Getting rhythm: how do babies do it?

OBJECTIVES: To investigate the emergence of biological rhythms in the first months of life in human infants, by measuring age-related changes in core body temperature during night-time sleep, hormones (cortisol and 6-sulfatoxymelatonin) and the expression of a clock-controlled gene H3f3b in oral epithelial cells. DESIGN: Observational longitudinal study. SETTING: We measured overnight core body temperature, actigraphy, day-night urinary cortisol and 6-sulfatoxymelatonin, as well as circadian gene expression, in infants at home from March 2007 to July 2008 in Leicester. PARTICIPANTS: We recruited 35 healthy Caucasian infants who were born at term. They were monitored from 6 to 18 weeks of age. RESULTS: At 8 weeks of age the day-night rhythm of cortisol secretion was the first to appear followed by 6-sulfatoxymelatonin 1 week later; at the same time that night-time sleep was established. At 10 weeks, the maximum fall in deep body temperature occurred with the onset of night-time sleep, followed at 11 weeks by the rhythmical expression of the H3f3b gene. CONCLUSIONS: In human infants, there is a clear sequential pattern for the emergence of diurnal biological rhythms between 6 and 18 weeks of postnatal age, led by the secretion of cortisol and linked with the establishment of consolidated night-time sleep. It is likely that this represents part of a maturation and adaption process as infants gain equilibrium with their external environment after birth.

STARS is essential to maintain cardiac development and function in vivo via a SRF pathway.

BACKGROUND: STARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein expressed early in cardiac development that acts as an acute stress sensor for pathological remodeling. However the role of STARS in cardiac development and function is incompletely understood. Here, we investigated the role of STARS in heart development and function in the zebrafish model and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: Expression of zebrafish STARS (zSTARS) first occurs in the somites by the 16 somite stage [17 hours post fertilization (hpf)]. zSTARS is expressed in both chambers of the heart by 48 hpf, and also in the developing brain, jaw structures and pectoral fins. Morpholino-induced knockdown of zSTARS alters atrial and ventricular dimensions and decreases ventricular fractional shortening (measured by high-speed video microscopy), with pericardial edema and decreased or absent circulation [abnormal cardiac phenotypes in 126/164 (77%) of morpholino-injected embryos vs. 0/152 (0%) of control morpholino embryos]. Co-injection of zsrf (serum response factor) mRNA rescues the cardiac phenotype of zSTARS knockdown, resulting in improved fractional shortening and ventricular end-diastolic dimensions. Ectopic over-expression of STARS in vitro activates the STARS proximal promoter, which contains a conserved SRF site. Chromatin immunoprecipitation demonstrates that SRF binds to this site in vivo and the SRF inhibitor CCG-1423 completely blocks STARS proximal reporter activity in H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study demonstrates for the first time that STARS deficiency severely disrupts cardiac development and function in vivo and revealed a novel STARS-SRF feed-forward autoregulatory loop that could play an essential role in STARS regulation and cardiac function.

Cardiac expression of ms1/STARS, a novel gene involved in cardiac development and disease, is regulated by GATA4.

Ms1/STARS is a novel muscle-specific actin-binding protein that specifically modulates the myocardin-related transcription factor (MRTF)-serum response factor (SRF) regulatory axis within striated muscle. This ms1/STARS-dependent regulatory axis is of central importance within the cardiac gene regulatory network and has been implicated in cardiac development and postnatal cardiac function/homeostasis. The dysregulation of ms1/STARS is associated with and causative of pathological cardiac phenotypes, including cardiac hypertrophy and cardiomyopathy. In order to gain an understanding of the mechanisms governing ms1/STARS expression in the heart, we have coupled a comparative genomic in silico analysis with reporter, gain-of-function, and loss-of-function approaches. Through this integrated analysis, we have identified three evolutionarily conserved regions (ECRs), α, SINA, and DINA, that act as cis-regulatory modules and confer differential cardiac cell-specific activity. Two of these ECRs, α and DINA, displayed distinct regulatory sensitivity to the core cardiac transcription factor GATA4. Overall, our results demonstrate that within embryonic, neonatal, and adult hearts, GATA4 represses ms1/STARS expression with the pathologically associated depletion of GATA4 (type 1/type 2 diabetic models), resulting in ms1/STARS upregulation. This GATA4-dependent repression of ms1/STARS expression has major implications for MRTF-SRF signaling in the context of cardiac development and disease.

Melatonin synthesis in retina: cAMP-dependent transcriptional regulation of chicken arylalkylamine N-acetyltransferase by a CRE-like sequence and a TTATT repeat motif in the proximal promoter.

Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4 kb of 5'-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the ∼4 kb promoter construct and by all 5'-deletion constructs except the smallest, Aanat (-217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (-484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT8) and a CRE-like sequence. Electrophoretic mobility shift assays, luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CRE-like sequence and TTATT8 elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat.

Myocyte stress 1 plays an important role in cellular hypertrophy and protection against apoptosis.

Myocyte stress 1 (MS1) is a recently described striated muscle actin-binding protein that is up-regulated in the early stages of pressure overload left ventricular hypertrophy. The aim of this study was to determine whether MS1 induces cellular hypertrophy and protects against apoptosis. Over-expressed MS1 co-localized with actin in H9c2 cells and altered expression of genes of the myocardin-related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathways and in addition the apoptosis repressor with caspase recruitment domain (Nol3) gene. The size of cells over-expressing MS1 was significantly increased by 55% and over-expression of MS1 dramatically inhibited staurosporine-induced apoptosis by 89%. These findings suggest the involvement of MS1 in cellular hypertrophy and protection against apoptosis.

The canonical E-box motif: a target for glucocorticoid action that drives rhythmic mouse Pai-1 transcription in vitro.

