP response element or CRE, located in the promoter region of target genes. CREB constitutively binds to the CRE sequence as a heterodimer or a homodimer. The transcriptional activity of CREB is in part regulated by phosphorylation of the protein at a specific residue, serine 133. Stimulation of myeloid cells by granulocyte– macrophage colony stimulating factor leads to the phosphorylation of CREB at serine 133 through an MEKK and pp90RSK (ribosomal S6 kinase)-dependent pathway. CREB has previously been shown to be upregulated in the majority of acute leukemias, both at diagnosis and relapse. In contrast, CREB expression was found to be lower in bone marrow cells of patients with acute leukemia in remission and in the marrow of healthy donors. We previously studied the biological consequences of CREB-overexpression in myeloid leukemia cells and identified CREB as a proto-oncogene involved in transformation by promoting abnormal proliferation and survival of myeloid cells. Transgenic mice in which CREB is overexpressed in the myeloid lineage developed myeloproliferative disease/myelodysplastic syndrome after 1 year. Although CREB is implicated in the development of leukemia, the mechanism by which CREB-overexpression leads to a malignant phenotype remains elusive and most likely involves multiple target genes. It is plausible that the overabundance of CREB, and thus phosphorylated CREB, leads to enhanced binding of CREB to CRE sequences in the promoter regions of genes involved in cell proliferation and survival. To this end, we sought to investigate possible CREB target genes to understand the downstream pathways mediating transformation by CREB in hematopoietic cells. In this paper, we report that microarray analysis identified Meis1 as a significantly upregulated gene in CREB-overexpressing myeloid cells. Meis1 is a member of the TALE (three amino-acid loop extension) family of transcription factors. It is closely related to the homeodomain-containing transcription factor family known as the hox genes. This diverse family of transcription factors, including Meis1, HoxA9 and Pbx1, are normally expressed in primitive bone marrow cells. Meis1 forms multimeric complexes with HoxA9, and sometimes Pbx1, which then bind to target sequences of DNA to activate gene transcription. Although the expression of these transcription factors is critical for normal development of myeloid cells, the aberrant expression of these proteins has been implicated in the development of leukemia. During myelopoiesis, Meis1 expression decreases as cells differentiate. Meis1 is expressed in immature CD34þ lineage negative hematopoietic cells but not in more differentiated CD34 lineage positive cells. Meis1 overexpression promotes self-renewal of the progenitor cell and inhibits G-CSFdependent differentiation into granulocytes. The Meis1 promoter contains a CRE half-site element. CREB-overexpression may disrupt the requisite decline in Meis1 protein expression that allows the differentiation of myeloid progenitor cells in response to cytokines. Here, we report that Meis1 is regulated downstream of CREB in normal hematopoietic cells, leukemia cell lines and primary leukemia cells. To identify potential downstream target genes, we performed microarray analysis with RNA from K562 cells stably expressing CREB. CREB signature genes were identified by comparing the gene expression data of CREB-overexpressing cells and control parental cells (Figure 1). Hierarchical clustering of 896 genes was determined from three microarray experiments performed in triplicate with RNA from CREB-overexpressing K562 cells. The analysis demonstrated genes that clearly separated into two groups, upregulated genes and downregulated genes. Approximately 896 genes were found to be differentially expressed in the CREB-overexpressing cells compared to control parental cells. Of 896 differentially expressed genes, 702 were upregulated and they included two members of the MEIS1 family of genes and two members of the PBX1 family of genes, which have been reported to be critical for hematopoietic stem cell