As the mathematical model developed by Woller et al. (1) also considers the role of SIRT1 in circadian clock, we regret not citing this work. However, we feel the inferences drawn in Furlan et al.’s (2) letter conflate 2 distinct studies with disparate purposes and methods. In Foteinou et al. (3), we address the role of SIRT1 in cell-autonomous clock function. We undertook this experimentation and modeling to resolve the conflicting reports by Asher et al. (4) and Nakahata et al. (5), elucidating … [↵][1]5To whom correspondence may be addressed. Email: frank_doyle{at}seas.harvard.edu. [1]: #xref-corresp-1-1

The circadian clock orchestrates 24-h rhythms in physiology in most living organisms. At the molecular level, the dogma is that circadian oscillations are based on a negative transcriptional feedback loop. Recent studies found the NAD+-dependent histone deacetylase, SIRT1, directly regulates acetylation status of clock components and influences circadian amplitude in cells. While Nakahata et al. [Nakahata Y, Kaluzova M (2008) Cell 134:329-340] reported that loss of SIRT1 increases amplitude through BMAL1 acetylation, Asher et al. [Asher G, Gatfield D (2008) Cell 134:317-328] reported that loss of SIRT1 decreases amplitude through an increase in acetylated PER2. To address this SIRT1 paradox, we developed a circadian enzymatic model. Predictions from this model and experimental validation strongly align with the findings of Asher et al., with PER2 as the primary target of SIRT1. Further, the model suggested SIRT1 influences BMAL1 expression through actions on PGC1α. We validated this finding experimentally. Thus, our computational and experimental approaches suggest SIRT1 positively regulates clock function through actions on PER2 and PGC1α.

Precise control of the relative ratio of retinal neurons and glia generated during development is essential for visual function. We show that Lhx2, which encodes a LIM-homeodomain transcription factor essential for specification and differentiation of retinal Müller glia, also plays a critical role in the development of retinal neurons. Overexpression of Lhx2, and its transcriptional coactivator Ldb1, triggers cell cycle exit and inhibits both Notch signaling and retinal gliogenesis. Lhx2/Ldb1 overexpression also induced the formation of wide-field amacrine cells (wfACs). In contrast Rnf12, which encodes a negative regulator of LDB1, is necessary for the initiation of retinal gliogenesis. We also show that LHX2 protein binds upstream of multiple neurogenic bHLH factors including Ascl1 and Neurog2, which are necessary for suppression of gliogenesis and wfAC formation respectively, and activates their expression. Finally, we demonstrate that the relative level of the LHX2-LDB1 complex in the retina decreases in tandem with the onset of gliogenesis. These findings show that control of Lhx2 function by Ldb1 and Rnf12 acts as a molecular mechanism underpinning the coordinated differentiation of neurons and Müller glia in postnatal retina. Significance Statement The molecular mechanisms that control the ratio neurons and glia that are generated by neuronal progenitors remain unclear. Here we show that Lhx2, a transcription factor essential for retinal gliogenesis, also controls development of retinal neurons. The Lhx2 coactivator Ldb1 promotes Lhx2-dependent neurogenesis, while the Lhx2 corepressor Rnf12 is necessary and sufficient for retinal gliogenesis. Furthermore, Lhx2 directly regulates expression of bHLH factors that promote neural development, which are necessary for Lhx2-dependent neurogenesis. Finally, we show that levels of the LHX2-LDB1 complex, which activates transcription, drop as gliogenesis begins. Dynamic regulation of Lhx2 activity by Ldb1 and Rnf12 thus controls the relative levels of retinal neurogenesis and gliogenesis, and may have similar functions elsewhere in the developing nervous system.

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.

A key component to overcoming the reproducibility crisis in biomedical research is the development of readily available, rigorously validated and renewable protein affinity reagents. As part of the NIH Protein Capture Reagents Program (PCRP), we have generated a collection of 1406 highly validated, immunoprecipitation (IP) and/or immunoblotting (IB) grade, mouse monoclonal antibodies (mAbs) to 736 human transcription factors. We used HuProt™ human protein microarrays to identify mAbs that recognize their cognate targets with exceptional specificity. Using an integrated production and validation pipeline, we validated these mAbs in multiple experimental applications, and have distributed them to the Developmental Studies Hybridoma Bank (DSHB) and several commercial suppliers. This study allowed us to perform a meta-analysis that identified critical variables that contribute to the generation of high quality mAbs. We find that using full-length antigens for immunization, in combination with HuProt™ analysis, provides the highest overall success rates. The efficiencies built into this pipeline ensure substantial cost savings compared to current standard practices.

