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Circadian Control of Histone Methylation Dynamics through the Fine-tuning of Methionine Metabolic Flux

Periodic Reporting for period 1 - MetEpiClock (Circadian Control of Histone Methylation Dynamics through the Fine-tuning of Methionine Metabolic Flux)

Reporting period: 2017-11-01 to 2019-10-31

The circadian clock is an endogenous, time-tracking system that directs multiple metabolic and physiological functions required for homeostasis. The master or “central” clock is located within the suprachiasmatic nucleus (SCN) in the hypothalamus; it functions autonomously and can be reset by environmental cues such as light. The central clock governs peripheral clocks present in all systemic tissues, keeping them synchronized with each other and with the solar cycle, therefore ensuring temporally coordinated physiology. The importance of a functional clock in organismal homeostasis is provided by the evidence that genetic models with disrupted circadian rhythms show many features of metabolic syndrome, including obesity, diabetes, steatosis, cardiovascular diseases, and accelerated aging. About 10% of all mammalian transcripts display circadian oscillation in expression in a tissue-specific manner. At the molecular level, the core circadian clock machinery relies on coupled feedback loops of transcriptional and translational control. Circadian transcription is driven by the DNA binding transcription factors, CLOCK and BMAL1, which heterodimerize and drive the transcription of a large number of core clock and clock-controlled genes (CCGs) by binding to E-box sequences within their promoters. Dynamic changes in chromatin structure play an essential role in the proper timing and extent of circadian gene expression.

Chromatin plasticity relies on a variety of enzymes that are dependent on intermediary metabolites, thereby coupling metabolic pathways to epigenetic modifications and gene regulation. A feature of the metabolic-chromatin axis is the translocation of some metabolic enzymes into the nucleus and their contribution to localized availability of metabolites involved in epigenetic regulation. The methionine metabolic pathway intermediates S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH), influence gene expression through epigenetic mechanisms as they activate and inhibit, respectively, the activity of enzymes that drive DNA and histone methylation.

The project aims at investigating a possible direct link between cellular metabolism, epigenetic dynamics and circadian rhythms. Results from MetEpiClock will uncover a functional crosstalk between the molecular clock and methionine metabolism.

Objectives:

1. To determine if key enzymes and metabolites of the methionine cycle display circadian expression and are clock controlled.
2. To elucidate if disruption of cellular methionine homeostasis affects circadian rhythms.
2.1 To investigate the effect of methionine metabolic perturbation on circadian gene expression in vitro.
2.2 To investigate the effect of methionine metabolic perturbation of circadian rhythms and metabolic homeostasis in vivo.
Mass spectrometry analyses of immunoprecipitated BMAL1, a core clock regulator, identified the SAH hydrolase enzyme AHCY as a BMAL1-associated protein. By defining the rate of SAH hydrolysis AHCY controls the SAM/SAH ratio and thereby the methylation potential of the cell. Presence of AHCY in the BMAL1 complex was confirmed by direct immunoprecipitation, which revealed that the interaction is rhythmic. By performing subcellular fractionation, we found that the interaction occurs at chromatin, suggesting that AHCY may function as a regulator of circadian transcription. To investigate whether AHCY is implicated in circadian regulation, we used CRISPR-Cas9 mediated gene editing to delete AHCY in mouse embryonic fibroblasts (MEFs) and performed RNA-sequencing at 6 circadian times (CT). RNA-sequencing of MEFs treated with the inhibitor of AHCY Deazaneplanocin A (DZnep), confirmed results obtained with AHCY mutant cells. U2OS cells stably transfected with a Bmal1-luciferase reporter construct (Bmal:luc) were utilized to study circadian rhythms in cellular bioluminescence following acute silencing of Ahcy expression. Chromatin immunoprecipitation (ChIP) was carried out to study the effects of AHCY deletion or inhibition on rhythmic histone methylation at clock-regulated promoters.
Circadian rhythms direct almost all aspects of diurnal physiology, including metabolism. Current evidences suggest that environmental disruption of circadian rhythms (i.e. shift working, dietary regimens and irregular feeding timing) leads to several pathological conditions associated with altered metabolism. Given the intimate link that exists between circadian clocks, metabolism and chromatin dynamics, understanding whether these components act as a coordinated network in response to environmental cues may give us novel insights into how these factors contribute to normal physiology and disease. Indeed, dissecting the nodes that tie together circadian regulation, metabolism and epigenetics could have profound impacts in multiple diseases including cancer, metabolic disorders and cardiac pathologies providing novel strategies for both dietary and therapeutic interventions.
Summary of MetEpiClock results