scholarly journals Quantification of circadian interactions and protein abundance defines a mechanism for operational stability of the circadian clock

2021 ◽  
Author(s):  
James S Bagnall ◽  
Alex A Koch ◽  
Nicola J Smyllie ◽  
Nicola Begley ◽  
Antony Adamson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Circadian Clock ◽  
Single Cells ◽  
Stochastic Modelling ◽  
Correlation Spectroscopy ◽  
Peripheral Tissues ◽  
Target Sites ◽  
Quantitative Understanding ◽  
Substantial Control ◽  
Affinity Interaction

The mammalian circadian clock exerts substantial control of daily gene expression through cycles of DNA binding. Understanding of mechanisms driving the circadian clock is hampered by lack of quantitative data, without which predictive mathematical models cannot be developed. Here we develop a quantitative understanding of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We have used fluorescent correlation spectroscopy (FCS) to track dynamic changes in CRISPR-modified fluorophore-tagged proteins in time and space in single cells across SCN and peripheral tissues. We determine the contribution of multiple rhythmic processes in coordinating BMAL1 DNA binding, including the roles of cycling molecular abundance, binding affinities and two repressive modes of action. We find that nuclear BMAL1 protein numbers determine corresponding nuclear CLOCK concentrations through heterodimerization and define a DNA residence time of 2.6 seconds for this complex. Repression of CLOCK:BMAL1 is in part achieved through rhythmic changes to BMAL1:CRY1 affinity as well as a high affinity interaction between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling of these data reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

scholarly journals Single-cell analysis of circadian dynamics in tissue explants

2015 ◽  
Vol 26 (22) ◽  
pp. 3940-3945 ◽  
Author(s):  
Laura Lande-Diner ◽  
Jacob Stewart-Ornstein ◽  
Charles J. Weitz ◽  
Galit Lahav
Keyword(s):  
Circadian Clock ◽  
Single Cell ◽  
Cell Tracking ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Tissue Imaging ◽  
Wide Field ◽  
Isolated Cells ◽  
Peripheral Tissues

Tracking molecular dynamics in single cells in vivo is instrumental to understanding how cells act and interact in tissues. Current tissue imaging approaches focus on short-term observation and typically nonendogenous or implanted samples. Here we develop an experimental and computational setup that allows for single-cell tracking of a transcriptional reporter over a period of >1 wk in the context of an intact tissue. We focus on the peripheral circadian clock as a model system and measure the circadian signaling of hundreds of cells from two tissues. The circadian clock is an autonomous oscillator whose behavior is well described in isolated cells, but in situ analysis of circadian signaling in single cells of peripheral tissues is as-yet uncharacterized. Our approach allowed us to investigate the oscillatory properties of individual clocks, determine how these properties are maintained among different cells, and assess how they compare to the population rhythm. These experiments, using a wide-field microscope, a previously generated reporter mouse, and custom software to track cells over days, suggest how many signaling pathways might be quantitatively characterized in explant models.

Quantifying transcription factor–DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy

2017 ◽  
Vol 12 (7) ◽  
pp. 1458-1471 ◽  
Author(s):  
Ziqing Winston Zhao ◽  
Melanie D White ◽  
Yanina D Alvarez ◽  
Jennifer Zenker ◽  
Stephanie Bissiere ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Fluorescence Correlation Spectroscopy ◽  
Single Cells ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

scholarly journals Circadian Rhythm: Potential Therapeutic Target for Atherosclerosis and Thrombosis

2021 ◽  
Vol 22 (2) ◽  
pp. 676
Author(s):  
Andy W. C. Man ◽  
Huige Li ◽  
Ning Xia
Keyword(s):  
Circadian Rhythm ◽  
Cardiovascular Diseases ◽  
Circadian Rhythms ◽  
Circadian Clock ◽  
Therapeutic Target ◽  
Environmental Changes ◽  
Clock Genes ◽  
Vascular System ◽  
Peripheral Tissues ◽  
Inflammatory Processes

