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Biomedical subjects

Shinji Kondo

Publications and source records attributed to Shinji Kondo.

At least 19 recordsLinked to original sources
PubMed2006

Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6).

Transcription factor paralogs may share a common role in staged or overlapping expression in specific tissues, as in the Hox family. In other cases, family members have distinct roles in a range of embryologic, differentiation or response pathways (as in the Tbx and Pax families). For the interferon regulatory factor (IRF) family of transcription factors, mice deficient in Irf1, Irf2, Irf3, Irf4, Irf5, Irf7, Irf8 or Irf9 have defects in the immune response but show no embryologic abnormalities. Mice deficient for Irf6 have not been reported, but in humans, mutations in IRF6 cause two mendelian orofacial clefting syndromes, and genetic variation in IRF6 confers risk for isolated cleft lip and palate. Here we report that mice deficient for Irf6 have abnormal skin, limb and craniofacial development. Histological and gene expression analyses indicate that the primary defect is in keratinocyte differentiation and proliferation. This study describes a new role for an IRF family member in epidermal development.

Animals↗
PubMed2006

A RecA-mediated exon profiling method.

We have developed a RecA-mediated simple, rapid and scalable method for identifying novel alternatively spliced full-length cDNA candidates. This method is based on the principle that RecA proteins allow to carry radioisotope-labeled probe DNAs to their homologous sequences, resulting in forming triplexes. The resulting complex is easily detected by mobility difference on electrophoresis. We applied this exon profiling method to four selected mouse genes as a feasibility study. To design probes for detection, the information on known exonic regions was extracted from public database, RefSeq. Concerning the potentially transcribed novel exonic regions, RNA mapping experiment using Affymetrix tiling array was performed. As a result, we were able to identify alternative splice variants of Thioredoxin domain containing 5, Interleukin1beta, Interleukin 1 family 6 and glutamine-rich hypothetical protein. In addition, full-length sequencing demonstrated that our method could profile exon structures with >90% accuracy. This reliable method can allow us to screen novel splice variants from a huge number of cDNA clone set effectively.

Alternative Splicing↗
PubMed2006

Histone H3 acetylated at lysine 9 in promoter is associated with low nucleosome density in the vicinity of transcription start site in human cell.

Nucleosome depletion in the promoters has been indicated in yeasts, suggesting that nucleosome depletion in promoter might be a fundamental feature of eukaryotic transcriptional regulation. We compared the relationship between histone H3 acetylation at lysine 9 (K9) in promoter, gene expression level, and nucleosome density in the vicinity of the transcription start site (TSS), in HepG2 cells (human hepatocellular liver carcinoma cells). We found that the density of nucleosome is relatively low in the close vicinity of TSS flanked by H3 K9 significantly acetylated promoter, compared with that for genes without marked H3 K9 acetylation in promoter, regardless of their transcriptional activation status. Our results imply that the relative nucleosome depletion in the vicinity of TSS is not necessarily associated with active transcription, but with histone H3 K9 acetylation in promoter.

Acetylation↗
PubMed2006

Expression of the Snurf-Snrpn IC transcript in the oocyte and its putative role in the imprinting establishment of the mouse 7C imprinting domain.

The human chromosome 15q11-q13, or mouse chromosome 7C, is an imprinting domain controlled by bipartite imprinting centers (ICs): Prader-Willi syndrome (PWS)-IC and Angelman syndrome (AS)-IC. PWS-IC functions to maintain the paternal epigenotype on the paternal chromosome in somatic cells, while AS-IC plays a role in the establishment of the maternal epigenetic mark at PWS-IC in the female germline or early embryos. Several alternative exons and promoters of Snurf-Snrpn (SNRPN upstream reading frame-small nuclear ribonucleoprotein polypeptide N) are expressed as "IC transcripts". Previous studies have shown that IC-transcript expression is restricted to the brain. We studied expression of the mouse IC-transcript in tissues including brain and oocytes as well as in cultured neurons and glia cells by RT-PCR and by in situ hybridization (ISH) in oocytes. The IC transcript was strongly expressed in brain (especially in neurons) and ovary (especially in oocytes and granulosa cells), while no expression was found in other tissues. This was confirmed by quantitative analysis and ISH. Expression levels in the brain were 7-fold higher compared to those in ovaries. ISH signals were observed in oocytes and granulosa cells of the secondary and developing follicles. These findings, together with previous data, suggest that the IC transcript may be associated with the establishment of PWS-IC methylation on the maternal chromosome as an AS-IC cis-acting element.

