Milota Kaluzová

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.

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.

Transcriptional control of the tumor- and hypoxia-marker carbonic anhydrase 9: A one transcription factor (HIF-1) show?

Transcriptional activation by hypoxia is mediated by the hypoxia-inducible factor (HIF) via binding to the hypoxia-responsive element (HRE). Hypoxia in solid tumors associates with poorer outcome of the disease and reliable cellular markers of tumor hypoxia would represent a valuable diagnostic marker and a potential therapeutic target. In this category, carbonic anhydrase IX (CAIX) is one of the most promising candidates. Here, we summarize the knowledge about transcriptional regulation of CA9. The HRE is the central regulatory element in the CA9 promoter, whereas other elements are limited to lesser roles of amplification of signals received at the HRE. The analysis of known mechanisms of activation of CA9 reveals the prominent role of the HIF-1 pathway. Experimental paradigms with uncoupled HIF-1alpha stability and transcriptional activity (pericellular hypoxia, proteasomal inhibitor) provide evidence that CA9 expression monitors transcriptional activity of HIF-1, rather than the abundance of HIF-1alpha. Furthermore, these paradigms could provide a corollary to some of the apparently discordant cases (CAIX+, HIF-1alpha-) or (CAIX-, HIF-1alpha+) observed in vivo. In conclusion, the existing data support the notion that CA9, due to the unique structure of its promoter, is one of the most sensitive endogenous sensors of HIF-1 activity.

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.

Regulation of gene expression by hypoxia: integration of the HIF-transduced hypoxic signal at the hypoxia-responsive element

Cells experiencing lowered O(2) levels (hypoxia) undergo a variety of biological responses in order to adapt to these unfavorable conditions. The master switch, orchestrating the cellular response to low O(2) levels, is the transcription factor, termed hypoxia-inducible factor (HIF). The alpha subunits of HIF are regulated by 2-oxoglutarate-dependent oxygenases that, in the presence of O(2), hydroxylate specific prolyl and asparaginyl residues of HIF-alpha, inducing its proteasome-dependent degradation and repression of transcriptional activity, respectively. Hypoxia inhibits oxygenases, stabilized HIF-alpha translocates to the nucleus, dimerizes with HIF-beta, recruits the coactivators p300/CBP, and induces expression of its transcriptional targets via binding to hypoxia-responsive elements (HREs). HREs are composite regulatory elements, comprising a conserved HIF-binding sequence and a highly variable flanking sequence that modulates the transcriptional response. In summary, the transcriptional response of a cell is the end product of two major functions. The first (trans-acting) is the level of activation of the HIF pathway that depends on regulation of stability and transcriptional activity of the HIF-alpha. The second (cis-acting) comprises the characteristics of endogenous HREs that are determined by the availability of transcription factors cooperating with HIF and/or individual HIF-alpha isoforms.

Rational design of minimal hypoxia-inducible enhancers.

The hypoxia-inducible factor (HIF) activates transcription via binding to the highly variable hypoxia-responsive elements (HREs). All hypoxia-inducible constructs described to date utilize multimers of naturally occurring HREs. Here, we describe the rational design of minimal hypoxia-inducible enhancers, conceptually equivalent to using an optimized HIF-binding site (HBS) as the building block. Optimizations of the HBS, spacing between HBSs, the distance from the minimal promoter, and orientation of HBSs allowed us to design constructs with high hypoxic activity. Activation of the 4xopt HBS (36bp) construct by hypoxia or HIF-1alpha and HIF-2alpha was comparable with that of the 4xEPO HRE (208bp) construct. The strong synergism between the properly arranged optimized HBSs was due to stimulation of high affinity HIF binding. Our data prove, for the first time, that it is possible to assemble artificial hypoxia-inducible enhancers from a single type of regulatory element-optimized HBS.

Does inhibition of degradation of hypoxia‐inducible factor (HIF) α always lead to activation of HIF? lessons learnt from the effect of proteasomal inhibition on HIF activity

At the cellular level hypoxia induces transcriptional response that is mediated by the transcription factor hypoxia‐inducible factor (HIF). HIF is regulated at the level of its α subunit by 2‐oxoglutarate (2OG)‐dependent oxygenases that hydroxylate specific prolyl and asparaginyl residues of HIF‐α, affecting its stability and activity, respectively. In the presence of O2, the α subunit is degraded in a complex process with several distinct steps. In the first step, the degradation process is initiated by prolyl hydroxylases (PHDs). In the second step, the von Hippel‐Lindau (VHL)/E3 ligase complex recognizes the hydroxylated HIF‐α and mediates its polyubiquitylation by the ubiquitin‐conjugating enzyme E2. In the third step, the polyubiquitylated HIF‐α is translocated to the proteasome where it is degraded. Degradation of HIF‐α can be inhibited at any of the three levels either by various pharmacological inhibitors or due to inactivation of genes whose products regulate the HIF system. The emerging data about inactivation of HIF under conditions of proteasomal inhibition prompted us to provide an overview contrasting the outcome of inhibition at various stages of the degradative pathway for HIF activity. J. Cell. Biochem. 104: 536–544, 2008.

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.

The role of extracellular signal-regulated protein kinase in transcriptional regulation of the hypoxia marker carbonic anhydrase IX.

In the present study, we investigated the role of the extracellular signal‐regulated protein kinase (ERK) in regulation of the hypoxia marker, carbonic anhydrase IX (CAIX). U0126, a specific inhibitor of MEK1/2, downregulated CAIX expression induced by true hypoxia and cell density. CA9 promoter activity was similarly affected. Mapping of the U0126 effect revealed that both critical elements within the CA9 promoter, the hypoxia response element and the juxtaposed SP1‐binding PR1, were inhibited. This confirmed that ERK signaling modulates CA9 promoter activity via its effects on hypoxia inducible factor‐1 (HIF‐1) and SP1. Further analysis of the U0126 effect on HIF‐1‐dependent transcription in MCF‐7 cells identified p300, a transcriptional co‐activator of HIF‐1, as the target of ERK. Constitutively increased ERK activity in isogenic fibrosarcoma cell lines did not cause increased cell density‐dependent CAIX expression/CA9 promoter activity. In HeLa cells, an inverse correlation between cell density‐induced CAIX expression and ERK activation was observed: sparse cultures did not express CAIX and displayed high ERK activation, whereas CAIX expression in dense cultures was associated with low ERK activation. Collectively, our data do not support any quantitative relationship between ERK activation and CAIX expression. Thus, although ERK signaling is required for optimal CAIX expression, our data are consistent with a model in which constitutive basal ERK activity plays an auxiliary role in CA9 promoter transactivation by modulating activity of the transcription factor SP1 and the transcriptional co‐activator p300.

High cell density induces expression from the carbonic anhydrase 9 promoter

Hypoxia Hypes Gene Expression The control of levels and timing of expression of specific genes is of considerable interest to studies of oncogenesis. Expression of the carbonic anhydrase IX (CA9) g...