A Genome-wide RNAi Screen for Modifiers of the Circadian Clock in Human Cells

Zhang, Eric Erquan; Liu, Andrew C.; Hirota, Tsuyoshi; Miraglia, Loren; Welch, Genevieve; Pongsawakul, Pagkapol Y.; Liu, Xianzhong; Atwood, Ann; Huss, Jon W.; Janes, Jeff; Su, Andrew I.; Hogenesch, John B.; Kay, Steve A.

doi:10.1016/j.cell.2009.08.031

Cited by 404 publications

(461 citation statements)

References 44 publications

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“…5D). Especially, it has been reported that Dbp gene expression level directly reflects BMAL1/CLOCK-mediated transcriptional activity via Ebox enhancer elements (29). Therefore, these results suggest dysfunction of the BMAL1/CLOCK-regulated canonical circadian feedback loops in ES cells.…”

Section: Expression Of Clock Genes In Es Cells Ips Cells and Differmentioning

confidence: 71%

Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro

Yagita

Horie²,

Koinuma³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

203

202

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The molecular oscillations underlying the generation of circadian rhythmicity in mammals develop gradually during ontogenesis. However, the developmental process of mammalian cellular circadian oscillator formation remains unknown. In differentiated somatic cells, the transcriptional-translational feedback loops (TTFL) consisting of clock genes elicit the molecular circadian oscillation. Using a bioluminescence imaging system to monitor clock gene expression, we show here that the circadian bioluminescence rhythm is not detected in the mouse embryonic stem (ES) cells, and that the ES cells likely lack TTFL regulation for clock gene expression. The circadian clock oscillation was induced during the differentiation culture of mouse ES cells without maternal factors. In addition, reprogramming of the differentiated cells by expression of Sox2, Klf4, Oct3/4, and c-Myc genes, which were factors to generate induced pluripotent stem (iPS) cells, resulted in the re-disappearance of circadian oscillation. These results demonstrate that an intrinsic program controls the formation of the circadian oscillator during the differentiation process of ES cells in vitro. The cellular differentiation and reprogramming system using cultured ES cells allows us to observe the circadian clock formation process and may help design new strategies to understand the key mechanisms responsible for the organization of the molecular oscillator in mammals.circadian clock | induced pluripotent stem cells | real-time monitor T he circadian rhythm is a fundamental biological system in mammals involved in the regulation of various physiological functions such as the sleep-wake cycle, energy metabolism, and the endocrine system (1, 2). These physiological rhythms develop gradually in the first year of life in humans (3). It is well known that the human sleep-wake rhythm is generated within a few months after birth. However, a weak circadian rhythm of core body temperature is present immediately after birth, suggesting that the development of the human circadian rhythms starts during fetal life. In fact, recent studies in rodents have suggested the appearance of circadian molecular rhythms in the suprachiasmatic nucleus (SCN) a few days before birth (4). However, little information is available on the development of the mammalian cellular circadian oscillator.In mammals, molecular oscillation of the circadian clock consists of interlocked positive and negative transcription/translation feedback loops (TTFL) involving a set of clock genes and clock-controlled output genes that link the oscillator to the clock-controlled processes (5). CLOCK and BMAL1 are basic-helix-loop-helix (bHLH) PAS transcription factors that heterodimerize and transactivate the core clock genes such as Period (Per1, -2, and -3), Cryptochrome (Cry1 and Cry2), and Rev-Erbα (2, 5, 6). PER and CRY proteins suppress the activity of the CLOCK/BMAL1, whereas REV-ERBα suppresses Bmal1 gene expression.In this study, we focused on the development of the mammalian circadian oscillator du...

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Section: Expression Of Clock Genes In Es Cells Ips Cells and Differmentioning

confidence: 71%

Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro

Yagita

Horie²,

Koinuma³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

203

202

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“…S5 D-F), the "master" oscillator in mammals that coordinates circadian rhythms throughout the body (33). To establish whether these genes influence clock function in human U2 OS cells, we used data from the BioGPS portal (34,35). Interestingly, two independent RNAi knockdown experiments of LSM7 generated a long period phenotype similar to that observed when knocking down the CRY2 core clock gene (Fig.…”

