Paolo Sassone-Corsi

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.

Signaling to Chromatin through Histone Modifications

We wish to thank all members of the Allis and Sassone-Corsi labs for exchange and discussion of many of the ideas presented in this review. Also we thank Craig Mizzen for assistance with figures and multiple rounds of proofreading, and Steve Cheung, for critical reading of the manuscript.

Synergistic coupling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stimulation.

Histone acetylation and phosphorylation have separately been suggested to affect chromatin structure and gene expression. Here we report that these two modifications are synergistic. Stimulation of mammalian cells by epidermal growth factor (EGF) results in rapid and sequential phosphorylation and acetylation of H3, and these dimodified H3 molecules are preferentially associated with the EGF-activated c-fos promoter in a MAP kinase-dependent manner. In addition, the prototypical histone acetyltransferase Gcn5 displays an up to 10-fold preference for phosphorylated (Ser-10) H3 over nonphosphorylated H3 as substrate in vitro, suggesting that H3 phosphorylation can affect the efficiency of subsequent acetylation reactions. Together, these results illustrate how the addition of multiple histone modifications may be coupled during the process of gene expression.

Circadian Control of the NAD+ Salvage Pathway by CLOCK-SIRT1

Circadian Oscillations The 24-hour day-night cycle plays an important role in mammalian physiology and behavior and, as most travelers are well aware, there is an intimate link between our in-built circadian clocks and metabolic rhythms. This link is in part forged by the protein deacetylase SIRT1, which regulates the clocks molecular circuitry. SIRT1 uses as a cofactor the cellular metabolite NAD+, which is synthesized through a salvage pathway that includes the enzyme nicotinamide phosphoribosyltransferase (NAMPT) (see the Perspective by Wijnen). Ramsey et al. (p. 651; published online 19 March) and Nakahata et al. (p. 654, published online 12 March) now show that NAMPT and NAD+ levels oscillate during the daily 24-hour cycle and that this oscillation is regulated by the circadian clock. Furthermore, the oscillations in NAD+ modulate the activity of SIRT1 feeding back into the circadian clock. A transcriptional-enzymatic feedback loop controls interactions between metabolism and circadian rhythms in mouse cells. Many metabolic and physiological processes display circadian oscillations. We have shown that the core circadian regulator, CLOCK, is a histone acetyltransferase whose activity is counterbalanced by the nicotinamide adenine dinucleotide (NAD+)–dependent histone deacetylase SIRT1. Here we show that intracellular NAD+ levels cycle with a 24-hour rhythm, an oscillation driven by the circadian clock. CLOCK:BMAL1 regulates the circadian expression of NAMPT (nicotinamide phosphoribosyltransferase), an enzyme that provides a rate-limiting step in the NAD+ salvage pathway. SIRT1 is recruited to the Nampt promoter and contributes to the circadian synthesis of its own coenzyme. Using the specific inhibitor FK866, we demonstrated that NAMPT is required to modulate circadian gene expression. Our findings in mouse embryo fibroblasts reveal an interlocked transcriptional-enzymatic feedback loop that governs the molecular interplay between cellular metabolism and circadian rhythms.

Circadian regulator CLOCK is a histone acetyltransferase.

The molecular machinery that governs circadian rhythmicity comprises proteins whose interplay generates time-specific transcription of clock genes. The role of chromatin remodeling in a physiological setting such as the circadian clock is yet unclear. We show that the protein CLOCK, a central component of the circadian pacemaker, has histone acetyltransferase (HAT) activity. CLOCK shares homology with acetyl-coenzyme A binding motifs within the MYST family of HATs. CLOCK displays high sequence similarity to ACTR, a member of SRC family of HATs, with which it shares also enzymatic specificity for histones H3 and H4. BMAL1, the heterodimerization partner of CLOCK, enhances HAT function. The HAT activity of CLOCK is essential to rescue circadian rhythmicity and activation of clock genes in Clock mutant cells. Identification of CLOCK as a novel type of DNA binding HAT reveals that chromatin remodeling is crucial for the core clock mechanism and identifies unforeseen links between histone acetylation and cellular physiology.