The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.

The time-of-day effect on cognitive performance has been investigated by various behavioral tools and is supported by anecdotal evidence. However, the molecular details of the interaction between the circadian and cognitive systems remain underexplored. Recent studies suggest an endogenous slaveoscillator in the hippocampus, the primary seat of all learning and memory processes. Furthermore, in mammals Arntl (Bmal1) is the only indispensable transcription factor in the core transcriptional and translational feedback loop (TTFL) of the circadian-clock. Global deletion of the Bmal1 locus (Bmal1 ) leads to arrhythmic locomotor activity but also many other behavioral and physiological deficits, complicating the relationship between the clock and these phenotypes. We hypothesized that conditional tissue-specific deletion of Bmal1 in the hippocampus will function as a proxy for disabling the ‘hippocampal-clock’ leading to learning and memory deficits. To test this, we ablated the Bmal1 loci in post-natal mouse hippocampi using the CamKII-Cre driver line (referred hereafter as Bmal1). The circadian locomotor activity rhythms of Bmal1 are comparable to wild-type mice. However, the behavioral performance of Bmal1 in hippocampal-dependent tasks like classical-fear conditioning and spatial-object recognition was significantly impaired compared to littermate controls. We also observed differences in the hippocampal molecular-network of Bmal1 and wild-type mice that provides some insight into the molecular correlates of this performance.

The NIH Protein Capture Reagents Program (PCRP): a standardized protein affinity reagent toolbox

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.

The MYC oncogene encodes MYC, a transcription factor that binds the genome through sites termed E-boxes (5'-CACGTG-3'), which are identical to the binding sites of the heterodimeric CLOCK-BMAL1 master circadian transcription factor. Hence, we hypothesized that ectopic MYC expression perturbs the clock by deregulating E-box-driven components of the circadian network in cancer cells. We report here that deregulated expression of MYC or N-MYC disrupts the molecular clock in vitro by directly inducing REV-ERB alpha to dampen expression and oscillation of BMAL1, and this could be rescued by knockdown of REV-ERB. REV-ERB alpha expression predicts poor clinical outcome for N-MYC-driven human neuroblastomas that have diminished BMAL1 expression, and re-expression of ectopic BMAL1 in neuroblastoma cell lines suppresses their clonogenicity. Further, ectopic MYC profoundly alters oscillation of glucose metabolism and perturbs glutaminolysis. Our results demonstrate an unsuspected link between oncogenic transformation and circadian and metabolic dysrhythmia, which we surmise to be advantageous for cancer.

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 in vitro 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.

