By analyzing 1,780,295 5Ј-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to DDBJ under accession nos. DA000001-DA999999, DB000001-DB294747, DB294748-DB384947, BP192706-BP383670, AU279383-AU280837, and AU116788-U160826.]One of the most striking findings revealed by the Human Genome Project is that the human genome contains only 20,000-25,000 kinds of protein-coding genes (International Human Genome Sequencing Consortium 2004). This number is unexpectedly small compared with the total gene numbers in yeast, fly, and worm genomes, which are estimated to be 6,000, 14,000, and 19,000, respectively (Goffeau et al. 1996;C. elegans Sequencing Consortium 1998;Adams et al. 2000). It is supposed that there must be other factors in addition to mere gene numbers to satisfy the prerequisites that enable the human genome to fabricate such highly elaborated systems as the brain and immune systems. To explain this, it has been hypothesized that multifaceted use of the genes should play a pivotal role in functional

Experiences during brain development may influence the pathogenesis of developmental disorders. Thus, social isolation (SI) rearing after weaning is a useful animal model for studying the pathological mechanisms of such psychiatric diseases. In this study, we examined the effect of SI on neurogenesis in the hippocampal dentate gyrus (DG) relating to memory and emotion-related behaviors. When newly divided cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) before SI, the number of BrdU-positive cells and the rate of differentiation into neurons were significantly decreased after 4-week SI compared with those in group-housed mice. Repeated treatment of fluoxetine prevented the SI-induced impairment of survival of newly divided cells and ameliorated spatial memory impairment and part of aggression in SI mice. Furthermore, we investigated the changes in gene expression in the DG of SI mice by using DNA microarray and real-time PCR. We finally found that SI reduced the expression of development-related genes Nurr1 and Npas4. These findings suggest that communication in juvenile is important in the survival and differentiation of newly divided cells, which may be associated with memory and aggression, and raise the possibility that the reduced expression of Nurr1 and/or Npas4 may contribute to the impairment of neurogenesis and memory and aggression induced by SI.

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lungspecific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for ␣-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and L-xylulose, and were inhibited competitively by nbutyric acid. Therefore, the proteins are designated as dicarbonyl/L-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and L-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC 1.1.1.5) are identical to Lxylulose reductase (EC 1.1.1.10), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.

DBTSS is a database of transcriptional start sites, based on our unique collection of precise, experimentally determined 5′-end sequences of full-length cDNAs. Since its first release in 2002, several major updates have been made. In this update, we expanded the human transcriptional start site dataset by 19 million uniquely mapped, and RefSeq-associated, 5′-end sequences, which were generated by a newly introduced Solexa sequencer. Moreover, in order to provide means for interpreting those massive TSS data, we implemented two new analytical tools: one for connecting expression information with predicted transcription factor binding sites; the other for examining evolutionary conservation or species-specificity of promoters and transcripts, which can be browsed by our own comparative genome viewer. With the expanded dataset and the enhanced functionalities, DBTSS provides a unique platform that enables in-depth transcriptome analyses. DBTSS is accessible at http://dbtss.hgc.jp/.

Osteoarthritis (OA) is the most prevalent joint disease and is characterized by pain and functional loss of the joint. However, the pathogenic mechanism of OA remains unclear, and no drug therapy for preventing its progress has been established. To identify genes related to the progress of OA, the gene expression profiles of paired intact and damaged cartilage obtained from OA patients undergoing joint substitution were compared using oligo microarrays. Using functional categorization combined with gene ontology and a statistical analysis, five genes were found to be highly expressed in damaged cartilage (HBEGF, ASUS, CRLF1, LOX, CDA), whereas three genes were highly expressed in intact tissues (CHST2, PTPRD, CPAN6). Among these genes, the upregulated expression of CRLF1 was reconfirmed using real-time PCR, and the in vivo expression of CRLF1 was detected in clusters of chondrocytes and fibrocartilage-like cells in damaged OA cartilages using in situ hybridization. In vitro, the transcriptional level of CRLF1 was positively regulated by TGF-beta1 in the mouse chondrogenic cell line ATDC5. Additionally, the CRLF1/CLC complex promoted the proliferation of ATDC5 cells and suppressed the expression level of aggrecan and type II collagen. Our data suggest that the CRLF1/CLC complex disrupts cartilage homeostasis and promotes the progress of OA by enhancing the proliferation of chondrocytes and suppressing the production of cartilage matrix. A component of the complex, CRLF1, may be useful as a biomarker of OA; and the corresponding receptor is a potential new drug target for OA.