ATF4 Is a Substrate of RSK2 and an Essential Regulator of Osteoblast Biology:Implication for Coffin-Lowry Syndrome

Coffin-Lowry Syndrome (CLS) is an X-linked mental retardation condition associated with skeletal abnormalities. The gene mutated in CLS, RSK2, encodes a growth factor-regulated kinase. However, the cellular and molecular bases of the skeletal abnormalities associated with CLS remain unknown. Here, we show that RSK2 is required for osteoblast differentiation and function. We identify the transcription factor ATF4 as a critical substrate of RSK2 that is required for the timely onset of osteoblast differentiation, for terminal differentiation of osteoblasts, and for osteoblast-specific gene expression. Additionally, RSK2 and ATF4 posttranscriptionally regulate the synthesis of Type I collagen, the main constituent of the bone matrix. Accordingly, Atf4-deficiency results in delayed bone formation during embryonic development and low bone mass throughout postnatal life. These findings identify ATF4 as a critical regulator of osteoblast differentiation and function, and indicate that lack of ATF4 phosphorylation by RSK2 may contribute to the skeletal phenotype of CLS.

A Web of Circadian Pacemakers

The mammalian circadian timing system is composed of almost as many individual clocks as there are cells. These countless oscillators have to be synchronized by a central pacemaker to coordinate temporal physiology and behavior. Recently, there has been some progress in understanding the relationship and communication mechanisms between central and peripheral clocks.

Inducibility and negative autoregulation of CREM: An alternative promoter directs the expression of ICER, an early response repressor

cAMP-responsive element modulator (CREM) expression is tissue specific and developmentally regulated. Here we report that CREM is unique within the family of cAMP-responsive promoter element (CRE)-binding factors since it is inducible by activation of the cAMP signaling pathway. The kinetic of expression is characteristic of an early response gene. The induction is transient and cell specific, does not involve increased transcript stability, and does not require protein synthesis. Significantly, the subsequent decline in CREM expression requires de novo protein synthesis. The induced transcript encodes a novel repressor, inducible cAMP early repressor (ICER), and is generated from an alternative intronic promoter. A cluster of four CREs in this promoter directs cAMP inducibility. ICER binds to these elements and thereby represses the activity of its own promoter, thus constituting a negative autoregulatory loop.

A reliable method for the recovery of DNA fragments from agarose and acrylamide gels

Abstract We describe a simple and efficient method for the recovery of DNA fragments from agarose or acrylamide gels. The procedure involves electrophoresis of the fragments onto strips of DEAE-cellulose paper inserted in the gel between bands visualized by ethidium bromide fluorescence. The electrophoretic transfer is achieved in the original resolving gel using the same apparatus, thereby enabling purification of DNA fragments up to 20 kb in size with a yield of 60–80%. DNA which has been recovered in this way can be used for restriction enzyme cleavage, nick translation, end labeling by T4 polynucleotide kinase, DNA sequencing, and electron microscopy. The DNA fragments can be recloned in pBR322 using either blunt-end or cohesive-end ligation. Thus our method is useful for the recovery of biologically active DNA from both agarose and high-percentage polyacrylamide gels.

CREM gene: Use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcription

We isolated a gene from a mouse pituitary cDNA library that encodes a protein highly homologous to nuclear factor CREB, an activator of cAMP-responsive promoter elements (CREs). We demonstrate that while CREB is expressed uniformly in several cell types, this gene, termed CREM, shows cell-specific expression. CREM has a remarkable organization, since down-stream of the stop codon there is a second, out-of-frame DNA-binding domain. Using PCR and RNAase protection analysis, we have identified three mRNA isoforms that appear to be obtained by differential cell-specific splicing. Sequencing of the isoforms demonstrated alternative usage of the two DNA-binding domains. CREM proteins reveal the same efficiency and specificity of binding to CRE sequences as CREB, but in contrast to CREB, CREM acts as a down-regulator of cAMP-induced transcription.