Michael Brunner

Genome-Wide and Phase-Specific DNA-Binding Rhythms of BMAL1 Control Circadian Output Functions in Mouse Liver

Temporal mapping during a circadian day of binding sites for the BMAL1 transcription factor in mouse liver reveals genome-wide daily rhythms in DNA binding and uncovers output functions that are controlled by the circadian oscillator.

Assignment of circadian function for the Neurospora clock gene frequency

Circadian clocks consist of three elements: entrainment pathways (inputs), the mechanism generating the rhythmicity (oscillator), and the output pathways that control the circadian rhythms. It is difficult to assign molecular clock components to any one of these elements. Experiments show that inputs can be circadianly regulated and outputs can feed back on the oscillator,. Mathematical simulations indicate that under- or overexpression of a gene product can result in arrhythmicity, whether the protein is part of the oscillator or substantially part of a rhythmically expressed input pathway. To distinguish between these two possibilities, we used traditional circadian entrainment protocols, on a genetic model system, Neurospora crassa.

Transcriptional Feedback of Neurospora Circadian Clock Gene by Phosphorylation-Dependent Inactivation of Its Transcription Factor

The circadian clock protein Frequency (FRQ) feedback-regulates its own expression by inhibiting its transcriptional activator, White Collar Complex (WCC). We present evidence that FRQ regulates the bulk of WCC through modulation of its phosphorylation status rather than via direct complex formation. In the absence of FRQ, WCC is hypophosphorylated and transcriptionally active, while WCC is hyperphosphorylated and transcriptionally inactive when FRQ is expressed. The phosphorylation status of WCC changes rhythmically over a circadian cycle. Dephosphorylation and activation of WCC depend on protein phosphatase 2A (PP2A), and WCC is a substrate of PP2A in vitro. Hypophosphorylated WCC binds to the clock box of the frq promoter even in the presence of FRQ, while binding of hyperphosphorylated WCC is compromised even when FRQ is depleted. We propose that negative feedback in the circadian clock of Neurospora is mediated by FRQ, which rhythmically promotes phosphorylation of WCC, functionally equivalent to a cyclin recruiting cyclin-dependent kinase to its targets.

Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex

ABSTRACT Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora crassa

FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST‐like elements in FRQ, PEST‐1 and PEST‐2, are phosphorylated in vitro by recombinant CK‐1a and CK‐1b, two newly identified Neurospora homologs of casein kinase 1ϵ. CK‐1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST‐1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frqΔPEST‐1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frqΔPEST‐1 RNA and FRQΔPEST‐1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST‐1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components.

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

Import of preproteins into the mitochondrial matrix is driven by the ATP‐dependent interaction of mt‐Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co‐chaperone of mt‐Hsp70, plays a key role in the ATP‐dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt‐Hsp70‐Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt‐Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt‐Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt‐Hsp70 to the preprotein in transit. Subsequently, the release of mt‐Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt‐Hsp70. Rebinding of ATP to mt‐Hsp70 completes the reaction cycle.

The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation

Unfolding and import of preproteins into mitochondria are facilitated by a molecular motor in which heat shock protein 70 (Hsp70) in the matrix plays an essential role. Here we present two different experimental approaches to analyze mechanisms underlying this function of Hsp70. First, preproteins containing stretches of glutamic acid (polyE) or glycine (polyG) repeats in front of folded domains were imported into mitochondria. This occurred although Hsp70 cannot pull on these stretches to unfold the folded domains, since it does not bind to polyE and polyG. Secondly, preproteins containing titin immunoglobulin (Ig)‐like domains were imported into mitochondria, despite the fact that forces of >200 pN are required to mechanically unfold these domains. Since molecular motors generate forces of ∼5 pN, Hsp70 could not promote unfolding of the Ig‐like domains by mechanical pulling. Our observations suggest that Hsp70 acts as an element of a Brownian ratchet, which mediates unfolding and translocation of preproteins across the mitochondrial membranes.

Photoadaptation in Neurospora by Competitive Interaction of Activating and Inhibitory LOV Domains

Light responses and photoadaptation of Neurospora depend on the photosensory light-oxygen-voltage (LOV) domains of the circadian transcription factor White Collar Complex (WCC) and its negative regulator VIVID (VVD). We found that light triggers LOV-mediated dimerization of the WCC. The activated WCC induces expression of VVD, which then disrupts and inactivates the WCC homodimers by the competitive formation of WCC-VVD heterodimers, leading to photoadaptation. During the day, expression levels of VVD correlate with light intensity, allowing photoadaptation over several orders of magnitude. At night, previously synthesized VVD serves as a molecular memory of the brightness of the preceding day and suppresses responses to light cues of lower intensity. We show that VVD is essential to discriminate between day and night, even in naturally ambiguous photoperiods with moonlight.

The C66W Mutation in the Deafness Dystonia Peptide 1 (DDP1) Affects the Formation of Functional DDP1·TIM13 Complexes in the Mitochondrial Intermembrane Space

Mohr-Tranebjaerg syndrome is a progressive, neurodegenerative disorder caused by loss-of-function mutations in theDDP1/TIMM8A gene. DDP1 belongs to a family of evolutionary conserved proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. They mediate the import and insertion of hydrophobic membrane proteins into the mitochondrial inner membrane. All of them share a conserved Cys4 metal binding site proposed to be required for the formation of zinc fingers. So far, the only missense mutation known to cause a full-blown clinical phenotype is a C66W exchange directly affecting this Cys4motif. Here, we show that the mutant human protein is efficiently imported into mitochondria and sorted into the intermembrane space. In contrast to wild-type DDP1, it does not complement the function of its yeast homologue Tim8. The C66W mutation impairs binding of Zn2+ ions via the Cys4 motif. As a consequence, the mutated DDP1 is incorrectly folded and loses its ability to assemble into a hetero-hexameric 70-kDa complex with its cognate partner protein human Tim13. Thus, an assembly defect of DDP1 is the molecular basis of Mohr-Tranebjaerg syndrome in patients carrying the C66W mutation.

Interlocked feedback loops of the circadian clock of Neurospora crassa

Circadian clocks drive daily rhythms in physiology and behaviour, and thus allow organisms to better adapt to rhythmic changes in the environment. Circadian oscillators are cell‐autonomous systems, which generate via transcriptional, post‐transcriptional, translational and post‐translational control mechanisms a daily activity‐rhythm of a circadian transcription factor complex. According to recent models, this complex of transcription factors controls directly or indirectly expression of a large number of genes, and thus generates the potential to modulate physiological processes in a rhythmic fashion. The basic principles of the generation of circadian oscillation are similar in all eukaryotic systems. The circadian clock of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Focusing on the molecular properties, interactions and post‐translational modifications of the core Neurospora clock proteins WHITE COLLAR‐1, WHITE COLLAR‐2, FREQUENCY and VIVID, this review summarizes our knowledge of the molecular basis of circadian time keeping in Neurospora. Moreover, we discuss the mechanisms by which environmental cues like light and temperature entrain and reset this circadian system.