Every organism has an intrinsic biological rhythm that orchestrates biological processes in adjusting to daily environmental changes. Circadian rhythms are maintained by networks of molecular clocks throughout the core and peripheral tissues, including immune cells, blood vessels, and perivascular adipose tissues. Recent findings have suggested strong correlations between the circadian clock and cardiovascular diseases. Desynchronization between the circadian rhythm and body metabolism contributes to the development of cardiovascular diseases including arteriosclerosis and thrombosis. Circadian rhythms are involved in controlling inflammatory processes and metabolisms, which can influence the pathology of arteriosclerosis and thrombosis. Circadian clock genes are critical in maintaining the robust relationship between diurnal variation and the cardiovascular system. The circadian machinery in the vascular system may be a novel therapeutic target for the prevention and treatment of cardiovascular diseases. The research on circadian rhythms in cardiovascular diseases is still progressing. In this review, we briefly summarize recent studies on circadian rhythms and cardiovascular homeostasis, focusing on the circadian control of inflammatory processes and metabolisms. Based on the recent findings, we discuss the potential target molecules for future therapeutic strategies against cardiovascular diseases by targeting the circadian clock.

Identification of a 150-kilodalton polypeptide that copurifies with yeast TFIIIC and binds specifically to tRNA genes

1989 ◽  
Vol 9 (5) ◽  
pp. 2018-2024
Author(s):  
D L Johnson ◽  
S L Wilson
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Iii ◽  
Deae Cellulose ◽  
Binding Activity ◽  
Trna Genes ◽  
Dna Binding Activity ◽  
Transcription In Vitro ◽  
Affinity Interaction ◽  
Dna Complex

The transcription in vitro of eucaryotic tRNA genes by RNA polymerase III requires two transcription factors, designated TFIIIB and TFIIIC. One of the critical functions of TFIIIC in the transcription of tRNA genes is that it interacts directly and specifically with the two internal promoter elements of these genes. We have partially purified Saccharomyces cerevisiae TFIIIC by chromatography on Bio-Rex 70, DEAE-cellulose, and phosphocellulose resins. A 150-kilodalton (kDa) DNA-binding polypeptide copurified with TFIIIC activity. This 150-kDa protein coeluted with the DNA-binding activity of TFIIIC after rechromatography of TFIIIC on phosphocellulose and its elution with a linear salt gradient. The stable and high-affinity interaction of this protein with tRNA genes was demonstrated by the maintenance of a protein-DNA complex under conditions of high ionic strength. Finally, we showed by two criteria that the interaction of this protein with tRNA genes was specific. First, the protein-DNA complex was competed with only by DNA-containing tRNA genes; second, the protein preferentially bound to DNA fragments containing a tRNA gene. These results strongly suggest that the DNA-binding domain of the yeast TFIIIC is contained within this 150-kDa polypeptide.

scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

scholarly journals Modeling and analysis of the impacts of jet lag on circadian rhythm and its role in tumor growth

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4877 ◽  
Author(s):  
Azka Hassan ◽  
Jamil Ahmad ◽  
Hufsah Ashraf ◽  
Amjad Ali
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Clock Genes ◽  
Damage Control ◽  
Vital Role ◽  
Biochemical Networks ◽  
Jet Lag ◽  
Peripheral Tissues ◽  
Modeling And Analysis ◽  
Circadian Clock Proteins

Circadian rhythms maintain a 24 h oscillation pattern in metabolic, physiological and behavioral processes in all living organisms. Circadian rhythms are organized as biochemical networks located in hypothalamus and peripheral tissues. Rhythmicity in the expression of circadian clock genes plays a vital role in regulating the process of cell division and DNA damage control. The oncogenic protein, MYC and the tumor suppressor, p53 are directly influenced by the circadian clock. Jet lag and altered sleep/wake schedules prominently affect the expression of molecular clock genes. This study is focused on developing a Petri net model to analyze the impacts of long term jet lag on the circadian clock and its probable role in tumor progression. The results depict that jet lag disrupts the normal rhythmic behavior and expression of the circadian clock proteins. This disruption leads to persistent expression of MYC and suppressed expression of p53. Thus, it is inferred that jet lag altered circadian clock negatively affects the expressions of cell cycle regulatory genes and contribute in uncontrolled proliferation of tumor cells.

