Benedetto Grimaldi

The NAD+-Dependent Deacetylase SIRT1 Modulates CLOCK-Mediated Chromatin Remodeling and Circadian Control

Circadian rhythms govern a large array of metabolic and physiological functions. The central clock protein CLOCK has HAT properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at Lys537, an event essential for circadian function. We show that the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner, correlating with rhythmic acetylation of BMAL1 and H3 Lys9/Lys14 at circadian promoters. SIRT1 associates with CLOCK and is recruited to the CLOCK:BMAL1 chromatin complex at circadian promoters. Genetic ablation of the Sirt1 gene or pharmacological inhibition of SIRT1 activity lead to disturbances in the circadian cycle and in the acetylation of H3 and BMAL1. Finally, using liver-specific SIRT1 mutant mice we show that SIRT1 contributes to circadian control in vivo. We propose that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock.

CLOCK-mediated acetylation of BMAL1 controls circadian function

Regulation of circadian physiology relies on the interplay of interconnected transcriptional–translational feedback loops. The CLOCK–BMAL1 complex activates clock-controlled genes, including cryptochromes (Crys), the products of which act as repressors by interacting directly with CLOCK–BMAL1. We have demonstrated that CLOCK possesses intrinsic histone acetyltransferase activity and that this enzymatic function contributes to chromatin-remodelling events implicated in circadian control of gene expression. Here we show that CLOCK also acetylates a non-histone substrate: its own partner, BMAL1, is specifically acetylated on a unique, highly conserved Lys 537 residue. BMAL1 undergoes rhythmic acetylation in mouse liver, with a timing that parallels the downregulation of circadian transcription of clock-controlled genes. BMAL1 acetylation facilitates recruitment of CRY1 to CLOCK–BMAL1, thereby promoting transcriptional repression. Importantly, ectopic expression of a K537R-mutated BMAL1 is not able to rescue circadian rhythmicity in a cellular model of peripheral clock. These findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.

PER2 controls lipid metabolism by direct regulation of PPARγ

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity, and inflammatory response. PER2-deficient mice display altered lipid metabolism with drastic reduction of total triacylglycerol and nonesterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated with altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the proadipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention.

Chromatin remodeling, metabolism and circadian clocks: The interplay of CLOCK and SIRT1

Circadian rhythms govern a wide variety of physiological and metabolic functions in almost all organisms. These are controlled by the circadian clock machinery, which is mostly based on transcriptional-translational feedback loops. Importantly, 10-15% of the mammalian transcripts oscillate in a circadian manner. The complex program of gene expression that characterizes circadian physiology is possible through dynamic changes in chromatin transitions. These remodeling events are therefore of great importance to insure the proper timing and extent of circadian regulation. Recent advances in the field have revealed unexpected links between circadian regulators, chromatin remodeling and cellular metabolism. Specifically, the central clock protein CLOCK has HAT enzymatic properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at K537, an event essential for circadian function. In addition, the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner. It has been proposed that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock. Thus, a specialized program of chromatin remodeling appears to be at the core of the circadian machinery.

Circadian clock regulates the host response to Salmonella

Organisms adapt to day–night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics

Mycorrhizal ascomycetes are ecologically and commercially important fungi that have proved impervious to genetic transformation so far. We report here on the successful transient transformation of Tuber borchii, an ectomycorrhizal ascomycete that colonizes a variety of trees and produces highly prized hypogeous fruitbodies known as “truffles”. A hypervirulent Agrobacterium tumefaciens strain bearing the binary plasmid pBGgHg was used for transformation. The genes for hygromycin resistance and the enhanced green fluorescent protein (EGFP), both under the control of vector-borne promoters, were employed as selection markers. Patches of dark and fluorescent hyphae were observed upon fluorescence microscopic examination of hygromycin-resistant mycelia. The presence of EGFP was confirmed by both confocal microscopy and PCR analysis. The lack in the transformed mycelia of the DNA coding for kanamicin resistance (a trait encoded by a vector-borne gene located outside of the T-DNA region) indicates that Agrobacterium-mediated gene transfer correctly occurred in T. borchii.

The Peripheral Clock Regulates Human Pigmentation

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

A meeting of two chronobiological systems: Circadian proteins period1 and bmal1 modulate the human hair cycle clock

The hair follicle (HF) is a continuously remodeled mini organ that cycles between growth (anagen), regression (catagen), and relative quiescence (telogen). As the anagen-to-catagen transformation of microdissected human scalp HFs can be observed in organ culture, it permits the study of the unknown controls of autonomous, rhythmic tissue remodeling of the HF, which intersects developmental, chronobiological, and growth-regulatory mechanisms. The hypothesis that the peripheral clock system is involved in hair cycle control, i.e., the anagen-to-catagen transformation, was tested. Here we show that in the absence of central clock influences, isolated, organ-cultured human HFs show circadian changes in the gene and protein expression of core clock genes (CLOCK, BMAL1, and Period1) and clock-controlled genes (c-Myc, NR1D1, and CDKN1A), with Period1 expression being hair cycle dependent. Knockdown of either BMAL1 or Period1 in human anagen HFs significantly prolonged anagen. This provides evidence that peripheral core clock genes modulate human HF cycling and are an integral component of the human hair cycle clock. Specifically, our study identifies BMAL1 and Period1 as potential therapeutic targets for modulating human hair growth.

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.

Chromatin Remodeling and Circadian Control: Master Regulator CLOCK Is an Enzyme

The molecular machinery that governs circadian rhythmicity is based on clock gene products organized in regulatory feedback loops. Recently, we have shown that CLOCK, a master circadian regulator, has histone acetyltransferase activity essential for clock gene expression. The Lys-14 residue of histone H3 is a preferential target of CLOCK-mediated acetylation. As the role of chromatin remodeling in eukaryotic transcription is well recognized, this finding identified unforeseen links between histone acetylation and cellular physiology. Indeed, we have shown that the enzymatic function of CLOCK drives circadian control. We reasoned that CLOCKs acetyltransferase activity could also target nonhistone proteins, a feature displayed by other HATs. Indeed, CLOCK also acetylates a nonhistone substrate: its own partner, BMAL1. This protein undergoes rhythmic acetylation in the mouse liver, with a timing that parallels the down-regulation of circadian transcription of clock-controlled genes. BMAL1 is specifically acetylated on a unique, highly conserved Lys-537 residue. This acetylation facilitates recruitment of the repressor CRY1 to BMAL1, indicating that CLOCK may intervene in negative circadian regulation. Our findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.