Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3.
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.
Verlinden
Lieve
L
Laboratorium voor Experimentele Geneeskunde en Endocrinologie, Katholieke Universiteit Leuven, Belgium.
Eelen
Guy
G
Beullens
Ine
I
Van Camp
Mark
M
Van Hummelen
Paul
P
Engelen
Kristof
K
Van Hellemont
Ruth
R
Marchal
Kathleen
K
De Moor
Bart
B
Foijer
Floris
F
Te Riele
Hein
H
Beullens
Monique
M
Bollen
Mathieu
M
Mathieu
Chantal
C
Bouillon
Roger
R
Verstuyf
Annemieke
A
D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D016000:Cluster Analysis; D015536:Down-Regulation / Q000187:drug effects*; D050684:E2F Transcription Factors / Q000378:metabolism*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D005810:Multigene Family; D020411:Oligonucleotide Array Sequence Analysis; D000075223:Poly-ADP-Ribose Binding Proteins; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*
D002868:Chromosomal Proteins, Non-Histone; D050684:E2F Transcription Factors; C506906:Ncapd2 protein, mouse; D000075223:Poly-ADP-Ribose Binding Proteins; C108524:Melk protein, mouse; D017346:Protein Serine-Threonine Kinases; D002117:Calcitriol