The lysine demethylase dKDM2 is non-essential for viability, but regulates circadian rhythms in Drosophila

AbstractPost-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive, depending on specific pathological contexts. However, the role of KDM2 proteins during development in the whole organisms remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, we have generated two dKdm2 null alleles using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately one hour shorter than the control. Interestingly, opposite defects are observed when dKDM2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of how dKDM2 and its orthologs in vertebrates regulate circadian rhythms will advance our understanding of the epigenetic regulations of circadian clocks.

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Arginine-Vasopressin Expressing Neurons in the Murine Suprachiasmatic Nucleus Exhibit a Circadian Rhythm in Network Coherence in vivo

10.1101/2021.12.07.471437 ◽

2021 ◽

Author(s):

Adam Stowie ◽

Zhimei Qiao ◽

Daniella Do Carmo Buonfiglio ◽

J. Christopher Ehlen ◽

Morris Benveniste ◽

...

Keyword(s):

Circadian Rhythms ◽

Suprachiasmatic Nucleus ◽

Arginine Vasopressin ◽

Single Cells ◽

Circadian Variation ◽

Population Level ◽

Correlational Analysis ◽

Constant Darkness ◽

Peak Time

AbstractThe Suprachiasmatic Nucleus (SCN) is composed of functionally distinct sub-populations of GABAergic neurons such as vasoactive intestinal polypeptide (VIP)-, arginine vasopressin (AVP)-, gastrin releasing peptide (GRP)-, and neuromedin S (NMS)-expressing neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons or neuronal sub-populations and which aspects result from neuronal interaction within a network. In this study, we address this question utilizing in vivo miniaturized microscopy to measure fluorescent GCaMP-mediated calcium dynamics in AVP neurons in the intact SCN of awake, behaving mice. This approach permits analysis of rhythms of single cells, populations, and correlational analysis among groups of AVP neurons in a field of view across the circadian and diurnal day and night. We report that AVP neurons in the murine SCN exhibit a periodic oscillatory increase in calcium of approximately 14 seconds across the day and night, in both constant darkness and under a 12:12 light-dark (LD) cycle. Using in vivo optogentically-targeted single unit activity recording, we demonstrated that these slow calcium waves are likely the result of burst-firing characteristic of AVP neurons previously reported for other brain regions. Rhythmicity analysis of several fluorescence measures suggests that individual AVP neurons exhibit unstable and stochastic rhythms, with approximately 30% of the neurons rhythmic during any given day across lighting conditions, and weak or absent rhythmicity at the population level. Network-level cross-correlational analysis revealed that coherence among neuron pairs also exhibited stochastic rhythms with about 25% of pairs rhythmic at any time. Notably, this analysis revealed a stronger rhythm at the population level than was observed in single cell analysis. The peak time of maximal coherence among AVP neuronal pairs occurs between CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity with the SCN as a whole. These results are the first to demonstrate robust circadian variation in the coordination between apparently weakly rhythmic or arrhythmic neurons suggesting that, for AVP neurons, interactions between neurons in the SCN are more influential than individual or single subpopulation activity in the regulation of mammalian circadian rhythms.

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The Saccharomyces cerevisiae Histone Demethylase Jhd1 Fine-Tunes the Distribution of H3K36me2

Molecular and Cellular Biology ◽

10.1128/mcb.00127-07 ◽

2007 ◽

Vol 27 (13) ◽

pp. 5055-5065 ◽

Cited By ~ 26

Author(s):

Jia Fang ◽

Gregory J. Hogan ◽

Gaoyang Liang ◽

Jason D. Lieb ◽

Yi Zhang

Keyword(s):

Saccharomyces Cerevisiae ◽

Histone Methylation ◽

Histone Demethylase ◽

Lysine Methylation ◽

Chromatin Dynamics ◽

Histone Lysine Methylation ◽

Jmjc Domain ◽

Steady State Levels ◽

Microarray Studies

ABSTRACT Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady-state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in Saccharomyces cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and α-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation-coupled microarray studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units.

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The elongation factors Pandora/Spt6 and Foggy/Spt5 promote transcription in the zebrafish embryo

Development ◽

10.1242/dev.129.7.1623 ◽

2002 ◽

Vol 129 (7) ◽

pp. 1623-1632 ◽

Cited By ~ 2

Author(s):

Brian R. Keegan ◽

Jessica L. Feldman ◽

Diana H. Lee ◽

David S. Koos ◽

Robert K. Ho ◽

...

