Mari Nakamura

Genome-wide analysis of mammalian promoter architecture and evolution

Mammalian promoters can be separated into two classes, conserved TATA box–enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3′ UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

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.

Tiny RNAs associated with transcription start sites in animals

It has been reported that relatively short RNAs of heterogeneous sizes are derived from sequences near the promoters of eukaryotic genes. As part of the FANTOM4 project, we have identified tiny RNAs with a modal length of 18 nt that map within −60 to +120 nt of transcription start sites (TSSs) in human, chicken and Drosophila. These transcription initiation RNAs (tiRNAs) are derived from sequences on the same strand as the TSS and are preferentially associated with G+C-rich promoters. The 5′ ends of tiRNAs show peak density 10–30 nt downstream of TSSs, indicating that they are processed. tiRNAs are generally, although not exclusively, associated with highly expressed transcripts and sites of RNA polymerase II binding. We suggest that tiRNAs may be a general feature of transcription in metazoa and possibly all eukaryotes.

CAGE: cap analysis of gene expression.

1Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), Yokohama Institute 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. 2 Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa Wako, Saitama, 351-0198, Japan. 3 K.K. Dnaform, Tsukuba Branch, 3-1 Chuo 8-chome, Ami Machi, Inashiki Gun, Ibaraki, 300-0332, Japan. 4Present address: Vaxine Pty Ltd., Department of Diabetes and Endocrinology, Flinders Medical Centre, Bedford Park, Southern Australia 5042, Australia. Correspondence should be addressed to Y.H. (rgscerg@gsc.riken.jp), P.C. (rgscerg@gsc.riken.jp) or M.H. (matthias. harbers@dnaforminc.com).

Hidden layers of human small RNAs

BackgroundSmall RNA attracts increasing interest based on the discovery of RNA silencing and the rapid progress of our understanding of these phenomena. Although recent studies suggest the possible existence of yet undiscovered types of small RNAs in higher organisms, many studies to profile small RNA have focused on miRNA and/or siRNA rather than on the exploration of additional classes of RNAs.ResultsHere, we explored human small RNAs by unbiased sequencing of RNAs with sizes of 19–40 nt. We provide substantial evidences for the existence of independent classes of small RNAs. Our data shows that well-characterized non-coding RNA, such as tRNA, snoRNA, and snRNA are cleaved at sites specific to the class of ncRNA. In particular, tRNA cleavage is regulated depending on tRNA type and tissue expression. We also found small RNAs mapped to genomic regions that are transcribed in both directions by bidirectional promoters, indicating that the small RNAs are a product of dsRNA formation and their subsequent cleavage. Their partial similarity with ribosomal RNAs (rRNAs) suggests unrevealed functions of ribosomal DNA or interstitial rRNA. Further examination revealed six novel miRNAs.ConclusionOur results underscore the complexity of the small RNA world and the biogenesis of small RNAs.

Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.

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.

Growth Phase- and Nutrient Limitation-Associated Transcript Abundance Regulation in Bordetella pertussis

ABSTRACT To survive in a host environment, microbial pathogens must sense local conditions, including nutrient availability, and adjust their growth state and virulence functions accordingly. No comprehensive investigation of growth phase-related gene regulation in Bordetella pertussis has been reported previously. We characterized changes in genome-wide transcript abundance of B. pertussis as a function of growth phase and availability of glutamate, a key nutrient for this organism. Using a Bordetella DNA microarray, we discovered significant changes in transcript abundance for 861 array elements during the transition from log phase to stationary phase, including declining transcript levels of many virulence factor genes. The responses to glutamate depletion exhibited similarities to the responses induced by exit from log phase, including decreased virulence factor transcript levels. However, only 23% of array elements that showed at least a fourfold growth phase-associated difference in transcript abundance also exhibited glutamate depletion-associated changes, suggesting that nutrient limitation may be one of several interacting factors affecting gene regulation during stationary phase. Transcript abundance patterns of a Bvg+ phase-locked mutant revealed that the BvgAS two-component regulatory system is a key determinant of growth phase- and nutrient limitation-related transcriptional control. Several adhesin genes exhibited lower transcript abundance during stationary phase and under glutamate restriction conditions. The predicted bacterial phenotype was confirmed: adherence to bronchoepithelial cells decreased 3.3- and 4.4-fold at stationary phase and with glutamate deprivation, respectively. Growth phase and nutrient availability may serve as cues by which B. pertussis regulates virulence according to the stage of infection or the location within the human airway.

Electronic Health Record Adoption by Children's Hospitals in the United States

OBJECTIVE To assess adoption of electronic health records (EHRs) and clinical functionalities, involvement in health information exchange, and barriers to and facilitators of adoption among childrens hospitals in the United States. DESIGN Survey presented as an information technology supplement to the American Hospital Associations annual member survey. SETTING General acute care childrens hospitals in 2008, identified using the membership directory of the National Association of Childrens Hospitals and Related Institutions. PARTICIPANTS Chief information officers or equivalent hospital leaders. MAIN EXPOSURES Potential barriers to or facilitators of EHR adoption. MAIN OUTCOME MEASURES Rates of EHR adoption, determined using expert-formulated definitions based on presence of essential functionalities, and rates of implementation for individual functionalities and participation in health information exchange. RESULTS Of 155 childrens hospitals, 108 (69.7%) responded to the survey. Only 2.8% had a comprehensive EHR, whereas an additional 17.9% had a basic system. Adoption of individual functionalities varied widely; comprehensive implementations of computerized provider order entry for medications and many forms of decision support were reported by fewer than half. In all, 15.7% of hospitals exchanged health information electronically. Hospital characteristics were not associated with EHR adoption or participation in health information exchange. Hospitals identified financing as the most important target for policy strategies. CONCLUSIONS Most childrens hospitals lack the minimum functionalities needed for a basic EHR. Ensuring access to adequate financial resources will be critical for inclusion of childrens hospitals in efforts to expand EHR use.

Influenza Vaccination in Adolescents With High-Risk Conditions

OBJECTIVES. We assessed influenza vaccination rates from 1992 to 2002, individual continuity of vaccination, and missed opportunities for vaccination in adolescents with high-risk conditions. METHODS. We performed a retrospective observational study of 18 703 adolescents with high-risk conditions who were enrolled in a large health maintenance organization and received care at a multisite practice for ≥1 influenza season and the preceding year, between 1992 and 2002, was performed. Subjects were identified as having a high-risk condition if they had ≥1 visit with an associated International Classification of Diseases, Ninth Revision, Clinical Modification code during the season or previous year. Influenza vaccination rates were compared by season in logistic regression analyses, using generalized estimating equations for repeated measurements of subjects enrolled for multiple seasons. Vaccination continuity was measured for adolescents who were enrolled for 4 consecutive seasons (1999–2002) as the number of seasons during which vaccine was received. Missed opportunities were defined as visits during the first 4 months of influenza season at which an unvaccinated adolescent did not receive vaccine. RESULTS. For adolescents with high-risk conditions, influenza vaccination rates varied from 8.3% to 15.4%. Rates improved significantly from 1992 to 1993, from 8.3% to 12.8%, and again in 2001, reaching 15.4%. Only 11.1% of those enrolled continuously from 1999 to 2002 received vaccine during all 4 seasons. According to season from 1992 to 2002, 45.7% to 53.6% of unvaccinated subjects had ≥1 missed opportunity. CONCLUSIONS. Influenza vaccination rates in adolescents with high-risk conditions improved from 1992 to 2002 but were still low in recent years. Individual vaccination continuity was poor. Numerous opportunities already exist for improving coverage.