Abstract
p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression. We previously showed that p400/mDomino is essential for embryonic development and primitive hematopoiesis. Here we generated a conditional knock-out mouse for the p400/mDomino gene and investigated the role of p400/mDomino in adult bone marrow hematopoiesis and in the cell-cycle progression of embryonic fibroblasts. The Mx1-Cre- mediated deletion of p400/mDomino resulted in an acute loss of nucleated cells in the bone marrow, including committed myeloid and erythroid cells as well as hematopoietic progenitor and stem cells. A hematopoietic colony assay revealed a drastic reduction in colony-forming activity after the deletion of p400/mDomino. Moreover, the loss of p400/mDomino in mouse embryonic fibroblasts (MEFs) resulted in strong growth inhibition. Cell-cycle analysis revealed that the mDomino-deficient MEFs exhibited a pleiotropic cell-cycle defect at the S and G(2)/M phases, and polyploid and multi-nucleated cells with micronuclei emerged. DNA microarray analysis revealed that the p400/mDomino deletion from MEFs caused the impaired expression of many cell-cycle-regulatory genes, including G(2)/M-specific genes targeted by the transcription factors FoxM1 and c-Myc. These results indicate that p400/mDomino plays a key role in cellular proliferation by controlling the expression of cell-cycle-regulatory genes.
Highlights
From the ‡Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, the §Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, and the ¶Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitima 332-0012, Japan p400/mDomino is an ATP-dependent chromatin-remodeling protein that catalyzes the deposition of histone variant H2A.Z into nucleosomes to regulate gene expression
These results indicated that mDomino plays a key role in cellular proliferation by regulating the expression of genes involved in cell-cycle progression
The deletion of the floxed exon 15 of the mDomfl allele was inefficient in the tail, an efficient deletion of the floxed allele (50 –70%) was observed in the spleen, and almost complete deletion was achieved in the liver and bone marrow (BM) in the mDom⌬/fl;Mx1-Cre and the mDomϩ/fl;Mx1-Cre mice (Fig. 2A)
Summary
Construction of Targeting Vectors and Generation of Conditional Knock-out Mice—FLPe transgenic mice [25] were kindly provided by Dr S. To delete the mDomfl allele from mDomfl/fl:CreER MEFs, the cells were treated with 7.5 nM 4-hydroxytamoxifen (OHT) for 8 h, washed with the culture medium, and further incubated in SEPTEMBER 24, 2010 VOLUME 285 NUMBER 39. Microarray Analysis—For DNA microarray analysis, RNA was extracted from Domfl/fl;CreER MEFs that were left untreated or treated with 7.5 nM OHT for 8 h and cultured for 2 days. In the assessment of down-regulated genes, genes presenting with a negative value in at least one of the three profiles or with an average intensity of Ͻ100 were deleted in the profiles of OHT-untreated MEFs. Real-time PCR for the Quantitative Analysis of mRNA—For the reverse-transcribed (RT)-PCR reaction, cDNA was synthesized from DNase I-treated total RNA (0.5 g) using an oligo(dT) primer and Superscript III (Invitrogen) in a 10-l reaction mixture. Expressed as copy numbers of target mRNA per nanogram of total RNA
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