Abstract MYC, which is overexpressed in a variety of human cancers, drives aerobic glycolysis, also called the Warburg effect. The Warburg effect results in regeneration of NAD+ from increased conversion of pyruvate to lactate, such that decreased lactate dehydrogenase A (LDHA) activity results in NAD+ depletion and can result in cell death. Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme that is involved in the NAD+ salvage pathway, was reported to be directly upregulated by Myc. Increased NAD+ synthesis thus helps to replenish NAD+ along with LDHA. In this regard, we have previously reported the synergy of inhibiting LDHA and NAMPT in causing lymphoma xenograft regression. Targeting NAMPT by its inhibitor FK866 has been tested in clinical trials. However, the toxicity of FK866 in normal cells is not negligible, with thrombocytopenia being reported. Toxicity, however, could be diminished if therapy is administered at specific times of the day (termed chronotherapy) when normal cells are least dependent on NAMPT, whose levels oscillate every 24 hours. We have previously found that Myc upregulates Rev-erbα, which inhibits and disrupts oscillation of the circadian rhythm gene ARNTL (protein name Bmal1) in the osteosarcoma cell line U2OS. However, although NAMPT was shown oscillating in normal mouse liver and other tissues, how the oscillation of NAMPT is affected in cancer especially under Myc overexpression is still poorly understood. Here we show Myc induces Nampt mRNA and protein in mouse hepatocellular carcinoma (mHCC) cell line that has been engineered tetracycline inducible Myc transgene expression vector. Inhibition of NAMPT by FK866 induces perturbation of circadian gene expression in mHCC cells, including Rev-erbα, Per1 and Cry1. The alteration of circadian gene expression can be rescued by nicotinamide, suggesting that the circadian clock is affected by NAD+/NADH ratio. Interestingly, Nampt mRNA oscillates in mHCC cell lines in the absence of Myc and the oscillation was lost when Myc is induced, suggesting a therapeutic opportunity could exist in the window between circadian regulation of the ebb-and-flow of normal cell NAMPT level and the sustained, non-circadian cancer cell NAMPT level. Our observation that Myc disrupts the oscillation its target gene NAMPT provides a conceptual framework for metabolic chronotherapy that could potentially lead to better cancer treatment strategies that reduce side effects. We thank the following founding sources: NIH R01CA57341, LLS 6106-14. Citation Format: Annie L. Hsieh, Brian J. Altman, Anand Venkataraman, David I. Bellovin, Dean W. Felsher, John B. Hogenesch, Chi V. Dang. Oncogenic Myc disrupts NAMPT circadian oscillation in mouse hepatocellular carcinoma cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1419. doi:10.1158/1538-7445.AM2014-1419

Abstract Circadian rhythms are regulated by feedback loops comprising a network of factors that regulate Clock-associated genes. Chronotherapy seeks to take advantage of altered circadian rhythms in some cancers to better time administration of treatments to increase efficacy and reduce toxicity. While many cancers have perturbed expression of core circadian rhythm genes, the molecular basis underlying these perturbations and their functional implications in oncogenesis are still poorly understood, and so it is impossible to predict which cancers have altered circadian rhythms and would best benefit from chronotherapy. We have observed in cancer cell models of osteosarcoma, hepatocellular carcinoma, and neuroblastoma that the c-Myc and N-Myc oncogenic transcription factors disrupt oscillation of the circadian clock by specifically upregulating the circadian rhythm gene and nuclear hormone receptor NR1D1 (Rev-erbα). Interestingly, while Rev-erbα has not been previously recognized as an oncogene, data from The Cancer Genome Atlas revealed that it is amplified in many forms of human cancer, and we also observed that Rev-erbα was upregulated in primary human neuroblastoma and associated with poor prognosis. Therefore, we hypothesized that Rev-erbα is a novel oncogene downstream of Myc and is important for cancer cell growth. Here we show that Rev-erbα is specifically essential for the growth of Myc-driven hepatocellular carcinoma cells, as the related protein Rev-erbβ did not strongly influence growth. While knockdown of Rev-erbα expression by siRNA slowed growth, it did not cause cell death or canonical cell cycle arrest. Rev-erbα modulates circadian rhythm by downregulating the central circadian regulatory protein Bmal1, but this pathway did not play a central role in Rev-erbα control of cell growth. Additionally, while Rev-erbα has a well-described role in heme metabolism and subsequent support of mitochondria respiration, this pathway was not directly altered in Myc-driven liver cancer cells. Rather, knockdown of Rev-erbα was associated with decreased glycolytic activity characterized by a decrease in intracellular lactate and extracellular lactate production as well as an increase in certain glycolytic intermediates. In addition to these glycolytic changes, the maximum respiratory capacity of cells lacking Rev-erbα increased, as measured by oxygen consumption. These data suggest a novel role for Rev-erbα in promoting the growth of cancer cells through modulation of glucose metabolism and a shift towards increased respiration, and imply that cancers with upregulated Myc and Rev-erbα may be good candidates for chronotherapy. We thank the following funding sources: NIH R01CA57341, LLS 6106-14. Citation Format: Brian J. Altman, Annie Hsieh, Arvin M. Gouw, Zachary E. Stine, Anand Venkataraman, David I. Bellovin, Sharon J. Diskin, Wenyun Lu, Sisi Zhang, Dean W. Felsher, John M. Maris, Mitchell A. Lazar, Joshua D. Rabinowitz, John B. Hogenesch, Chi V. Dang. Rev-erbα modulates Myc-driven cancer cell growth and altered metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2953. doi:10.1158/1538-7445.AM2014-2953

