We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.

Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca 2؉ /calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis.Chondrogenesis is an important biological event for endochondral bone development, skeletogenesis, and tissue patterning (1, 2). The first step in chondrogenesis is the aggregation of mesenchymal cells into prechondrogenic condensations. These condensations start to express cartilage-specific genes and further differentiate into mature chondrocytes. In the growth plate, chondrocytes proliferate and further differentiate into hypertrophic chondrocytes. The control of chondrogenic differentiation and hypertrophy plays a pivotal role in the process. Dysregulation of either step leads to severe skeletal dysplasia in both mice and humans (3).The transcription factor Sox9 (SRY (sex-related Y)-type high mobility group box), which contains a SRY-related high mobility group box, has an essential role in the chondrocyte differentiation pathway (4, 5). Sox9 regulates the transcription of cartilage-specific extracellular matrix molecules, such as collagen type II (6), IX (7), and XI (8) and aggrecan (9). Heterozygous mutations in the SOX9 gene cause campomelic dysplasia characterized by severe chondrodysplasia (10). Sox9 heterozygous mutant mice and mice lacking SOX9 function show impaired endochondral bone formation (4, 5). Sox9 is also involved in the expression of Sox5 and So...

We attempted to find Escherichia coli proteins which preferentially bind to a curved DNA sequence even in the presence of an excess amount of a non-curved DNA sequence as a competitor, mainly by means of a DNA-binding gel retardation assay. Since the two sequences used had nearly the same nucleotide compositions, including consecutive dA5 stretches, we reasoned that this strategy would allow us to identify proteins which preferentially recognize an overall DNA curvature. We purified such a protein from E. coli. Its preferential binding to the curved DNA was found to be inhibited by distamycin, which removes the curvature from appropriate DNA sequences. The purified protein was identified as the E. coli nucleoid protein, H-NS.

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54 nrb ) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54 nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54 nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54 nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54 nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54 nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54 nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54 nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54 nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

In addition to the set of curved DNA segments isolated previously from Escherichia coli, another set of curved DNA segments has now been isolated. To gain an insight into the functional significance of these curved DNA sequences, systematic analyses were carried out, which included not only mapping of the precise locations of the segments on the E. coli chromosome but also clarification of the gene organization in the chromosomal regions surrounding the curved DNA sequences. It was demonstrated that most of the curved DNA sequences, which have been characterized so far, appear to be located immediately upstream of the coding sequences of adjacent genes. It was also demonstrated that an E. coli histone-like protein, named H-NS (or H1a), exhibits a strong affinity for naturally occurring curved DNA sequences in regions upstream promoters.