Keyword(s):

Transcription Elongation ◽

Elongation Factor ◽

Cardiac Differentiation ◽

Null Alleles ◽

Pcr Analysis ◽

Regulation Of Transcription ◽

Developmental Defects ◽

Transcription Elongation Factor ◽

On Line

Precise temporal and spatial control of transcription is a fundamental component of embryonic development. Regulation of transcription elongation can act as a rate-limiting step during mRNA synthesis. The mechanisms of stimulation and repression of transcription elongation during development are not yet understood. We have identified a class of zebrafish mutations (pandora, sk8 and s30) that cause multiple developmental defects, including discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. We demonstrate that the pandora gene encodes a protein similar to Spt6, a proposed transcription elongation factor. Additionally, the sk8 and s30 mutations are null alleles of the foggy/spt5 locus, which encodes another transcription elongation factor. Through real-time RT-PCR analysis, we demonstrate that Spt6 and Spt5 are both required for efficient kinetics of hsp70 transcription in vivo. Altogether, our results suggest that Spt6 and Spt5 play essential roles of comparable importance for promoting transcription during embryogenesis. This study provides the first genetic evidence for parallel functions of Spt6 and Spt5 in metazoans and establishes a system for the future analysis of transcription elongation during development. Supplemental figure available on-line

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Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development

Development ◽

10.1242/dev.124.2.313 ◽

1997 ◽

Vol 124 (2) ◽

pp. 313-326 ◽

Cited By ~ 11

Author(s):

P. Kastner ◽

M. Mark ◽

N. Ghyselinck ◽

W. Krezel ◽

V. Dupe ◽

...

Keyword(s):

Null Alleles ◽

Mouse Development ◽

Functional Specificity ◽

Developmental Defects ◽

Retinoid Receptors ◽

Alpha Beta ◽

Functional Units ◽

Dependent Processes ◽

Mutant Combination

We describe here the analysis of congenital malformations in compound mutant fetuses bearing null alleles in one RXR (alpha, beta or gamma) and one RAR (alpha, beta or gamma) isotype gene. A marked synergy was observed between the effects of mutations in RXR alpha and RARs, as a large number of developmental defects previously found mainly in RAR single and compound mutants were recapitulated in specific RXR alpha/RAR compound mutants. Several malformations were seen only in one type of RXR alpha/RAR mutant combination, whereas others were seen in several types of RXR alpha/RAR double mutants. No synergy was observed between the effects of mutations of either RXR beta or RXR gamma mutations and those of any of the RAR mutations. These genetic data suggest that RXR/RAR heterodimers are the functional units transducing the retinoid signal for a large number of RA-dependent processes, and furthermore, that RXR alpha is the main RXR implicated in the developmental functions of RARs. The significance of these observations is discussed with respect to the problem of functional specificity and redundancy among retinoid receptors in vivo.

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The Circadian Protein PER1 Modulates the Cellular Response to Anticancer Treatments

International Journal of Molecular Sciences ◽

10.3390/ijms22062974 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2974

Author(s):

Marina Maria Bellet ◽

Claudia Stincardini ◽

Claudio Costantini ◽

Marco Gargaro ◽

Stefania Pieroni ◽

...

Keyword(s):

Circadian Rhythms ◽

Circadian Clock ◽

Cancer Cells ◽

Cellular Response ◽

Molecular Mechanisms ◽

Antitumor Agents ◽

Pharmacological Therapy ◽

Drug Induced

The circadian clock driven by the daily light–dark and temperature cycles of the environment regulates fundamental physiological processes and perturbations of these sophisticated mechanisms may result in pathological conditions, including cancer. While experimental evidence is building up to unravel the link between circadian rhythms and tumorigenesis, it is becoming increasingly apparent that the response to antitumor agents is similarly dependent on the circadian clock, given the dependence of each drug on the circadian regulation of cell cycle, DNA repair and apoptosis. However, the molecular mechanisms that link the circadian machinery to the action of anticancer treatments is still poorly understood, thus limiting the application of circadian rhythms-driven pharmacological therapy, or chronotherapy, in the clinical practice. Herein, we demonstrate the circadian protein period 1 (PER1) and the tumor suppressor p53 negatively cross-regulate each other’s expression and activity to modulate the sensitivity of cancer cells to anticancer treatments. Specifically, PER1 physically interacts with p53 to reduce its stability and impair its transcriptional activity, while p53 represses the transcription of PER1. Functionally, we could show that PER1 reduced the sensitivity of cancer cells to drug-induced apoptosis, both in vitro and in vivo in NOD scid gamma (NSG) mice xenotransplanted with a lung cancer cell line. Therefore, our results emphasize the importance of understanding the relationship between the circadian clock and tumor regulatory proteins as the basis for the future development of cancer chronotherapy.

