Characterization of the Condensin Component Cnap1 and Protein Kinase Melk as Novel E2F Target Genes Down-regulated by 1,25-Dihydroxyvitamin D3

Lieve Verlinden,Guy Eelen,Ine Beullens,Mark Van Camp,Paul Van Hummelen,Kristof Engelen,Ruth Van Hellemont,Kathleen Marchal,Bart De Moor,Floris Foijer,Hein Te Riele,Monique Beullens,Mathieu Bollen,Chantal Mathieu,Roger Bouillon,Annemieke Verstuyf

doi:10.1074/jbc.m503587200

Abstract

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has potent antiproliferative effects characterized by a hampered G(1)/S transition. cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)(2)D(3). Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)(2)D(3) and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)(2)D(3)-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Cnap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)(2)D(3) was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)(2)D(3) depended on the pocket proteins p107 and p130 because 1,25(OH)(2)D(3) failed to repress these E2F target genes and lost its antiproliferative action in p107(-/-);p130(-/-) cells but not in pRb(-/-) cells.

Highlights

Active complexes between cyclin D and cyclin-dependent kinases 4/6 regulate the transition through the G1/S restriction point by phosphorylation of the retinoblastoma protein3 and other members of the pocket protein family, p107 and p130
Down-regulation started at 12–24 h after treatment, and the degree of down-regulation at 36 h ranged from 1.5- to 4.3-fold. 1,25(OH)2D3 decreased the expression of genes that are involved in cell cycle regulation and DNA replication and that of genes that mediate checkpoint control, DNA repair, chromosome transactions, and mitosis
An additional 20% of the genes was found to contain E2F binding sites. Four of these genes were selected for further study based on the highly conserved E2F binding sites in their promoter (Cnap1, enhancer of Zeste homolog 2 (Ezh2), and retroviral integration site 2 (Ris2)), on the one hand, and on their overexpression in undifferentiated cancers, on the other hand (Cnap1, Ezh2, and maternal embryonic leucine zipper kinase (Melk)) [23]