Deciphering the Duality of Clock and Growth Metabolism in a Cell Autonomous System Using NMR Profiling of the Secretome

Oscillations in circadian metabolism are crucial to the well being of organism. Our understanding of metabolic rhythms has been greatly enhanced by recent advances in high-throughput systems biology experimental techniques and data analysis. In an in vitro setting, metabolite rhythms can be measured by time-dependent sampling over an experimental period spanning one or more days at sufficent resolution to elucidate rhythms. We hypothesized that cellular metabolic effects over such a time course would be influenced by both oscillatory and circadian-independent cell metabolic effects. Here we use nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling of mammalian cell culture media of synchronized U2 OS cells containing an intact transcriptional clock. The experiment was conducted over 48 h, typical for circadian biology studies, and samples collected at 2 h resolution to unravel such non-oscillatory effects. Our data suggest specific metabolic activities exist that change continuously over time in this settting and we demonstrate that the non-oscillatory effects are generally monotonic and possible to model with multivariate regression. Deconvolution of such non-circadian persistent changes are of paramount importance to consider while studying circadian metabolic oscillations

CRY1‐CBS binding regulates circadian clock function and metabolism

Circadian rhythms are generated by interlocked transcription-translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR-tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR-tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR-tail interactions to reversibly control their activity

Machine Learning Helps Identify CHRONO as a Circadian Clock Component

<div><p>Over the last decades, researchers have characterized a set of “clock genes” that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate <i>in vitro</i> cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.</p></div

Integration of core clock features.

<p>(A) List of exemplar core clock genes used as example models of core clock components. (B–E) Metric functions describing core clock features were generated from published data. Distributions of these metrics among nonclock genes (left panel) and exemplar clock genes (center panel) were used to construct evidence factors (right panel). (B) Cycling was evaluated using time-course microarray data from liver, pituitary, and NIH 3T3 cells. (C) Circadian disturbance metric quantifies the influence of RNAi-mediated gene knockdown on circadian dynamics in the U2OS model system. (D) The interaction metric counts the number of interactions inferred between each gene and the exemplar set of core clock genes. (E) The tissue ubiquity scores were taken from an EST database. (F) List of 20 genes most likely to have a core circadian function as determined by evidence factor integration. Genes highlighted in blue were included in the exemplar training set. Genes highlighted in purple were not in the training set but have been identified as having a role in the circadian clock. <i>Gm129</i> was selected for further characterization.</p

CHRONO interacts with the C-terminus of BMAL1 but not BMAL2.

<p>(A) Overexpression of either BMAL1 or BMAL2, along with CLOCK, activates <i>Per1</i>:Luciferase reporter activity. Both are repressed by overexpression of CRY1. CHRONO specifically represses BMAL1-induced reporter activity. (B) BMAL1 and BMAL2 have similar structures with conserved bHLH DNA binding domains and PAS A and B interaction domains. BMAL1 contains a unique C-terminal region. Chimeric proteins were constructed by swapping corresponding domains from each protein as shown. Two-hybrid screening in HEK 293T cells demonstrates that BMAL1 truncation mutants (C) and chimeric proteins (D) that contain the 487–586 region of BMAL1 bind CHRONO and induce UAS:Luc reporter expression. This region is adjacent to but distinct from the annotated CRY1 binding site. (E) All BMAL1–BMAL2 constructs induce <i>Per1</i>-luc reporter activity in HEK 293T cells. In all constructs, reporter signal is repressed by the addition of CRY1. Functional repression by CHRONO is limited to BMAL constructs containing the implicated binding domain. (F) In cells overexpressing MYC–CHRONO along with BMAL1, BMAL2, or a chimeric BMAL2–BMAL1 construct, co-IP confirms complex formation between CHRONO and proteins containing the implicated BMAL1 C-terminal region.</p

CHRONO interferes with BMAL1–CBP binding.

<p>(A) BiFC was used to observe BMAL1–CBP interactions in the nuclei of HEK 293T cells. Co-expression of intact or S-tagged CHRONO reduced the complementation signal. Expression of the 212–385 CHRONO truncation mutant had no discernable effect. (B) IP confirms CHRONO-mediated interference in BMAL1/CBP complex formation. Endogenous protein was immunoprecipitated with anti-CBP antibody followed by immunoblotting as indicated. (C) ChIP qPCR analysis was used to evaluate the effect of CHRONO on the acetylation of histone H3–K9 near the <i>Per1</i> promoter E-box region. Schematic diagram of the human <i>Per1</i> promoter and primers used for ChIP assay are shown. Lysates obtained from control U2OS cells and those stably expressing CHRONO were collected 24 and 36 h after dexamethasone synchronization. ChIP DNA samples were quantified by quantitative real-time RT-PCR. Data are mean ± standard error of biological triplicates. (D) Various S-tagged, N-, and C-terminal CHRONO truncation mutants were generated. (E) Percent of cell nuclei demonstrating complementation after overexpression of various CHRONO constructs. (F) Per1:luciferase reporter signal in unsynchronized cells overexpressing BMAL1/CLOCK is enhanced by the transient overexpression of CBP. The effect of the overexpression of CHRONO constructs on reporter activity is shown.</p

<i>Chrono</i> transcript demonstrates circadian oscillations in peripheral tissues.

<p>qPCR was used to measure transcript abundance of <i>Chrono</i>, <i>Per2</i>, and <i>Nr1d1</i> in (A) liver, (B) skeletal muscle, and (C) adipose tissue. Circadian variation is observed in each tissue with the amplitude of <i>Chrono</i> oscillations comparable to that of <i>Per2</i> and <i>Nr1d1</i>. Data shown are the average of 3–4 biological replicates.</p

A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

Summary: The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform. : Finn et al. describe the development of a transient, biodegradable LNP-based CRISPR/Cas9 delivery system that achieves >97% knockdown of serum TTR levels following a single administration. Editing levels were stable for 12 months, despite the transient nature of the delivery system and the editing components. Keywords: CRISPR, Cas9, genome editing, LNP, lipid nanoparticle, TTR, CRISPR/Cas9, liver delivery, gene therapy, sgRN