Selma Masri

Plasticity and specificity of the circadian epigenome

Circadian clocks control a variety of neuronal, behavioral and physiological responses, via transcriptional regulation of an appreciable portion of the genome. We describe the complex communication network between the brain-specific central clock and the tissue-specific peripheral clocks that serve to synchronize the organism to both external and internal demands. In addition, we discuss and speculate on how epigenetic processes are involved in creating transcriptional environments that are permissive to tissue-specific gene expression programs, which work in concert with the circadian machinery. Accumulating data show that chromatin remodeling events may be critical for providing specificity and plasticity in circadian regulation, and metabolic cues may be involved in directing such epigenetic events. A detailed understanding of the communication cues between the central and peripheral clocks is crucial for a more complete understanding of the circadian system and the several levels of control that are implicated in maintaining biological timekeeping.

Circadian acetylome reveals regulation of mitochondrial metabolic pathways

The circadian clock is constituted by a complex molecular network that integrates a number of regulatory cues needed to maintain organismal homeostasis. To this effect, posttranslational modifications of clock proteins modulate circadian rhythms and are thought to convert physiological signals into changes in protein regulatory function. To explore reversible lysine acetylation that is dependent on the clock, we have characterized the circadian acetylome in WT and Clock-deficient (Clock−/−) mouse liver by quantitative mass spectrometry. Our analysis revealed that a number of mitochondrial proteins involved in metabolic pathways are heavily influenced by clock-driven acetylation. Pathways such as glycolysis/gluconeogenesis, citric acid cycle, amino acid metabolism, and fatty acid metabolism were found to be highly enriched hits. The significant number of metabolic pathways whose protein acetylation profile is altered in Clock−/− mice prompted us to link the acetylome to the circadian metabolome previously characterized in our laboratory. Changes in enzyme acetylation over the circadian cycle and the link to metabolite levels are discussed, revealing biological implications connecting the circadian clock to cellular metabolic state.

Genome-Wide Analysis of Aromatase Inhibitor-Resistant, Tamoxifen-Resistant, and Long-Term Estrogen-Deprived Cells Reveals a Role for Estrogen Receptor

Acquired resistance to either tamoxifen or aromatase inhibitors (AI) develops after prolonged treatment in a majority of hormone-responsive breast cancers. In an attempt to further elucidate mechanisms of acquired resistance to AIs, MCF-7aro cells resistant to letrozole (T+LET R), anastrozole (T+ANA R), and exemestane (T+EXE R), as well as long-term estrogen deprived (LTEDaro) and tamoxifen-resistant (T+TAM R) lines were generated. This is the first complete panel of endocrine therapy-resistant cell lines, which were generated as multiple independent biological replicates for unbiased genome-wide analysis using affymetrix microarrays. Although similarities are apparent, microarray results clearly show gene signatures unique to AI-resistance were inherently different from LTEDaro and T+TAM R gene expression profiles. Based on hierarchical clustering, unique estrogen-responsive gene signatures vary depending on cell line, with some genes up-regulated in all lines versus other genes up-regulated only in the AI-resistant lines. Characterization of these resistant lines showed that LTEDaro, T+LET R, and T+ANA R cells contained a constitutively active estrogen receptor (ER)alpha that does not require estrogen for activation. This ligand-independent activation of ER was not observed in the parental cells, as well as T+EXE R and T+TAM R cells. Further characterization of these resistant lines was performed using cell cycle analysis, immunofluorescence experiments to visualize ER subcellular localization, as well as cross-resistance studies to determine second-line inhibitor response. Using this well-defined model system, our studies provide important information regarding differences in resistance mechanisms to AIs, TAM, and LTEDaro, which are critical in overcoming resistance when treating hormone-responsive breast cancers.

The circadian clock: a framework linking metabolism, epigenetics and neuronal function

The circadian clock machinery is responsible for biological timekeeping on a systemic level. The central clock system controls peripheral clocks through a number of output cues that synchronize the system as a whole. There is growing evidence that changing cellular metabolic states have important effects on circadian rhythms and can thereby influence neuronal function and disease. Epigenetic control has also been implicated in the modulation of biological timekeeping, and cellular metabolism and epigenetic state seem to be closely linked. We discuss the idea that cellular metabolic state and epigenetic mechanisms might work through the circadian clock to regulate neuronal function and influence disease states.

Sirtuins and the circadian clock: Bridging chromatin and metabolism

The sirtuin family of protein deactylases contributes to the regulation of circadian functions. Gloss Emerging data on the regulation of the circadian clock by protein acetylation reveal unexpected links with fundamental cellular functions. This review with three figures and 81 references highlights the recent advances in our understanding of the roles of the sirtuin family of protein deactylases in the control of circadian genetics, epigenetics, and metabolism. New strategies for treatment of various metabolic and aging-related disorders may become possible with the development and characterization of drugs that modulate sirtuin activity. The circadian clock is a finely tuned system of transcriptional and translational regulation that is required for daily synchrony of organismal physiological processes. Additional layers of complexity that contribute to efficient clock function involve posttranslational modifications and enzymatic feedback loops. SIRT1, the founding member of the sirtuin family of protein deacetylases, was the first sirtuin to be reported to modulate circadian function. SIRT1 affects the circadian clock by its actions in the nucleus. Moreover, recent data implicate SIRT3 and SIRT6 in controlling mitochondrial and nuclear circadian functions, revealing previously unappreciated roles that extend to various subcellular domains, including fatty acid metabolism in the mitochondria. This review focuses on the roles of sirtuins in directing circadian functions in diverse organelles and speculates on the endogenous signals that may mediate the segregated roles of this family of enzymes.

