Stefan Taubert

Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans

Many conditions that shift cells from states of nutrient utilization and growth to states of cell maintenance extend lifespan. We have carried out a systematic lifespan analysis of conditions that inhibit protein synthesis. We find that reducing the levels of ribosomal proteins, ribosomal‐protein S6 kinase or translation‐initiation factors increases the lifespan of Caenorhabditis elegans. These perturbations, as well as inhibition of the nutrient sensor target of rapamycin (TOR), which is known to increase lifespan, all increase thermal‐stress resistance. Thus inhibiting translation may extend lifespan by shifting cells to physiological states that favor maintenance and repair. Interestingly, different types of translation inhibition lead to one of two mutually exclusive outputs, one that increases lifespan and stress resistance through the transcription factor DAF‐16/FOXO, and one that increases lifespan and stress resistance independently of DAF‐16. Our findings link TOR, but not sir‐2.1, to the longevity response induced by dietary restriction (DR) in C. elegans, and they suggest that neither TOR inhibition nor DR extends lifespan simply by reducing protein synthesis.

MYC recruits the TIP60 histone acetyltransferase complex to chromatin

The transcription factor MYC binds specific DNA sites in cellular chromatin and induces the acetylation of histones H3 and H4. However, the histone acetyltransferases (HATs) that are responsible for these modifications have not yet been identified. MYC associates with TRRAP, a subunit of distinct macromolecular complexes that contain the HATs GCN5/PCAF or TIP60. Although the association of MYC with GCN5 has been shown, its interaction with TIP60 has never been analysed. Here, we show that MYC associates with TIP60 and recruits it to chromatin in vivo with four other components of the TIP60 complex: TRRAP, p400, TIP48 and TIP49. Overexpression of enzymatically inactive TIP60 delays the MYC‐induced acetylation of histone H4, and also reduces the level of MYC binding to chromatin. Thus, the TIP60 HAT complex is recruited to MYC‐target genes and, probably with other other HATs, contributes to histone acetylation in response to mitogenic signals.

E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1.

ABSTRACT E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G1 following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.

Function of the c-Myc oncoprotein in chromatin remodeling and transcription

Deregulated expression of the c-myc proto-oncogene contributes to malignant progression of a variety of tumors. The c-Myc protein (or Myc) is a transcription factor that positively or negatively regulates expression of distinct sets of target genes. Transcriptional activation by Myc is mediated through dimerization with Max and binding to the DNA consensus sequence CA(C/T)GTG (the E-box). Transcriptional inhibition is mediated through distinct DNA elements, and may be due to functional interference with factors that transactivate via these sequences. We review here our current knowledge on these transcriptional activities of Myc and their relationship to its biological function. The findings that Myc interacts with subunits of histone acetyl-transferase (HAT) complexes and of the ATP-dependent chromatin remodeling complex, SWI/SNF, suggest that localized changes in chromatin structure may mediate Myc function. We present a working hypothesis for the concerted action of HAT and SWI/SNF complexes in Myc-activated transcription and argue that this model should prompt re-thinking of the experimental strategies and criteria used to identify Myc target genes.

Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor

In wild‐type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3′,5′ monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse‐labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ‐tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross‐feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 μM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co‐ordinate antibiotic biosynthesis.

The gauge including magnetically induced current method

An overview of applications of the recently developed gauge including magnetically induced current method (GIMIC) is presented. The GIMIC method is used to obtain magnetically induced current densities in molecules. It provides detailed information about electron delocalization, aromatic character, and current pathways in molecules. The method has been employed in aromaticity studies on hydrocarbons, complex multi-ring organic nanorings, Möbius twisted molecules, inorganic and all-metal molecular rings and open-shell species. Recent studies on hydrogen-bonded molecules indicate that GIMIC can also be used to estimate hydrogen-bond strengths without fragmentation of the system. Preliminary results are presented on the applicability of GIMIC for investigating current transport in molecules attached to clusters simulating molecular conductivity measurements. Advantages and limitations of the GIMIC method are reviewed and discussed.

Magnetically Induced Current Densities in Aromatic, Antiaromatic, Homoaromatic, and Nonaromatic Hydrocarbons

The magnetically induced current densities for ring-shaped hydrocarbons are studied at the density functional theory (DFT) and second-order Møller-Plesset (MP2) levels using gauge-including atomic orbitals. The current densities are calculated using the gauge-including magnetically induced current approach. The calculations show that all studied hydrocarbon rings sustain strong diatropic and paratropic ring currents when exposed to an external magnetic field, regardless whether they are unsaturated or not. For nonaromatic rings, the strength of the paratropic current flowing inside the ring is as large as the diatropic one circling outside it, yielding a vanishing net ring current. For aromatic molecules, the diatropic current on the outside of the ring is much stronger than the paratropic one inside, giving rise to the net diatropic ring current that is typical for aromatic molecules. For antiaromatic molecules, the paratropic ring-current contribution inside the ring dominates. For homoaromatic molecules, the diatropic current circles at the periphery of the ring. The ring current is split at the CH(2) moiety; the main fraction of the current flow passes outside the CH(2) at the hydrogens, and some current flows inside the carbon atom. The diatropic current does not take the through-space short-cut pathway, whereas the paratropic current does take that route. Calculations of the ring-current profile show that the ring current of benzene is not transported by the pi electrons on both sides of the molecular ring. The strongest diatropic ring current flows on the outside of the ring and in the ring plane. A weaker paratropic current circles inside the ring with the largest current density in the ring plane. Due to the ring strain, small unconjugated and saturated hydrocarbon rings sustain a strong ring current which could be called ring-strain current. Nuclear magnetic shieldings calculated for 1,3,5-cycloheptatriene and homotropylium at the DFT and MP2 levels agree well with experimental values.