Alternative Splicing↗
PubMed2005

PAX9 and TGFB3 are linked to susceptibility to nonsyndromic cleft lip with or without cleft palate in the Japanese: population-based and family-based candidate gene analyses.

The prevalence of nonsyndromic cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO) are believed to be higher in the Japanese than in Americans, Europeans or Africans. The purpose of this study was to investigate, in a Japanese population, relationships between CL/P or CPO and seven candidate genes (TGFB3, DLX3, PAX9, CLPTM1, TBX10, PVRL1, TBX22) that showed positive associations in other populations and are expressed in the oral/lip region in developing mice. We first searched for mutations in these genes among 112 CL/P and 16 CPO patients, and found a heterozygous missense mutation (640A > G, S214G) in exon 3 of PAX9 in two sibs with CL/P and their phenotypically normal mother from a Japanese family. A population-based case-control analysis and a family-based transmission disequilibrium test (TDT), using single nucleotide polymorphisms (SNPs), and two-SNP haplotypes of the genes, between the 112 CL/P cases with their parents and 192 controls indicated a significant association at one SNP site, IVS1 + 5321, in TGFB3 with a P-value of 0.0016. Population-based haplotyping revealed that the association was most significant for haplotype "A/A" consisting of IVS1 + 5321 and IVS1 - 1572; TDT also gave a P-value of 0.0252 in this haplotype.

Amino Acid Sequence↗
PubMed2005

Mammary serine protease inhibitor (Maspin) binds directly to interferon regulatory factor 6: identification of a novel serpin partnership.

Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation.

Amino Acid Sequence↗
PubMed2004

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.

It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

Alternative Splicing↗
PubMed2004

Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.

BACKGROUND: Cleft lip or palate (or the two in combination) is a common birth defect that results from a mixture of genetic and environmental factors. We searched for a specific genetic factor contributing to this complex trait by examining large numbers of affected patients and families and evaluating a specific candidate gene. METHODS: We identified the gene that encodes interferon regulatory factor 6 (IRF6) as a candidate gene on the basis of its involvement in an autosomal dominant form of cleft lip and palate, Van der Woude's syndrome. A single-nucleotide polymorphism in this gene results in either a valine or an isoleucine at amino acid position 274 (V274I). We carried out transmission-disequilibrium testing for V274I in 8003 individual subjects in 1968 families derived from 10 populations with ancestry in Asia, Europe, and South America, haplotype and linkage analyses, and case-control analyses, and determined the risk of cleft lip or palate that is associated with genetic variation in IRF6. RESULTS: Strong evidence of overtransmission of the valine (V) allele was found in the entire population data set (P<10(-9)); moreover, the results for some individual populations from South America and Asia were highly significant. Variation at IRF6 was responsible for 12 percent of the genetic contribution to cleft lip or palate and tripled the risk of recurrence in families that had already had one affected child. CONCLUSIONS: DNA-sequence variants associated with IRF6 are major contributors to cleft lip, with or without cleft palate. The contribution of variants in single genes to cleft lip or palate is an important consideration in genetic counseling.

Cleft Lip↗
PubMed2003

Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage.

We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5' end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The high-throughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.

Animals↗
PubMed2003

Novel IRF6 mutations in Japanese patients with Van der Woude syndrome: two missense mutations (R45Q and P396S) and a 17-kb deletion.

Three Japanese families with Van der Woude syndrome (VWS) were screened for mutations in the interferon regulatory factor 6 gene (IRF6) by sequencing its entire coding region. Two novel missense mutations, R45Q in exon 3 and P396S in exon 9, were identified in families 1 and 2, respectively. In family 3, no causative base change was found by the sequencing analysis, but a deletion involving exons 4-9 was suggested by multiplex PCR analysis. To confirm the deletion and to determine its 5'- and 3'-boundaries, we amplified a DNA fragment containing a heterozygous polymorphic site in exon 2 by using a 5'-upstream forward PCR primer and eight different reverse primers located 3'-downstream of exon 2. The amplified product was subjected to nested PCR to generate a DNA fragment containing the polymorphic site. When a reverse primer located within the deletion was used for the first PCR amplification, only the nondeletion allele was detected after the second PCR. Repeated analyses with eight different reverse primers allowed us to map the boundaries of the deletion, and subsequently a heterozygous 17,162-bp deletion involving exons 4-9 was identified. Since IRF6 mutations in a significant portion of VWS patients remain undetected by conventional sequencing analysis, it may be important to search for a large deletion in those patients. Our simple methods to identify deletions and to determine the boundaries of a deletion would facilitate the identification of such patients.