Circadian clocks, epigenetics, and cancer.

Purpose of review The interplay between circadian rhythm and cancer has been suggested for more than a decade based on the observations that shift work and cancer incidence are linked. Accumulating evidence implicates the circadian clock in cancer survival and proliferation pathways. At the molecular level, multiple control mechanisms have been proposed to link circadian transcription and cell-cycle control to tumorigenesis. Recent findings The circadian gating of the cell cycle and subsequent control of cell proliferation is an area of active investigation. Moreover, the circadian clock is a transcriptional system that is intricately regulated at the epigenetic level. Interestingly, the epigenetic landscape at the level of histone modifications, DNA methylation, and small regulatory RNAs are differentially controlled in cancer cells. This concept raises the possibility that epigenetic control is a common thread linking the clock with cancer, though little scientific evidence is known to date. Summary This review focuses on the link between circadian clock and cancer, and speculates on the possible connections at the epigenetic level that could further link the circadian clock to tumor initiation or progression.

The Role of Amphiregulin in Exemestane-Resistant Breast Cancer Cells: Evidence of an Autocrine Loop

Exemestane-resistant breast cancer cell lines (i.e., ExeR), derived from MCF-7 cells expressing a high level of aromatase (MCF-7aro), were generated in our laboratory. The epidermal growth factor (EGF)-like protein amphiregulin (AREG) was highly expressed in ExeR cells based on cDNA microarray analysis. The high levels of AREG mRNA in ExeR cell lines were confirmed by real-time reverse transcription-PCR. The high levels of AREG protein in ExeR cell lysates and culture media were confirmed by Western blot analysis and ELISA, respectively. Furthermore, our Western blot analysis showed that whereas no AREG was detected in the DMSO control, overnight treatment of parental MCF-7aro cells with 1 micromol/L exemestane strongly induced the expression of AREG. This induction was totally blocked by 100 nmol/L of pure antiestrogen ICI 182,780, implying estrogen receptor (ER) dependence of exemestane-induced AREG expression. MCF-7aro cells were not able to proliferate in hormone-free medium, but were able to proliferate in conditioned medium from ExeR cells, similar to the treatment of recombinant human AREG. Small interference RNA targeting AREG inhibited ExeR proliferation, confirming that AREG is truly functioning as a growth factor of ExeR cells. The specific inhibitors to ER (ICI 182,780), EGF receptor (EGFR; AG1478), and mitogen-activated protein kinase (MAPK; U0126) all showed dose-dependent suppression of the proliferation of ExeR cells, indicating the involvement of the ER, EGFR, and MAPK pathways. Based on these findings, we propose a possible mechanism that underlies exemestane resistance: exemestane induces AREG in an ER-dependent manner. AREG then activates the EGFR pathway and leads to the activation of the MAPK pathway that drives cell proliferation.

The circadian clock transcriptional complex: metabolic feedback intersects with epigenetic control

Chromatin remodeling is a prerequisite for most nuclear functions, including transcription, silencing, and DNA replication. Accumulating evidence shows that many physiological processes require highly sophisticated events of chromatin remodeling. Recent findings have linked cellular metabolism, epigenetic state, and the circadian clock. The control of a large variety of neuronal, behavioral, and physiological responses follows diurnal rhythms. This is possible through a transcriptional regulatory network that governs a significant portion of the genome. The harmonic oscillation of gene expression is paralleled by critical events of chromatin remodeling that appear to provide specificity and plasticity in circadian regulation. Accumulating evidence shows that the circadian epigenome appears to share intimate links with cellular metabolic processes. These notions indicate that the circadian epigenome might integrate tissue specificity within biological pacemakers, bridging systems physiology to metabolic control. This review highlights several advances related to the circadian epigenome, the contribution of NAD+ as a critical signaling metabolite, and its effects on epigenetic state, followed by more recent reports on circadian metabolomics analyses.

Circadian Control of Fatty Acid Elongation by SIRT1 Protein-mediated Deacetylation of Acetyl-coenzyme A Synthetase 1

Background: Circadian clock regulates various aspects of metabolism. Results: Rhythmic acetylation of AceCS1 controls circadian oscillations in acetyl-CoA levels and in fatty acid elongation. Conclusion: A previously unrecognized regulation of acetyl-CoA provides additional evidence to circadian regulation of metabolism. Significance: Understanding of the role of acetyl-CoA in fatty acid elongation might provide therapeutic benefits for treating metabolic diseases and cancer. The circadian clock regulates a wide range of physiological and metabolic processes, and its disruption leads to metabolic disorders such as diabetes and obesity. Accumulating evidence reveals that the circadian clock regulates levels of metabolites that, in turn, may regulate the clock. Here we demonstrate that the circadian clock regulates the intracellular levels of acetyl-CoA by modulating the enzymatic activity of acetyl-CoA Synthetase 1 (AceCS1). Acetylation of AceCS1 controls the activity of the enzyme. We show that acetylation of AceCS1 is cyclic and that its rhythmicity requires a functional circadian clock and the NAD+-dependent deacetylase SIRT1. Cyclic acetylation of AceCS1 contributes to the rhythmicity of acetyl-CoA levels both in vivo and in cultured cells. Down-regulation of AceCS1 causes a significant decrease in the cellular acetyl-CoA pool, leading to reduction in circadian changes in fatty acid elongation. Thus, a nontranscriptional, enzymatic loop is governed by the circadian clock to control acetyl-CoA levels and fatty acid synthesis.

Circadian Reprogramming in the Liver Identifies Metabolic Pathways of Aging

The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD+-related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism, and aging.