The Mediator Subunit MDT-15 Confers Metabolic Adaptation to Ingested Material

In eukaryotes, RNA polymerase II (PolII) dependent gene expression requires accessory factors termed transcriptional coregulators. One coregulator that universally contributes to PolII-dependent transcription is the Mediator, a multisubunit complex that is targeted by many transcriptional regulatory factors. For example, the Caenorhabditis elegans Mediator subunit MDT-15 confers the regulatory actions of the sterol response element binding protein SBP-1 and the nuclear hormone receptor NHR-49 on fatty acid metabolism. Here, we demonstrate that MDT-15 displays a broader spectrum of activities, and that it integrates metabolic responses to materials ingested by C. elegans. Depletion of MDT-15 protein or mutation of the mdt-15 gene abrogated induction of specific detoxification genes in response to certain xenobiotics or heavy metals, rendering these animals hypersensitive to toxin exposure. Intriguingly, MDT-15 appeared to selectively affect stress responses related to ingestion, as MDT-15 functional defects did not abrogate other stress responses, e.g., thermotolerance. Together with our previous finding that MDT-15:NHR-49 regulatory complexes coordinate a sector of the fasting response, we propose a model whereby MDT-15 integrates several transcriptional regulatory pathways to monitor both the availability and quality of ingested materials, including nutrients and xenobiotic compounds.

Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo

Significance The unfolded protein response of the endoplasmic reticulum (UPRER) is induced by proteotoxic conditions. Lipid disequilibrium also activates the UPRER, but whether this activation is accompanied by disturbed proteostasis in vivo remains controversial. In this study, we show that in the nematode Caenorhabditis elegans compromised fatty acid desaturation and reduced phosphatidylcholine production activate the UPRER without overt proteostatic imbalance, as assessed by molecular, pharmacological, and genetic analyses. This finding suggests that membrane composition is a direct input able to activate UPRER signaling even when proteostasis in the ER is largely intact. The activation of the UPRER by independent inputs may reflect the central role of the ER in both lipid and protein biosynthesis. The Mediator is a conserved transcriptional coregulator complex required for eukaryotic gene expression. In Caenorhabditis elegans, the Mediator subunit mdt-15 is essential for the expression of genes involved in fatty acid metabolism and ingestion-associated stress responses. mdt-15 loss of function causes defects in reproduction and mobility and shortens lifespan. In the present study, we find that worms with mutated or depleted mdt-15 (mdt-15 worms) exhibit decreased membrane phospholipid desaturation, especially in phosphatidylcholine. Accordingly, mdt-15 worms exhibit disturbed endoplasmic reticulum (ER) homeostasis, as indicated by a constitutively activated ER unfolded protein response (UPRER). Activation of this stress response is only partially the consequence of reduced membrane lipid desaturation, implicating other mdt-15–regulated processes in maintaining ER homeostasis. Interestingly, mdt-15 inactivation or depletion of the lipid metabolism enzymes stearoyl-CoA-desaturases (SCD) and S-adenosyl methionine synthetase (sams-1) activates the UPRER without promoting misfolded protein aggregates. Moreover, these worms exhibit wild-type sensitivity to chemically induced protein misfolding, and they do not display synthetic lethality with mutations in UPRER genes, which cause protein misfolding. Therefore, the constitutively activated UPRER in mdt-15, SCD, and sams-1 worms is not the consequence of proteotoxic stress but likely is the direct result of changes in ER membrane fluidity and composition. Together, our data suggest that the UPRER is induced directly upon membrane disequilibrium and thus monitors altered ER homeostasis.

Functional modularity of nuclear hormone receptors in a Caenorhabditis elegans metabolic gene regulatory network

Gene regulatory networks (GRNs) provide insights into the mechanisms of differential gene expression at a systems level. GRNs that relate to metazoan development have been studied extensively. However, little is still known about the design principles, organization and functionality of GRNs that control physiological processes such as metabolism, homeostasis and responses to environmental cues. In this study, we report the first experimentally mapped metazoan GRN of Caenorhabditis elegans metabolic genes. This network is enriched for nuclear hormone receptors (NHRs). The NHR family has greatly expanded in nematodes: humans have 48 NHRs, but C. elegans has 284, most of which are uncharacterized. We find that the C. elegans metabolic GRN is highly modular and that two GRN modules predominantly consist of NHRs. Network modularity has been proposed to facilitate a rapid response to different cues. As NHRs are metabolic sensors that are poised to respond to ligands, this suggests that C. elegans GRNs evolved to enable rapid and adaptive responses to different cues by a concurrence of NHR family expansion and modular GRN wiring.