Cleft Lip↗
PubMed2003

A comprehensive search for HNF-3alpha-regulated genes in mouse hepatoma cells by 60K cDNA microarray and chromatin immunoprecipitation/PCR analysis.

To characterize the regulatory pattern by a specific transcription regulatory factor, we used a combination of expression analysis with the mouse cDNA microarray composed of 60,000 cDNA clones and cross-linking/chromatin immunoprecipitation (X-ChIP) followed by comparative PCR. Overexpression of mouse hepatocyte nuclear factor-3alpha (HNF-3alpha) in a mouse hepatoma cell line resulted in accompanied perturbed expression of more than 1500 genes. Search for HNF-3alpha consensus recognition sequences in the upstream regions of their coding sequences, which were mapped on the mouse genome, enabled us to mine 300 genes as the potential HNF-3alpha-regulated genes and classify 135 annotated ones into several functional categories. Further X-ChIP/PCR analysis demonstrated in vivo binding of HNF-3alpha to the 5(')-flanking sequences of 25 members selected out of these genes. Besides known HNF-3alpha-regulated genes such as albumin and alpha-fetoprotein genes, the genes newly identified as the HNF-3alpha-regulated ones include three encoding CDP-diacylglycerol-inositol 3-phosphatidyltransferase, phosphatidylserine decarboxylase, and phospholipase A2, which are located en suite in the lipid metabolic pathway in liver. The potential usefulness of the present approach to extensive characterization of gene expression framework directed by a specific transcription regulatory factor is discussed.

5' Flanking Region↗
PubMed2003

Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice.

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Alternative Splicing↗
PubMed2003

Transneuronal degeneration in patients with temporal lobe epilepsy: evaluation by MR imaging.

The aim of this study was to assess the MR imaging findings of transneuronal degeneration of limbic system in the patients with temporal lobe epilepsy, and to detect the influence of surgery on the anatomy of the limbic system. Axial and coronal T1- and T2-weighted MR images were retrospectively analyzed in 34 patients with temporal lobe epilepsy, focusing on transneuronal degeneration. In 17 of the 34 patients, MR images were also analyzed after selective amygdalo-hippocampectomy. Atrophy of the fornix, mamillary body, mamillothalamic tract (MTT), and thalamus ipsilateral to the epileptic focus was demonstrated on MR images in 14.7, 17.6, 8.8, and 11.8% of the 34 patients, respectively. Focal hyperintensity of the thalamus was found on T2-weighted images in 8.8% of the 34 patients. In 17 patients who were evaluated before and after surgery, transneuronal degeneration was seen more frequently after surgery: fornix (11.8 vs 29.4%), mamillary body (11.8 vs 52.9%), MTT (5.9 vs 11.8%), and thalamus (11.8 vs 11.8%). Transneuronal degeneration of the limbic system is clearly demonstrated by MR imaging in patients with temporal lobe epilepsy, and surgical intervention induces transneuronal degeneration more frequently.

Adolescent↗
PubMed2003

Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection.

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products-has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

Animals↗
PubMed2003

Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome.

We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleotide distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs.

Alternative Splicing↗
PubMed2003

Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.

We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Animals↗
PubMed2003

Antisense transcripts with FANTOM2 clone set and their implications for gene regulation.

We have used the FANTOM2 mouse cDNA set (60,770 clones), public mRNA data, and mouse genome sequence data to identify 2481 pairs of sense-antisense transcripts and 899 further pairs of nonantisense bidirectional transcription based upon genomic mapping. The analysis greatly expands the number of known examples of sense-antisense transcript and nonantisense bidirectional transcription pairs in mammals. The FANTOM2 cDNA set appears to contain substantially large numbers of noncoding transcripts suitable for antisense transcript analysis. The average proportion of loci encoding sense-antisense transcript and nonantisense bidirectional transcription pairs on autosomes was 15.1 and 5.4%, respectively. Those on the X chromosome were 6.3 and 4.2%, respectively. Sense-antisense transcript pairs, rather than nonantisense bidirectional transcription pairs, may be less prevalent on the X chromosome, possibly due to X chromosome inactivation. Sense and antisense transcripts tended to be isolated from the same libraries, where nonantisense bidirectional transcription pairs were not apparently coregulated. The existence of large numbers of natural antisense transcripts implies that the regulation of gene expression by antisense transcripts is more common that previously recognized. The viewer showing mapping patterns of sense-antisense transcript pairs and nonantisense bidirectional transcription pairs on the genome and other related statistical data is available on our Web site.

Animals↗