Cancer cells are known to have a metabolic advantage, but it is not known whether the circadian clock, which couples metabolic and cell cycles, restrains cancer cell metabolism and growth. We report that the deregulated expression of the MYC or MYCN oncogene disrupts the molecular clock by inducing NR1D1 (Rev-erbα) to diminish expression and dampen oscillation of ARNTL (Bmal1) that could be rescued by knockdown of Rev-erb. The expression of Rev-erbα is highly correlated with MYC expression in human B cell lymphoma, human T-cell acute lymphoblastic leukemia, murine hepatocellular carcinoma cells and human neuroblastoma cells. MYC or MYCN directly upregulates Rev-erbα expression by binding to its promoter. Suppression of Rev-erbα diminished hepatocellular cancer cell growth, illustrating Rev-erbα's role in MYC-induced tumorigenesis. Furthermore, Rev-erbα expression is a predictor of poor clinical outcome for human neuroblastoma and correlates with MYCN expression. Our results demonstrate an unsuspected link between oncogenic transformation and circadian arrhythmia, which may be metabolically advantageous for cancer cells. Having observed that Myc and N-Myc disrupt circadian rhythm in tumor cells, suggesting a therapeutic opportunity could exist in the window between circadian regulation of the ebb-and-flow of normal cell metabolism and the sustained, not oscillate non-circadian cancer metabolism. The understanding of these basic mechanisms from our work should lead to better cancer treatment strategies that reduce side effects and increase effectiveness.

Abstract Circadian rhythms are regulated by feedback loops comprising a network of factors that regulate Clock-associated genes. Chronotherapy seeks to take advantage of altered circadian rhythms in some cancers to better time administration of treatments to increase efficacy and reduce toxicity. Taking advantage of cancers that have substantially different circadian rhythms, or are ‘out of phase’, with normal tissues, could open a wide therapeutic window to make them vulnerable to chemotherapy or targeted drugs at different times than normal tissue. However, there is currently no basis to identify which cancers have disrupted circadian rhythms and would be amenable to chronotherapy. c- and N-Myc are oncogenic transcription factors translocated or amplified in many cancers. While the role of Myc in circadian rhythm is currently unknown, it may affect circadian rhythm by binding to the same E-box promoter regions used by the central regulators of circadian rhythm, Clock/Bmal1. Additionally, Myc increases NAD+ levels through upregulation of NAMPT, and NAD+ is a crucial cofactor in the activity of the circadian regulator Sirt1. Thus, we hypothesized that Myc may disrupt circadian rhythm through two mechanisms: inappropriate engagement of E-box promoters and also upregulation of NAMPT leading to dysregulated Sirt1 activity. Here we show in neuroblastoma, osteosarcoma, and hepatocellular carcinoma cells that overexpressed Myc specifically upregulated the negative circadian regulator Rev-erbα, which in turn decreased expression of Bmal1. Inhibition of NAMPT downstream of Myc upregulation also led to major perturbations in circadian gene expression, suggesting a role for NAD modulation downstream of Myc in disruption of circadian rhythm. Importantly, My-expressing cells showed dramatically disrupted circadian oscillations, which could be partially rescued by inhibiting expression of Rev-erbα. Together, these data suggest that Myc-driven cancers have altered circadian oscillation due to upregulation of Rev-erbα and NAMPT, and that cancers driven by Myc may thus be good candidates for chronotherapy. We thank the following funding sources: NIH R01CA051497, R01CA57341, LLS 636311 Citation Format: Brian J. Altman, Annie Hsieh, Arvin Gouw, Anand Venkataraman, Bo Li, David Bellovin, M. Celeste Simon, Dean Felsher, John Hogenesch, Chi V. Dang. Oncogenic c- and N-Myc disrupt circadian rhythm. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4616. doi:10.1158/1538-7445.AM2013-4616