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Hiroki R Ueda

Publications and source records attributed to Hiroki R Ueda.

16 recordsLinked to original sources
PubMed2006

The BMAL1 C terminus regulates the circadian transcription feedback loop.

The circadian clock is driven by cell-autonomous transcription/translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. Here, we present a molecular genetic screening assay for mutant circadian clock proteins that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, we identified a domain in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor CRYPTOCHROME 1 (CRY1), depending on the coexistence of CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression.

ARNTL Transcription Factors↗
PubMed2006

An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.

Animals↗
PubMed2006

Feedback repression is required for mammalian circadian clock function.

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

3T3 Cells↗
PubMed2006

Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Amino Acid Sequence↗
PubMed2005

System-level identification of transcriptional circuits underlying mammalian circadian clocks.

Mammalian circadian clocks consist of complexly integrated regulatory loops, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits. Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E' boxes, DBP/E4BP4 binding elements and RevErbA/ROR binding elements in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E' boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E' boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.

Animals↗
PubMed2004

Molecular-timetable methods for detection of body time and rhythm disorders from single-time-point genome-wide expression profiles.

Detection of individual body time (BT) via a single-time-point assay has been a longstanding unfulfilled dream in medicine, because BT information can be exploited to maximize potency and minimize toxicity during drug administration and thus will enable highly optimized medication. To achieve this dream, we created a "molecular timetable" composed of >100 "time-indicating genes," whose gene expression levels can represent internal BT. Here we describe a robust method called the "molecular-timetable method" for BT detection from a single-time-point expression profile. The power of this method is demonstrated by the sensitive and accurate detection of BT and the sensitive diagnosis of rhythm disorders. These results demonstrate the feasibility of BT detection based on single-time-point sampling, suggest the potential for expression-based diagnosis of rhythm disorders, and may translate functional genomics into chronotherapy and personalized medicine.

Animals↗
PubMed2004

Universality and flexibility in gene expression from bacteria to human.

Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but to explore for underlying governing principles. Recent analysis of the large-scale organization of gene expression has revealed its complex and dynamic nature. However, the underlying dynamics that generate complex gene expression and cellular organization are not yet understood. To comprehensively and quantitatively elucidate these underlying gene expression dynamics, we have analyzed genome-wide gene expression in many experimental conditions in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens. Here we demonstrate that the gene expression dynamics follows the same and surprisingly simple principle from E. coli to human, where gene expression changes are proportional to their expression levels, and show that this "proportional" dynamics or "rich-travel-more" mechanism can regenerate the observed complex and dynamic organization of the transcriptome. These findings provide a universal principle in the regulation of gene expression, show how complex and dynamic organization can emerge from simple underlying dynamics, and demonstrate the flexibility of transcription across a wide range of expression levels.

Animals↗
PubMed2002

The period gene and allochronic reproductive isolation in Bactrocera cucurbitae.

Clock genes that pleiotropically control circadian rhythm and the time of mating may cause allochronic reproductive isolation in the melon fly Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Flies with a shorter circadian period (ca. 22 h of locomotor activity rhythm) mated 5 h earlier in the day than those with a longer circadian period (ca. 30 h). Mate-choice tests demonstrated significant pre-mating isolation between populations with short and long circadian periods. Pre-mating isolation did not occur when the mating time was synchronized between the two populations by photoperiodic controls, indicating that reproductive isolation is due to variations in the time of mating and not any unidentified ethological difference between the two populations. We cloned the period (per) gene of B. cucurbitae that is homologous to the per gene in Drosophila. The relative level of per mRNA in the melon fly exhibited a robust daily fluctuation under light : dark conditions. The fluctuation of per expression under dark : dark conditions is closely correlated to the locomotor rhythm in B. cucurbitae. These results suggest that clock genes can cause reproductive isolation via the pleiotropic effect as a change of mating time.

Animals↗
PubMed2002

A transcription factor response element for gene expression during circadian night.

Mammalian circadian clocks consist of complex integrated feedback loops that cannot be elucidated without comprehensive measurement of system dynamics and determination of network structures. To dissect such a complicated system, we took a systems-biological approach based on genomic, molecular and cell biological techniques. We profiled suprachiasmatic nuclei and liver genome-wide expression patterns under light/dark cycles and constant darkness. We determined transcription start sites of human orthologues for newly identified cycling genes and then performed bioinformatical searches for relationships between time-of-day specific expression and transcription factor response elements around transcription start sites. Here we demonstrate the role of the Rev-ErbA/ROR response element in gene expression during circadian night, which is in phase with Bmal1 and in antiphase to Per2 oscillations. This role was verified using an in vitro validation system, in which cultured fibroblasts transiently transfected with clock-controlled reporter vectors exhibited robust circadian bioluminescence.

Animals↗
PubMed2002

Intercellular coupling mechanism for synchronized and noise-resistant circadian oscillators.

The circadian clock in multicellular organisms consists of multiple autonomous single-cell oscillators. These individual oscillator cells produce coherent oscillations even in the presence of internal noise associated with rhythm-generating reaction rates and in the absence of external time cues such as light and temperature. Thus, an intercellular coupling mechanism must synchronize the cells to induce coherent circadian oscillations. We propose the roles of a synchronizing factor that is secreted from individual cells during subjective day to induce light-pulse-type phase shifts in the neighboring cells or, alternatively, a factor that is secreted during subjective night to induce dark-pulse-type phase shifts. Here, we present our multicellular stochastic model of Drosophila circadian rhythms that emulates the intercellular coupling mechanism and suggest that the mechanism facilitates the constancy of the circadian rhythm with possible functional redundancy among different synchronizing factors.

Animals↗
PubMed2002

Genome-wide transcriptional orchestration of circadian rhythms in Drosophila.

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.

Animals↗
PubMed2002

[A systems-biological approach in drug discovery for circadian rhythm disorders].

A paradigm shift is occurring in all over the biological fields, from the molecular level to the system level. However, systematic and efficient method to dissect the complicated genetic network is still immature. To dissect such a complicated system, we took a systems-biological approach based on genomic, molecular and cell biological techniques and applied to mammalian circadian rhythms, one of the most complicated biological systems. We profiled suprachiasmatic nuclei (SCN) and liver genome-wide expression patterns under light/dark (LD) cycles and constant darkness (DD). We extensively determined transcription start sites (TSS) of human orthologues for newly identified cycling genes and then performed bioinformatical searches for relationships between time-of-day specific expression and transcription factor response elements around TSS. Here we demonstrate the role of the Rev-ErbA/ROR response element in gene expression during circadian night, which is in phase with Bmal1 and antiphase to Per2 oscillations. This role was verified using a newly developed in vitro validation system, in which cultured fibroblasts transiently transfected with clock-controlled reporter vectors exhibited robust circadian bioluminescence. The systems-biological approach used here enhances accurate and comprehensive measurement of circadian dynamics, accelerates extensive identification of clockwork circuits, and leads to systems-level understanding of mammalian circadian clocks.

ARNTL Transcription Factors↗