Section: A Mutation In Arabidopsis Lsm5 Confers a Long Period Phenotymentioning

confidence: 99%

Role for LSM genes in the regulation of circadian rhythms

Perez-Santángelo

Mancini

Francey

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Growing evidence suggests that core spliceosomal components differentially affect RNA processing of specific genes; however, whether changes in the levels or activities of these factors control specific signaling pathways is largely unknown. Here we show that some SM-like (LSM) genes, which encode core components of the spliceosomal U6 small nuclear ribonucleoprotein complex, regulate circadian rhythms in plants and mammals. We found that the circadian clock regulates the expression of LSM5 in Arabidopsis plants and several LSM genes in mouse suprachiasmatic nucleus. Further, mutations in LSM5 or LSM4 in Arabidopsis, or down-regulation of LSM3, LSM5, or LSM7 expression in human cells, lengthens the circadian period. Although we identified changes in the expression and alternative splicing of some core clock genes in Arabidopsis lsm5 mutants, the precise molecular mechanism causing period lengthening remains to be identified. Genome-wide expression analysis of either a weak lsm5 or a strong lsm4 mutant allele in Arabidopsis revealed larger effects on alternative splicing than on constitutive splicing. Remarkably, large splicing defects were not observed in most of the introns evaluated using RNA-seq in the strong lsm4 mutant allele used in this study. These findings support the idea that some LSM genes play both regulatory and constitutive roles in RNA processing, contributing to the fine-tuning of specific signaling pathways.posttranscriptional | alternative splicing | circadian clock | Arabidopsis | mammals C ircadian rhythms are persistent 24-h oscillations in biological processes that occur under constant environmental conditions. They allow organisms to coordinate multiple physiological processes with periodic or seasonal changes that occur in the environment. At the heart of the eukaryotic circadian system lies a complex set of interconnected transcriptional and translational feedback loops, in which a group of core clock genes regulate each other to ensure that their mRNA levels oscillate with a period of ∼24 h (1). The core oscillator in Arabidopsis thaliana involves two MYB domain-containing transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), that repress the expression of TIMING OF CAB2 EXPRESSION 1 (TOC1) at the beginning of the day. In turn, TOC1, a member of the PSEUDO-RESPONSE REGULATOR (PRR) family, represses CCA1/LHY expression at the end of the day (2). Other clock components expressed throughout the day form multiple interconnected transcriptional feedback loops (2).Mounting evidence indicates that alternative splicing (AS), the process by which pre-mRNA molecules are differentially spliced to yield multiple mRNA isoforms from a single gene, plays a key role in the regulation of circadian networks in a variety of organisms, including Drosophila melanogaster (3), Neurospora crassa (4-6), and Arabidopsis (7-11). For example, the core clock genes period in Drosophila and frequency in Neurospora give rise to different mRNA isoforms through AS, which helps t...

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“…Recently, genome-wide reverse genetic screens using RNAi libraries have identified a large number of possible modifiers of the circadian clock using human cell lines (Maier et al, 2009;Zhang et al, 2009).…”

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confidence: 99%

Rapid Assessment of Gene Function in the Circadian Clock Using Artificial MicroRNA in Arabidopsis Mesophyll Protoplasts

Kim

Somers

2010

Plant Physiology

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Rapid assessment of the effect of reduced levels of gene products is often a bottleneck in determining how to proceed with an interesting gene candidate. Additionally, gene families with closely related members can confound determination of the role of even a single one of the group. We describe here an in vivo method to rapidly determine gene function using transient expression of artificial microRNAs (amiRNAs) in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. We use a luciferasebased reporter of circadian clock activity to optimize and validate this system. Protoplasts transiently cotransfected with promoter-luciferase and gene-specific amiRNA plasmids sustain free-running rhythms of bioluminescence for more than 6 d. Using both amiRNA plasmids available through the Arabidopsis Biological Resource Center, as well as custom design of constructs using the Weigel amiRNA design algorithm, we show that transient knockdown of known clock genes recapitulates the same circadian phenotypes reported in the literature for loss-of-function mutant plants. We additionally show that amiRNA designed to knock down expression of the casein kinase II b-subunit gene family lengthens period, consistent with previous reports of a short period in casein kinase II b-subunit overexpressors. Our results demonstrate that this system can facilitate a much more rapid analysis of gene function by obviating the need to initially establish stably transformed transgenics to assess the phenotype of gene knockdowns. This approach will be useful in a wide range of plant disciplines when an endogenous cell-based phenotype is observable or can be devised, as done here using a luciferase reporter.

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A Genome-wide RNAi Screen for Modifiers of the Circadian Clock in Human Cells

Cited by 404 publications

References 44 publications

Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro

Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro

Role for LSM genes in the regulation of circadian rhythms

Rapid Assessment of Gene Function in the Circadian Clock Using Artificial MicroRNA in Arabidopsis Mesophyll Protoplasts

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