Yasufumi Shigeyoshi

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.

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

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.

Acute induction of Eya3 by late-night light stimulation triggers TSHβ expression in photoperiodism.

Living organisms detect seasonal changes in day length (photoperiod) [1-3] and alter their physiological functions accordingly to fit seasonal environmental changes. TSHβ, induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism [4, 5]. However, the upstream inducers of TSHβ expression remain unknown. Here we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSHβ expression response is preserved [6]. This analysis identified short-day and long-day genes, including TSHβ, and further predicted the acute induction of long-day genes by late-night light stimulation. We verified this by advancing and extending the light period by 8xa0hr, which induced TSHβ expression within one day. In the following genome-wide expression analysis under this acute long-day condition, we searched for candidate upstream genes by looking for expression that preceded TSHβs, and we identified the Eya3 gene. We demonstrated that Eya3 and its partner Six1 synergistically activate TSHβ expression and that this activation is further enhanced by Tef and Hlf. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of TSHβ expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system.

Analysis and synthesis of high-amplitude Cis-elements in the mammalian circadian clock

Mammalian circadian clocks consist of regulatory loops mediated by Clock/Bmal1-binding elements, DBP/E4BP4 binding elements, and RevErbA/ROR binding elements. As a step toward system-level understanding of the dynamic transcriptional regulation of the oscillator, we constructed and used a mammalian promoter/enhancer database (http://promoter.cdb.riken.jp/) with computational models of the Clock/Bmal1-binding elements, DBP/E4BP4 binding elements, and RevErbA/ROR binding elements to predict new targets of the clock and subsequently validated these targets at the level of the cell and organism. We further demonstrated the predictive nature of these models by generating and testing synthetic regulatory elements that do not occur in nature and showed that these elements produced high-amplitude circadian gene regulation. Biochemical experiments to characterize these synthetic elements revealed the importance of the affinity balance between transactivators and transrepressors in generating high-amplitude circadian transcriptional output. These results highlight the power of comparative genomics approaches for system-level identification and knowledge-based design of dynamic regulatory circuits.

Differential entrainment of peripheral clocks in the rat by glucocorticoid and feeding.

The suprachiasmatic nucleus is the master circadian clock and resets the peripheral clocks via various pathways. Glucocorticoids and daily feeding are major time cues for entraining most peripheral clocks. However, recent studies have suggested that the dominant timing factor differs among organs and tissues. In our current study, we reveal differences in the entrainment properties of the peripheral clocks in the liver, kidney, and lung through restricted feeding (RF) and antiphasic corticosterone (CORT) injections in adrenalectomized rats. The peripheral clocks in the kidney and lung were found to be entrained by a daily stimulus from CORT administration, irrespective of the meal time. In contrast, the liver clock was observed to be entrained by an RF regimen, even if daily CORT injections were given at antiphase. These results indicate that glucocorticoids are a strong zeitgeber that overcomes other entrainment factors regulating the peripheral oscillators in the kidney and lung and that RF is a dominant mediator of the entrainment ability of the circadian clock in the liver.

Quantitative Expression Profile of Distinct Functional Regions in the Adult Mouse Brain

The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ∼50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems.

Variations in PROKR2, But Not PROK2, Are Associated With Hypopituitarism and Septo-optic Dysplasia

Context: Loss-of-function mutations in PROK2 and PROKR2 have been implicated in Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia. Recent data suggest overlapping phenotypes/genotypes between KS and congenital hypopituitarism (CH), including septo-optic dysplasia (SOD). Objective: We screened a cohort of patients with complex forms of CH (n = 422) for mutations in PROK2 and PROKR2. Results: We detected 5 PROKR2 variants in 11 patients with SOD/CH: novel p.G371R and previously reported p.A51T, p.R85L, p.L173R, and p.R268C—the latter 3 being known functionally deleterious variants. Surprisingly, 1 patient with SOD was heterozygous for the p.L173R variant, whereas his phenotypically unaffected mother was homozygous for the variant. We sought to clarify the role of PROKR2 in hypothalamopituitary development through analysis of Prokr2−/− mice. Interestingly, these revealed predominantly normal hypothalamopituitary development and terminal cell differentiation, with the exception of reduced LH; this was inconsistent with patient phenotypes and more analogous to the healthy mother, although she did not have KS, unlike the Prokr2−/− mice. Conclusions: The role of PROKR2 in the etiology of CH, SOD, and KS is uncertain, as demonstrated by no clear phenotype-genotype correlation; loss-of-function variants in heterozygosity or homozygosity can be associated with these disorders. However, we report a phenotypically normal parent, homozygous for p.L173R. Our data suggest that the variants identified herein are unlikely to be implicated in isolation in these disorders; other genetic or environmental modifiers may also impact on the etiology. Given the phenotypic variability, genetic counseling may presently be inappropriate.