Achilles-Mediated and Sex-Specific Regulation of Circadian mRNA Rhythms in Drosophila

2019 ◽  
Vol 34 (2) ◽  
pp. 131-143 ◽  
Author(s):  
Jiajia Li ◽  
Renee Yin Yu ◽  
Farida Emran ◽  
Brian E. Chen ◽  
Michael E. Hughes
Keyword(s):  
Circadian Clock ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Clock Genes ◽  
Peripheral Tissues ◽  
Knock Down ◽  
Rhythmic Expression ◽  
The Core ◽  
Physiological Processes ◽  
Specific Regulation

The circadian clock is an evolutionarily conserved mechanism that generates the rhythmic expression of downstream genes. The core circadian clock drives the expression of clock-controlled genes, which in turn play critical roles in carrying out many rhythmic physiological processes. Nevertheless, the molecular mechanisms by which clock output genes orchestrate rhythmic signals from the brain to peripheral tissues are largely unknown. Here we explored the role of one rhythmic gene, Achilles, in regulating the rhythmic transcriptome in the fly head. Achilles is a clock-controlled gene in Drosophila that encodes a putative RNA-binding protein. Achilles expression is found in neurons throughout the fly brain using fluorescence in situ hybridization (FISH), and legacy data suggest it is not expressed in core clock neurons. Together, these observations argue against a role for Achilles in regulating the core clock. To assess its impact on circadian mRNA rhythms, we performed RNA sequencing (RNAseq) to compare the rhythmic transcriptomes of control flies and those with diminished Achilles expression in all neurons. Consistent with previous studies, we observe dramatic upregulation of immune response genes upon knock-down of Achilles. Furthermore, many circadian mRNAs lose their rhythmicity in Achilles knock-down flies, suggesting that a subset of the rhythmic transcriptome is regulated either directly or indirectly by Achilles. These Achilles-mediated rhythms are observed in genes involved in immune function and in neuronal signaling, including Prosap, Nemy and Jhl-21. A comparison of RNAseq data from control flies reveals that only 42.7% of clock-controlled genes in the fly brain are rhythmic in both males and females. As mRNA rhythms of core clock genes are largely invariant between the sexes, this observation suggests that sex-specific mechanisms are an important, and heretofore under-appreciated, regulator of the rhythmic transcriptome.

scholarly journals HERP, a Novel Heterodimer Partner of HES/E(spl) in Notch Signaling

2001 ◽  
Vol 21 (17) ◽  
pp. 6080-6089 ◽  
Author(s):  
Tatsuya Iso ◽  
Vittorio Sartorelli ◽  
Coralie Poizat ◽  
Simona Iezzi ◽  
Hung-Yi Wu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Notch Signaling ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
De Novo ◽  
Single Cells ◽  
Binding Activity ◽  
Bhlh Protein ◽  
Helix Loop Helix ◽  
Dna Binding Activity

ABSTRACT HERP1 and -2 are members of a new basic helix-loop-helix (bHLH) protein family closely related to HES/E(spl), the only previously known Notch effector. Like that of HES, HERP mRNA expression is directly up-regulated by Notch ligand binding without de novo protein synthesis. HES and HERP are individually expressed in certain cells, but they are also coexpressed within single cells after Notch stimulation. Here, we show that HERP has intrinsic transcriptional repression activity. Transcriptional repression by HES/E(spl) entails the recruitment of the corepressor TLE/Groucho via a conserved WRPW motif, whereas unexpectedly the corresponding—but modified—tetrapeptide motif in HERP confers marginal repression. Rather, HERP uses its bHLH domain to recruit the mSin3 complex containing histone deacetylase HDAC1 and an additional corepressor, N-CoR, to mediate repression. HES and HERP homodimers bind similar DNA sequences, but with distinct sequence preferences, and they repress transcription from specific DNA binding sites. Importantly, HES and HERP associate with each other in solution and form a stable HES-HERP heterodimer upon DNA binding. HES-HERP heterodimers have both a greater DNA binding activity and a stronger repression activity than do the respective homodimers. Thus, Notch signaling relies on cooperation between HES and HERP, two transcriptional repressors with distinctive repression mechanisms which, either as homo- or as heterodimers, regulate target gene expression.

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