A differential screen for ligand-regulated genes: identification of HoxA10 as a target of vitamin D3 induction in myeloid leukemic cells.
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the hormonal ligand for vitamin D3, is a potent inducer of myeloid-leukemic-cell differentiation. Such cells differentiate exclusively into monocytes/macrophages in response to this ligand. Since 1,25(OH)2D3 transduces its hormone signal through the vitamin D3 receptor (VDR), a ligand-modulated transcription factor and member of the nuclear hormone receptor superfamily, we sought to identify direct VDR target genes induced during this differentiation process. To do so, we applied a modified differential screen with a nascent-RNA purification strategy using biases for immediate-early-response genes induced by 1,25(OH)2D3 in the myelomonocytic cell line U937. Using this screen, we had previously identified p21Waf1/Cip1 as a gene transcriptionally induced by 1,25(OH)2D3 and demonstrated that this induction facilitates the differentiation of U937 cells into monocytes/macrophages (24). Here, we describe in detail our differential screen strategy and the identification and isolation of 20 1,25(OH)2D3-inducible genes or unknown cDNAs by means of this screen. One gene newly identified as a target of VDR regulation in myeloid cells is the homeobox HoxA10 gene. HoxA10 protein may act as a general regulator of cell growth, since overexpression of HoxA10 facilitated the differentiation of U937 cells into monocytes/macrophages independent of 1,25(OH)2D3 and acted to strongly inhibit the growth of the breast cancer cell line MCF-7 by arresting these cells in G1.
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Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Liu
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Anderson
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Freedman
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D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D002117:Calcitriol / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D016193:G1 Phase; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005801:Genes, Homeobox; D005821:Genetic Techniques; D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D018167:Receptors, Calcitriol / Q000502:physiology; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins; D008024:Ligands; D018167:Receptors, Calcitriol; C092119:Hoxa10 protein, mouse; D002117:Calcitriol