Alterations of circadian clockworks during differentiation and apoptosis of rat ovarian cells

Ovarian development is related to cell proliferation, differentiation, and apoptosis of granulosa cells and luteal cells under the control of various modulators, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth factors. In the present study, the expression of clock genes and the related regulation mechanism were analyzed in different ovarian cell types during differentiation and apoptosis. The authors focused on the circadian expression of Per2 as a core clock gene for the maintenance of circadian rhythms. By using a real-time monitoring system of the Per2 promoter activity, the circadian oscillation was analyzed in the granulosa and luteal cells from preantral follicles, antral follicles, and corpora lutea of immature Per2 promoter–destabilized luciferase transgenic rats that were primed with diethylstilbestrol, equine chorionic gonadotropin (eCG), and/or human CG. In addition, transcript levels of Per2, Bmal1, Clock, and Nampt were quantified by quantitative polymerase chain reaction (qPCR). Immunohistochemical studies revealed strong circadian rhythmicity of PER2 protein in the luteal cells, but apparently little rhythmicity in granulosa cells of both preantral and antral follicles. In vitro monitoring of promoter activity showed generation of several oscillations in luteal cells after exposure to dexamethasone (DXM), whereas oscillatory amplitudes of immature and mature granulosa cells were rapidly attenuating. The circadian rhythm of the Bmal1 transcript levels, but not the Per2 transcript, was very weak in the granulosa cells, as compared with that in luteal cells. Granulosa cells gained a strong circadian rhythm ability of the Per2 promoter activity after stimulation with FSH for 3 days. In contrast, LH had little effect on the circadian rhythm before stimulation of granulosa cells with FSH, probably owing to lack of LH receptor. In luteal cells, induction of apoptosis by inhibiting progesterone synthesis resulted in deregulation of Per2 circadian oscillation. Transcript levels of Bmal1 and Clock, but not Per2 and Nampt, were significantly decreased in apoptotic luteal cells. The Bmal1 transcript level was particularly reduced. Consequently, these results strongly suggest the circadian clockwork alters in ovarian cells during follicular development, luteinization, and apoptosis, and expression of Bmal1 may be related to the switch-on and switch-off of the circadian oscillation. (Author correspondence: mhattori@agr.kyushu-u.ac.jp)

Distinct localization of prokineticin 2 and prokineticin receptor 2 mRNAs in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the master circadian clock that regulates physiological and behavioral circadian rhythms in mammals. Prokineticin 2 (PK2) is highly expressed in the SCN, and its involvement in the generation of circadian locomotor activity has been reported previously. In the present study, using in situ hybridization methods, we investigated the localization of PK2 and prokineticin receptor 2 (PKR2), a specific receptor for PK2, in the rat SCN. In steady lightu2003:u2003dark (Lu2003:u2003Du2003=u200312u2003:u200312u2003h) and constant dark conditions, rPK2 mRNA displayed a robust circadian oscillation with a peak occurring during the day. Moreover, during peak expression, the rPK2 mRNA‐positive neurons were scattered in both the dorsomedial and ventrolateral SCN, which are two functionally and morphologically distinct subregions. Furthermore, double‐labeling in situ hybridization experiments revealed that greater than 50% of the rPK2 mRNA‐containing neurons co‐expressed either vasoactive intestinal peptide (VIP), gastrin‐releasing peptide (GRP) or arginine vasopressin (AVP) in the SCN. In contrast, the rPKR2 mRNA levels did not show significant diurnal alterations. rPKR2 mRNA‐containing neurons were also clustered in the dorsolateral part of the SCN, which shows negligible labeling of either rAVP, rVIP, rGRP or rPK2 transcripts. In addition, this region exhibited a delayed cycling of the rPer1 gene. These results suggest an intrinsic PK2 neurotransmission and functionally distinct roles for PKR2‐expressing neurons in the SCN.

Downregulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells

Ovarian circadian oscillators have been implicated in the reproductive processes of mammals. However, there are few reports regarding the detection of ovarian clock-controlled genes (CCGs). The present study was designed to unravel the mechanisms through which CCG ovarian circadian oscillators regulate fertility, primarily using quantitative RT-PCR and RNA interference against Bmal1 in rat granulosa cells. Mature granulosa cells were prepared from mouse Per2-destabilized luciferase (dLuc) reporter gene transgenic rats. A real-time monitoring system of Per2 promoter activity was employed to detect Per2-dLuc oscillations. The cells exposed to luteinizing hormone (LH) displayed clear Per2-dLuc oscillations and a rhythmic expression of clock genes (Bmal1, Per1, Per2, Rev-erbα, and Dbp). Meanwhile, the examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Lhcgr, and p53) showed rhythmic transcript profiles except for Hsd3b2, indicating that these rhythmic expression genes may be CCGs. Notably, Bmal1 small interfering (si)RNA treatment significantly decreased both the amplitude of Per2-dLuc oscillations and Bmal1 mRNA levels compared with nonsilencing RNA treatment in luteinizing granulosa cells. Depletion of Bmal1 by siRNA decreased the transcript levels of clock genes (Per1, Per2, Rev-erbα, and Dbp) and examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Hsd3b2, and Lhcgr). Accordingly, knockdown of Bmal1 also inhibited the synthesis of progesterone and prostaglandin E2, which are associated with crucial reproductive processes. Collectively, these data suggest that ovarian circadian oscillators regulate the synthesis of steroid hormones and prostaglandins through ovarian-specific CCGs in response to LH stimuli. The present study provides new insights into the physiologic significance of Bmal1 related to fertility in ovarian circadian oscillators.