The receptor activator of NF-B ligand (RANKL) induces various osteoclast-specific marker genes during osteoclast differentiation mediated by mitogen-activated protein (MAP) kinase cascades. However, the results of transcriptional programming of an osteoclastspecific cathepsin K gene are inconclusive. Here we report the regulatory mechanisms of RANKL-induced cathepsin K gene expression during osteoclastogenesis in a p38 MAP kinase-dependent manner. The reporter gene analysis with sequential 5-deletion constructs of the cathepsin K gene promoter indicates that limited sets of the transcription factors such as NFATc1, PU.1, and microphthalmia transcription factor indeed enhance synergistically the gene expression when overexpressed in RAW264 cells. In addition, the activation of p38 MAP kinase is required for the maximum enhancement of the gene expression. RANKL-induced NFATc1 forms a complex with PU.1 in nuclei of osteoclasts following the nuclear accumulation of NFATc1 phosphorylated by the activated p38 MAP kinase. These results suggest that the RANKL-induced cathepsin K gene expression is cooperatively regulated by the combination of the transcription factors and p38 MAP kinase in a gradual manner.

Like most eukaryotic rDNA promoters, the promoter for rDNA in Saccharomyces cerevisiae consists of two elements: a core element, which is essential, and an upstream element, which is not essential but is required for a high level of transcription. We have demonstrated that stimulation of transcription by the upstream element is mediated by a multiprotein transcription factor, UAF (upstream activation factor) which contains three proteins encoded by RRN5, RRN9, and RRN10 genes, respectively, and probably two additional uncharacterized proteins. The three genes were originally defined by mutants that show specific reduction in the transcription of rDNA. These genes were cloned and characterized. Epitope tagging of RRN5 (or RRN9), combined with immunoaffinity purification was used to purify UAF, which complemented all three (rrn5, rrn9, and rrn10) mutant extracts. Using rrn10 mutant extracts, a large stimulation by UAF was demonstrated for template containing both the core element and the upstream element but not for a template lacking the upstream element. In the absence of UAF, the mutant extracts showed the same weak transcriptional activity regardless of the presence or absence of the upstream element. We have also demonstrated that UAF alone makes a stable complex with the rDNA template, committing that template to transcription. Conversely, no such template commitment was observed with rrn10 extracts without UAF. By using a series of deletion templates, we have found that the region necessary for the stable binding of UAF corresponds roughly to the upstream element defined previously based on its ability to stimulate rDNA transcription. Differences between the yeast UAF and the previously studied metazoan UBF are discussed.

The 35S rRNA gene of the yeast Saccharomyces cerevisiae was fused to the GAL7 promoter. This hybrid gene, when present on a multicopy plasmid and induced by galactose, suppressed the growth defects of a temperaturesensitive RNA polymerase I (pol I) mutant and those of a mutant in which the gene for the second largest subunit of pol I was deleted. Analysis of pulse-labeled RNA directly demonstrated that rRNA synthesis in this deletion mutant is from the GAL7 promoter. These experiments show that the sole essential function of pol I is the transcription ofthe rRNA genes, that pol I is not absolutely required for the synthesis of rRNA and ribosomes or cell growth if 35S rRNA synthesis is achieved by some other means, and that the tandemly repeated structure of the chromosomal rRNA genes is also not absolutely required for the synthesis of rRNA and ribosomes. pCl/l carrying GAL7-35SrDNA (see Fig. 1) pNOY102 pAA7 carrying GAL7-35SrDNA (see Fig. 1) *NOY401 was constructed by crossing NOY265 (10) and W303-la (7) and then screening meitotic segregants for a temperature-sensitive (rpal90-3) and strongly galactose-positive phenotype.tNOY408-la, -lb, -lc, and -ld are four haploid segregants derived from a single ascospore produced by diploid strain NOY408 on galactose plates.

Previously, we have isolated mutants of Saccharomyces cerevisiae primarily defective in the transcription of 35S rRNA genes by RNA polymerase I and have identified a number of genes (RRN genes) involved in this process. We have now cloned the RRN6 and RRN7 genes, determined their nucleotide sequences, and found that they encode proteins of calculated molecular weights of 102,000 and 60,300, respectively. Extracts prepared from rrn6 and rrn7 mutants were defective in in vitro transcription of rDNA templates. We used extracts from strains containing epitope-tagged wild-type Rrn6 or Rrn7 proteins to purify protein components that could complement these mutant extracts. By use of immunoaffinity purification combined with biochemical fractionation, we obtained a highly purified preparation (Rrn6/7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparent molecular weight of 66,000, but which did not contain the TATA-binding protein (TBP). This complex complemented both rrn6 and rrn7 mutant extracts. Template commitment experiments carried out with this purified Rm6/7 complex and with rrn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is dependent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent preinitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.

Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34⌬ weakened the binding of Rpa49 to RNA polymerase. rpa34⌬ mutants were caffeine sensitive, and the rpa34⌬ mutation was lethal in a top1⌬ mutant and in rpa14⌬, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49⌬ mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34⌬ mutants, but strains carrying rpa49⌬ and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30°C, failed to grow at 25°C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49⌬ and rpa49-338::HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.Fast-growing Saccharomyces cerevisiae and Schizosaccharomyces pombe cells mobilize some 70% of their transcriptional capacity to produce the 6.8-kb precursor of the 18S, 5.8S, and 25S rRNAs by transcribing the highly repeated ribosomal DNA (rDNA) locus (44, 50). Genetic studies (43) have established that this is the only role or, at least, the only essential role of yeast RNA polymerase I (Pol I). rDNA transcription starts with the binding of Pol I to its specificity factor Rrn3 (7, 39-41, 48, 57). The Rrn3-Pol I dimer is then directed to a preinitiation complex residing at the rDNA promoter region. This complex combines the TATA box-binding protein (also operating in the Pol II and Pol III systems) with the core and upstream activation factors that are specific to the Pol I system (31, 32). In mammals, Pol I first binds transcription initiation factor IA, akin to Rrn3 and functionally interchangeable with that protein in vivo (7,40,41). The Pol I-transcription initiation factor IA dimer is then targeted to the rDNA promoter by interacting with SL1, a factor made of the TATA box-binding protein associated with four protein subunits, TAF I 95, TAF I 68, TAF I 48, and TAF I 41 (11,22,23,40,59); TAF I 68 is distantly related to the yeast core factor at the level of the core factor's Rrn7/TAF I 68 subunit (9). Pol I is also associated with the upstream binding factor (UBF; mammals) and Hmo1 (yeast) HMG box proteins, which stimulate rDNA t...