The correlation between mutations in the Werner's syndrome (WRN) gene and the haplotypes of surrounding markers was studied in Japanese patients. We have elucidated the genomic structure of WRN helicase, and found five additional mutations, designated mutations 6-10. Mutations 4 and 6 were found to be the two major mutations in this population; these mutations comprised 50.8% and 17.5%, respectively, of the total in a sample of 126 apparently unrelated chromosomes. Almost all the patients homozygous for mutation 4 shared a haplotype around the WRN gene, consistent with the view that they are derived from a single ancestor. This important advantage demonstrated in the identification of the WRN gene suggests that the Japanese present a unique population for the cloning of other disease genes. The conserved haplotype was observed across 19 loci, extending a distance estimated to be more than 1.4 Mbp around the WRN gene. This haplotype is rare among random Japanese individuals. Unexpectedly, all the nine patients homozygous for mutation 6 shared a haplotype that was identical to this haplotype at 18 of these 19 markers. These results suggest that mutations 4 and 6 arose independently in almost identical rare haplotypes. The remaining mutations (1, 5, 7, 8, 9, and 10) occurred rarely, and were each associated with different haplotypes.

The regulation of Werner's syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5 upstream region (2.8 kb) of WRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides ؊67 to ؉160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRN promoter-luciferase reporter (WRN-Luc) plasmids that contained the 5-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.Werner's syndrome (WS) is a rare autosomal recessive genetic disorder causing symptoms of premature aging, such as gray hair, baldness, cataracts, and osteoporosis (9, 15, 23), accompanied by rare cancers (14). In vitro studies of fibroblast growth characteristics also suggest that WS may be related to normal aging: the life span of WS fibroblasts as expressed by population doubling levels is much shorter than that of normal fibroblasts (10, 32). The hypermutator phenotype, such as represented by genetic instability, also occurs frequently in WS fibroblasts and lymphoid cells (27,31,33,41).The gene responsible for WS (WRN) has been identified by positional cloning from the 8p11-p12 region (48) and is composed of a total of 35 exons (25, 49) that generate mRNA with 5,189 nucleotide residues. We have recently demonstrated that the gene product of WRN is an active RecQ-type DNA helicase by expressing WRN in insect cells, and we postulated that defective DNA metabolism is involved in a complex process of premature aging in WS patients (40). DNA helicases are enzymes that unwind the energetically stable double-stranded structure of DNA to provide the single-stranded template for important cellular processes such as replication, recombination, and repair (42).Mutations occurring in more than 100 WS patients have been extensively investigated by us and others. The more than 19 diff...

The androgen receptor (AR) is implicated in prostate cancer growth, progression, and angiogenesis. Hypoxia-inducible factor-1 (HIF-1), which transcriptionally regulates hypoxia-inducible angiogenic factors, is up-regulated in prostate cancers compared with adjacent normal tissues. HIF-1 may be involved in prostate cancer as well as the AR, but the involvement of HIF-1 in prostate cancer angiogenesis and progression has not been fully elucidated. In the present study, we found that in prostate cancer LNCaP cells dihydrotestosterone enhanced the expression of GLUT-1, one of the HIF-1 target genes, and also that hypoxia enhanced the expression of prostate-specific antigen (PSA) that is one of the AR target genes and is involved in tumor invasion. Small interfering RNA that specifically inhibits HIF-1 reduced the expression levels of PSA as well as GLUT-1. Reporter gene analysis showed that dihydrotestosterone activated the HIF-1 -mediated gene expression and hypoxia enhanced the AR-induced promoter activity of human PSA gene. Deletion and site-directed mutation of the 5 ¶-flanking region of human PSA gene revealed that the sequence ACGTG between À3951 and À3947 was essential in the response to hypoxia. Furthermore, chromatin immunoprecipitation assay indicated that HIF-1 interacts with the AR on the human PSA gene promoter. These results indicated that in prostate cancers, HIF-1 might cooperate with the AR to activate the expression of several genes related to tumor angiogenesis, invasion, and progression. (Mol Cancer Res 2007;5(4):383 -91)

The profile of helicase gene mutations was studied in 89 Japanese Werner's syndrome (WRN) patients by examining the previously described mutations 1-4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89 patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes, including those carrying mutations of both types 1 and 4. The genotypes analysis of the markers surrounding the. WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single founders.

A unique gene, RBP-MS, spanning over 230 kb in the human chromosome 8pll-12 near the Werner syndrome gene locus is described. The single-copy RBP-MS gene is alternatively spliced, resulting in a family of at least 12 transcripts (average length of 1.5 kb). Nine different types of cDNAs that encode an RNA-binding motif at the N terminus and helix-rich sequences at the C terminus have been identified thus far. Among the 16 exons identified, four 5'-proximal exons contained sequences homologous to the RNAbinding domain of Drosophila couch potato gene. Northern blot analysis showed that the RBP-MS gene was expressed strongly in the heart, prostate, intestine, and ovary, and poorly in the skeletal muscle, spleen, thymus, brain, and peripheral leukocytes. The possible role of this gene in RNA metabolism is discussed.Several genes of medical importance map to the short arm of chromosome 8, including the tumor suppressor genes implicated in the pathogenesis of colorectal carcinoma and prostate cancer (1, 2) and the gene responsible for Werner syndrome (WS), a rare autosomal recessive disorder characterized by the premature aging (3-7). Recently, Yu and coworkers cloned a DNA helicase gene as a candidate for WS gene from the 8pl 1-12 region (8). As part of similar efforts to identify the WS gene as well as other disease-related genes, we established a 1.3-megabase physical map spanning the WS region and localized known and novel genes in this interval. These efforts facilitated the physical mapping of the sequence-tagged site and known genes such as those encoding for glutathione S-reductase (GSR), transcription factor IIE ,3 subunit (TFIIEf3), and protein phosphatase 2A f3 subunit (PP2A 4) on chromosome 8pll-12. In addition, analysis by a dual-color fluorescence in situ hybridization analysis enabled us to estimate the precise distances between the genomic markers and genes (9).The WS region was cloned in P1. Exon trapping was used to identify novel gene(s) occurring in this important region. Candidate exons were used in searches of the public data bases and also used to screen cDNA libraries. One novel gene obtained through the processes was of particular interest, as this gene is in close proximity to the closest genetic markers to the WS gene, D8S339 and D8S1055 (10,11). Further interest in this gene was strengthened by its unique features and complex genomic structure.Sequence analysis of cloned cDNAs and genomic DNA showed that this gene, referred to as RBP-MS (RNA-binding protein gene with multiple splicing), produces multiple transcripts by differential splicing and codes for a family of RNA-binding proteins. The RNA-binding motif region shared in all the predicted proteins is highly homologous to that of Drosophila couch potato proteins implicated in the regulation of peripheral nervous system development in the embryo (12). Initial studies in gene cloning and Northern analysis revealed that the transcripts were rather short (1.5 kb) despite the finding that the gene was as long as 230 kb.This paper desc...

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