Although recent studies have revealed that the majority of human genes are subject to regulation of alternative promoters, the biological relevance of this phenomenon remains unclear. We have also demonstrated that roughly half of the human RefSeq genes examined contain putative alternative promoters (PAPs). Here we report large-scale comparative studies of PAPs between human and mouse counterpart genes. Detailed sequence comparison of the 17,245 putative promoter regions (PPRs) in 5463 PAP-containing human genes revealed that PPRs in only a minor fraction of genes (807 genes) showed clear evolutionary conservation as one or more pairs. Also, we found that there were substantial qualitative differences between conserved and non-conserved PPRs, with the latter class being AT-rich PPRs of relative minor usage, enriched in repetitive elements and sometimes producing transcripts that encode small or no proteins. Systematic luciferase assays of these PPRs revealed that both classes of PPRs did have promoter activity, but that their strength ranges were significantly different. Furthermore, we demonstrate that these characteristic features of the non-conserved PPRs are shared with the PPRs of previously discovered putative non-protein coding transcripts. Taken together, our data suggest that there are two distinct classes of promoters in humans, with the latter class of promoters emerging frequently during evolution.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank under accession nos. BP870448-BP873619 and BP244227-BP249739.]With the completion of the human and mouse genome sequencing projects (Waterston et al. 2002; International Human Genome Sequencing Consortium 2004) as well as the large-scale compilation of full-length cDNA information (Zhang et al. 2000;Okazaki et al. 2002;Strausberg et al. 2002;Imanishi et al. 2004;Ota et al. 2004), it has gradually become clear that the genome systems in higher mammals are far more complex than previously thought. Now, the once-dominant static view that a single locus corresponds to only one transcript and one protein has been shown to be of very limited validity. Rather, it is more common for a single locus to produce several transcript variants. In about half of human genes, on average, four different transcripts are produced by alternative splicing and as a consequence translated into proteins of divergent biological functions (Modrek and Lee 2002;Imanishi et al. 2004). Similarly, recent studies also demonstrated that diversification via transcriptional regulation is no less common in human genes (Landry et al. 2003). By use of alternative promoters (APs), which consist of different modules of transcriptional regulatory elements, diversified transcriptional regulation is enabled within a single locus (Landry et al. 2003;Carninci et al. 2005;Cheng et al. 2005;Kim et al. 2005;Kimura et al. 2006).The functional diversification of a single gene enabled by the use of alternative splices (ASs) and APs ...

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In order to investigate the role of osteocytes in bone resorption, we examined the homogenate and conditioned medium from purified chick calvarial osteocytes in a pit-formation assay using unfractionated bone cells from mice. The osteocyte homogenate markedly inhibited pit formation, whereas the conditioned medium of osteocytes had no effect. This inhibitory activity was not the result of cytotoxicity of the homogenate. A novel bone-resorption-inhibitory protein was purified from collagenase-digested chick calvarial fragments enriched in osteocytes. The inhibitory protein, of molecular mass 18.5 kDa, showed significant dose-dependent inhibition of pit formation by unfractionated bone cells from mice and rabbits, and by human giant tumour cells. This protein also inhibited the bone-resorbing activity of purified osteoclasts in the pit-formation assay in the absence of other effector cells. Microinjection of the protein into osteoclasts caused disruption of the podosomes in the cells. The N-terminal 25-amino-acid sequence of the protein showed 68% identity to a part of Rho-GTP-dissociation inhibitor. Thus chick calvarial osteocytes may be involved in the regulation of bone resorption by osteoclasts.