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Machine Learning and In-Vivo Expression Analyses Reveal Circadian Clock Features Predictive of Anxiety in Humans

10.21203/rs.3.rs-1190238/v1 ◽

2021 ◽

Author(s):

Aziz Zafar ◽

Rebeccah Overton ◽

Ziad Attia ◽

Ahmet Ay ◽

Krista Ingram

Keyword(s):

Machine Learning ◽

Circadian Rhythms ◽

Circadian Clock ◽

Clock Gene ◽

Molecular Mechanisms ◽

Clock Genes ◽

Anxiety Symptoms ◽

Circadian Phase ◽

Functional Studies

Abstract Mood disorders, including anxiety, are associated with disruptions in circadian rhythms and are linked to polymorphisms in circadian clock genes. Molecular mechanisms underlying these connections may be direct—via transcriptional activity of clock genes on downstream mood pathways in the brain, or indirect—via clock gene influences on the phase and amplitude of circadian rhythms which, in turn, modulate physiological processes influencing mood. Employing machine learning combined with statistical approaches, we explored clock genotype combinations that predict risk for anxiety symptoms in a deeply phenotyped population. We identified multiple novel circadian genotypes predictive of anxiety, with the PER3B-AG/CRY1-CG genotype being the strongest predictor of anxiety risk in males. Molecular chronotyping, using clock gene expression oscillations, revealed that advanced circadian phase and robust circadian amplitudes are associated with high levels of anxiety symptoms. Further analyses revealed that individuals with advanced phases and pronounced circadian misalignment were at higher risk for severe anxiety symptoms. Our results support both direct and indirect influences of clock gene variants on mood: while sex-specific clock genotype combinations predictive of anxiety symptoms suggest direct effects on mood pathways, the mediation of PER3B effects on anxiety via diurnal preference measures and the association of circadian phase with anxiety symptoms provide evidence for indirect effects of the molecular clockwork on mood. Unraveling the complex molecular mechanisms underlying the links between circadian physiology and mood is essential to identifying the core clock genes to target in future functional studies, thereby advancing the development of non-invasive treatments for anxiety-related disorders.

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Transcriptional Profiling of the Chick Pineal Gland, a Photoreceptive Circadian Oscillator and Pacemaker

Molecular Endocrinology ◽

10.1210/me.2003-0121 ◽

2003 ◽

Vol 17 (10) ◽

pp. 2084-2095 ◽

Cited By ~ 71

Author(s):

Michael J. Bailey ◽

Phillip D. Beremand ◽

Rick Hammer ◽

Deborah Bell-Pedersen ◽

Terry L. Thomas ◽

...

Keyword(s):

Pineal Gland ◽

Molecular Mechanisms ◽

Clock Genes ◽

Transcriptional Profiling ◽

Complex Form ◽

Time Of Day ◽

Constant Darkness ◽

Immune Stress

Abstract The avian pineal gland contains both circadian oscillators and photoreceptors to produce rhythms in biosynthesis of the hormone melatonin in vivo and in vitro. The molecular mechanisms for melatonin biosynthesis are largely understood, but the mechanisms driving the rhythm itself or the photoreceptive processes that entrain the rhythm are unknown. We have produced cDNA microarrays of pineal gland transcripts under light-dark and constant darkness conditions. Rhythmic transcripts were classified according to function, representing diverse functional groups, including phototransduction pathways, transcription/translation factors, ion channel proteins, cell signaling molecules, and immune function genes. These were also organized relative to time of day mRNA abundance in light-dark and constant darkness. The transcriptional profile of the chick pineal gland reveals a more complex form of gene regulation than one might expect from a gland whose sole apparent function is the rhythmic biosynthesis of melatonin. The mRNAs encoding melatonin biosynthesis are rhythmic as are many orthologs of mammalian “clock genes.” However, the oscillation of phototransductive, immune, stress response, hormone binding, and other important processes in the transcriptome of the pineal gland, raises new questions regarding the role of the pineal gland in circadian rhythm generation, organization, and avian physiology.

