Eukaryotic transcription is regulated by multiple DNA elements, such as promoter-proximal elements, and is often combined with distal enhancer elements, which are recognized by several transcription factors. Core promoter regions often contain TATA, GC, and CCAAT boxes. The CCAAT box is one of the most typical elements for transcriptional activation. Several proteins, such as transcription factors of the C/EBP family and NF-I, have been shown to bind to CCAAT sequences. Nuclear factor Y (NF-Y) also binds to CCAAT boxes on many promoter regions (8). NF-Y is a well-conserved, ubiquitous transcription factor that consists of three subunits, NF-YA, NF-YB, and NF-YC, all of which are necessary for CCAAT box binding. NF-YB and NF-YC contain histone-folding motifs similar to H2A and H2B, respectively, and a tight complex of NF-YB and NF-YC associates with NF-YA (25). It has been reported that NF-Y associates with TATA box binding protein (TBP) (5) and several TBP-associated factors (TAFs) (11). NF-Y also interacts with p300/CBP, PCAF, and GCN5 and mediates histone acetylation on several promoters through these interactions (6,15,24,33).Osteoclast differentiation factor (ODF) is an essential factor for differentiation and activation of osteoclasts that is expressed on the osteoblast membrane (22,43). ODF is also known as TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine) and RANKL (receptor activator of NF-B ligand) (3, 39). ODF knockout mice exhibit typical osteoporosis with total occlusion of bone marrow space within endosteal bone (21) and also fail to develop mammary glands (10). In ODFϪ/Ϫ mice, mature osteoclasts are not found, whereas osteoclast progenitor cells are present. These observations indicate that ODF is an essential factor for developing mature osteoclasts and inducing bone resorption.ODF gene expression is induced by vitamin D3, parathyroid hormone (PTH), interleukin-1 (IL-1), TNF-␣, and PGE in primary osteoblasts or stromal cells (12,27,35,43). The mouse ODF promoter region contains a vitamin D3 response element (VDRE) (19,20). A heterodimer of vitamin D3 receptor (VDR) and retinoid X receptor (RXR) mediates vitamin D3 response (30). VDR-RXR transactivates target genes by recruiting transcriptional coactivators and mediators, such as the DRIP complex, which mediates transcriptional initiation, and the SRC-1/p300 complexes, which induce histone acetylation on target genes (13,31,32). The mouse ODF promoter also contains a CCAAT sequence near the transcriptional start site, but its significance is not known.In this report, we show that NF-Y is essential for transcriptional regulation of the ODF promoter. NF-Y exhibits a synergistic effect with vitamin D3 in activating this promoter. Knockdown analysis for an NF-Y subunit reveals that NF-Y is essential for the recruitment of RNAPII prior to induction but not for histone acetylation after vitamin D3 induction on the ODF promoter. Furthermore, NF-Y is also required for RNA-PII recruitment onto other CCAAT box-containing promoters, suc...

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein.Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.

Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-␤-D-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitt's lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.

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Mu-opioid receptor activation induces c-fos and junB expression and elevates AP-1-mediated transcriptional activities via the mitogen-activated protein kinase cascade.