Circadian (approximately 24 h) control impinges on an array of diverse physiological processes in many organisms, ranging from plants to human. Disruption of the mammalian circadian clockwork can lead to severe chronic illnesses such as cardiovascular disease, cancer progression and metabolic disorders. Transcriptional regulation of plasminogen activator inhibitor 1 (PAI-1) is of particular importance because of its crucial role in these pathological conditions. Pai-1 expression is partly regulated by the circadian clock, although direct mechanisms on Pai-1 rhythmicity are unknown. In the present study, we have identified a conserved functional E-box cis-element in the distal part of the mouse Pai-1 gene that is necessary and sufficient to drive circadian expression in Pai-1 activity after dexamethasone synchronisation in vitro. Mutagenesis and in vitro transfection analysis indicated this E-box provides a cognate binding site for cross-talk between clock and hypoxia factors, thus providing a potential cooperation mechanism between circadian and stress pathways, which is conserved in the human Pai-1 gene. Together, these results suggest that the canonical E-box is a target for glucocorticoid action, thus providing the molecular interface between gene transcription and drug action. The mechanism described has global impact on diverse dynamic biological processes governed by the neuroendocrine axis and the circadian clockwork to control complex coordination of gene cascades and biology.

Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte stress 1 gene promoter.

BACKGROUND: Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative in silico analysis coupled with an experimental promoter characterisation to delineate these mechanisms. RESULTS: Analysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in ms1 mRNA. An in silico comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with ms1 promoter/luciferase reporters and myogenic regulatory factor (MRF) over-expression plasmids revealed specific sensitivity of the ms1 promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2) within identified evolutionary conserved regions (ECRs, alpha and beta). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs alpha and beta correlates with the temporal induction in ms1 mRNA. CONCLUSION: These findings suggest that the tissue specific and differentiation dependent up-regulation in ms1 mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the ms1 5' upstream sequence. We believe, through its activation of ms1, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination, differentiation and regeneration.

Circadian clock genes cause activation of the human PAI-1 gene promoter with 4G/5G allelic preference.

Increased plasminogen activator inhibitor-1 (PAI-1) activity is associated with greater risk of myocardial infarction. PAI-1 expression is regulated by a 4G/5G promoter polymorphism. The 4G allele is associated with higher PAI-levels and greater circadian variation. Here we show that clock protein heterodimers BMAL/CLOCK cause greater activation (approximately 2-fold, P<0.05) of the 4G allele. Site-directed mutagenesis studies suggest that clock genes act on two canonical E-boxes to regulate PAI-1 promoter activity. These results identify a potential novel mechanism whereby allele-specific clock genes - mediated modulation of PAI-1 expression may contribute to circadian variation in cardiac risk.

Characterization of expressed sequence tags from a gallus gallus pineal gland cDNA library.

The pineal gland is the circadian oscillator in the chicken, regulating diverse functions ranging from egg laying to feeding. Here, we describe the isolation and characterization of expressed sequence tags (ESTs) isolated from a chicken pineal gland cDNA library. A total of 192 unique sequences were analysed and submitted to GenBank; 6% of the ESTs matched neither GenBank cDNA sequences nor the newly assembled chicken genomic DNA sequence, three ESTs aligned with sequences designated to be on the Z_random, while one matched a W chromosome sequence and could be useful in cataloguing functionally important genes on this sex chromosome. Additionally, single nucleotide polymorphisms (SNPs) were identified and validated in 10 ESTs that showed 98% or higher sequence similarity to known chicken genes. Here, we have described resources that may be useful in comparative and functional genomic analysis of genes expressed in an important organ, the pineal gland, in a model and agriculturally important organism.

Temporal-spatial characterization of chicken clock genes: circadian expression in retina, pineal gland, and peripheral tissues.

The molecular core of the vertebrate circadian clock is a set of clock genes, whose products interact to control circadian changes in physiology. These clock genes are expressed in all tissues known to possess an endogenous self-sustaining clock, and many are also found in peripheral tissues. In the present study, the expression patterns of two clock genes, cBmal1 and cMOP4, were examined in the chicken, a useful model for analysis of the avian circadian system. In two tissues which contain endogenous clocks--the pineal gland and retina--circadian fluctuations of both cBmal1 and cMOP4 mRNAs were observed to be synchronous; highest levels occurred at Zeitgeber time 12. Expression of these genes is also rhythmic in several peripheral tissues; however, the phases of these rhythms differ from those in the pineal gland and retina: in the liver the peaks of cMOP4 and cBmal1 mRNAs are delayed 4-8 h and in the heart they are advanced by 4 h, relative to those in the pineal gland and retina. These results provide the first temporal characterization of cBmal1 and cMOP4 mRNAs in avian tissues: their presence in avian peripheral tissues indicates they may influence temporal features of daily rhythms in biochemical, physiological, and behavioral functions at these sites.

ms1, a novel stress-responsive, muscle-specific gene that is up-regulated in the early stages of pressure overload-induced left ventricular hypertrophy.

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up-regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that ms1 may be important in striated muscle biology and the development of pressure-induced left ventricular hypertrophy.

Chickens' Cry2: molecular analysis of an avian cryptochrome in retinal and pineal photoreceptors.

We have identified and characterized an ortholog of the putative mammalian clock gene cryptochrome 2 (Cry2) in the chicken, Gallus domesticus. Northern blot analysis of gCry2 mRNA indicates widespread distribution in central nervous and peripheral tissues, with very high expression in pineal and retina. In situ hybridization of chick brain and retina reveals expression in photoreceptors and in visual and circadian system structures. Expression is rhythmic; mRNA levels predominate in late subjective night. The present data suggests that gCry2 is a candidate avian clock gene and/or photopigment and set the stage for functional studies of gCry2.