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Long-term in vivo recording of circadian rhythms in brains of freely moving mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1717735115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4276-4281 ◽

Cited By ~ 16

Author(s):

Long Mei ◽

Yanyan Fan ◽

Xiaohua Lv ◽

David K. Welsh ◽

Cheng Zhan ◽

...

Keyword(s):

Circadian Rhythms ◽

Clock Gene ◽

Constant Darkness ◽

Behavioral Rhythms ◽

Clock Gene Expression ◽

Hippocampal Ca1 ◽

Freely Moving ◽

Term Monitoring

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1−/−, and Cry2−/− mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

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Chronobiotics KL001 and KS15 Extend Lifespan and Modify Circadian Rhythms of Drosophila melanogaster

Clocks & Sleep ◽

10.3390/clockssleep3030030 ◽

2021 ◽

Vol 3 (3) ◽

pp. 429-441

Author(s):

Ilya A. Solovev ◽

Mikhail V. Shaposhnikov ◽

Alexey A. Moskalev

Keyword(s):

Drosophila Melanogaster ◽

Locomotor Activity ◽

Circadian Rhythms ◽

Molecular Mechanisms ◽

Fat Body ◽

Young Male ◽

Constant Darkness ◽

Starvation Resistance ◽

Positive Effects ◽

Median Lifespan

Chronobiotics are a group of drugs, which are utilized to modify circadian rhythms targeting clock-associated molecular mechanisms. The circadian clock is known as a controller of numerous processes in connection with aging. Hypothesis: KL001 and KS15 targeting CRY, affect lifespan, locomotor activity and circadian rhythm of Drosophila melanogaster. We observed a slight (2%, p < 0.001) geroprotective effect on median lifespan (5 µM solution of KL001 in 0.1% DMSO) and a 14% increase in maximum lifespan in the same group. KS15 10 µM solution extended males’ median lifespan by 8% (p < 0.05). The statistically significant positive effects of KL001 and KS15 on lifespan were not observed in female flies. KL001 5 µM solution improved locomotor activity in young male imagoes (p < 0.05), elevated morning activity peak in aged imagoes and modified robustness of their circadian rhythms, leaving the period intact. KS15 10 µM solution decreased the locomotor activity in constant darkness and minimized the number of rhythmic flies. KL001 5 µM solution improved by 9% the mean starvation resistance in male flies (p < 0.01), while median resistance was elevated by 50% (p < 0.0001). This phenomenon may suggest the presence of the mechanism associated with improvement of fat body glucose depos’ utilization in starvation conditions which is activated by dCRY binding KL001.

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Chronobiotics KL001 and KS15 Extend Lifespan and Modify Circadian Rhythms of Drosophila melanogaster

10.20944/preprints202107.0518.v1 ◽

2021 ◽

Author(s):

Ilya Solovev ◽

Mikhail Shaposhnikov ◽

Alexey Moskalev

Keyword(s):

Drosophila Melanogaster ◽

Locomotor Activity ◽

Circadian Rhythms ◽

Molecular Mechanisms ◽

Fat Body ◽

Young Male ◽

Constant Darkness ◽

Starvation Resistance ◽

Positive Effects ◽

Median Lifespan

Chronobiotics is a group of drugs utilized to modify circadian rhythms targeting clock-associated molecular mechanisms. The circadian clock is known as a controller of numerous processes standing behind aging. Hypothesis: KL001 and KS15 targeting CRY, affect lifespan, locomotor activity and circadian rhythm of Drosophila melanogaster. We observed a slight (2%, p<0.001) geroprotective effect on median lifespan (5 µM solution of KL001 in 0.1% DMSO) and a 14% increase in maximum lifespan in the same group. KS15 10 µM solution extended males’ median lifespan by 8% (p <0.05). The statistically significant positive effects of KL001 and KS15 on lifespan were not observed in female flies. KL001 5 µM solution improved locomotor activity in young male imagos (p<0.05) and elevated morning activity peak in aged imagos and modified robustness of circadian rhythms, leaving the period intact. KS15 10 µM solution decreased the locomotor activity in constant darkness and minimized the number of rhythmic flies. KL001 5 µM solution improved by 9% the mean starvation resistance in male flies (p<0.01), while median resistance was elevated by 50% (p<0.0001). This phenomenon may suggest the presence of the mechanism associated with improvement of fat body glucose depos’ utilization in starvation conditions which is activated by dCRY binding KL001.

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