Tobias Schafmeier

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.

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.

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo–cytoplasmic shuttling

The Neurospora clock protein FREQUENCY (FRQ) inhibits its transcriptional activator WHITE COLLAR COMPLEX (WCC) in a negative feedback loop and supports its accumulation in a positive loop. We show that positive feedback is a delayed effect of negative feedback underlying the same post-translational mechanisms: DNA-binding-competent active WCC commits rapidly to degradation. FRQ-dependent phosphorylation of WCC, which interferes with DNA binding (negative feedback), leads to reduced turnover and slow accumulation of newly expressed WCC (positive feedback). When DNA binding of WCC is compromised by mutation, its accumulation is independent of FRQ. Cycles of FRQ-dependent inactivation and PP2A-dependent reactivation of WCC occur in the minute range and are coupled to obligate rapid cycles of nucleo-cytoplasmic shuttling. WCC shuttling and activity cycles are modulated by FRQ in circadian fashion.

Light input and processing in the circadian clock of Neurospora

Circadian clocks are endogenous oscillators that use zeitgebers as environmental cues to synchronise with the exogenous day–night cycle. The role of light as a zeitgeber has been investigated intensively to date. In Neurospora crassa the transcription factor White Collar Complex (WCC) is directly activated by light, which resets the clock. In addition, a hierarchical cascade of transcription factors activates the light‐induced expression of hundreds of genes. Disturbance of the clock during the day through changes in light intensity should be prevented to ensure efficient synchronisation. This can be achieved by desensitisation to the ambient light (photoadaptation). Photoadaptation in Neurospora is dependent on the blue light receptor Vivid (VVD), which accumulates immediately after light activation and rapidly silences the expression of WCC‐dependent genes. Recent studies have elucidated the molecular mechanism of VVD‐mediated photoadaptation. Here we review the increasing knowledge about light‐dependent gene expression and photoadaptation in Neurospora and discuss their relevance for synchronisation of the circadian clock.

Phosphorylations: making the Neurospora crassa circadian clock tick

Various post‐translational modifications have been identified that play a role in the function of circadian clocks. Among these, phosphorylation has been investigated extensively. It was shown that phosphorylation influences half‐life, subcellular localisation, transcriptional activity and conformation of clock components over the course of a circadian day. Recent observations also indicate that time‐of‐day specific sequential phosphorylation of the Neurospora crassa clock protein FREQUENCY is crucial for measuring time and thus for establishing a robust circadian rhythm. The circadian clock of Neurospora is one of the best‐investigated molecular clocks to date. In this review, we summarise the data on what is known so far about the role of phosphorylation of proteins involved in the Neurospora circadian clock.

Browning of white adipose tissue uncouples glucose uptake from insulin signaling.

Presence of thermogenically active adipose tissue in adult humans has been inversely associated with obesity and type 2 diabetes. While it had been shown that insulin is crucial for the development of classical brown fat, its role in development and function of inducible brown-in-white (brite) adipose tissue is less clear. Here we show that insulin deficiency impaired differentiation of brite adipocytes. However, adrenergic stimulation almost fully induced the thermogenic program under these settings. Although brite differentiation of adipocytes as well as browning of white adipose tissue entailed substantially elevated glucose uptake by adipose tissue, the capacity of insulin to stimulate glucose uptake surprisingly was not higher in the brite state. Notably, in line with the insulin-independent stimulation of glucose uptake, our data revealed that brite recruitment results in induction of solute carrier family 2 (GLUT-1) expression in adipocytes and inguinal WAT. These results for the first time demonstrate that insulin signaling is neither essential for brite recruitment, nor is it improved in cells or tissues upon browning.

Activity of the circadian transcription factor White Collar Complex is modulated by phosphorylation of SP-motifs.

MINT‐7033885: WC‐2 (uniprotkb:P78714) physically interacts (MI:0218) with FRQ (uniprotkb:P19970) and WC‐1 (uniprotkb:Q01371) by cross‐linking studies (MI:0030)

Brown Adipose Tissue Harbors a Distinct Sub-Population of Regulatory T Cells

Regulatory T (Treg) cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT). Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress.

Transcriptional regulation and function of the Neurospora clock gene white collar 2 and its isoforms

FREQUENCY (FRQ) and the White Collar Complex (WCC), consisting of WC1 and WC2 subunits, are crucial components of positive and negative feedback loops of the circadian clock of Neurospora. In the positive limb, FRQ supports the accumulation of WC1 on a post‐translational level and activates transcription of wc2. We analysed the transcriptional regulation of wc2. The WCC indirectly inhibits wc2 by controlling expression of a putative repressor. FRQ activates wc2 transcription by inhibiting WCC. A putative transcriptional activator binds to the wc2 promoter and antagonizes the repressor function. Furthermore, an internal promoter in the wc2 coding region drives expression of an amino‐terminally shortened isoform, sWC2. Full‐length WC2 and sWC2 are expressed in an antagonistic manner; thus, sWC2 expression seems to be a fail‐safe mechanism that maintains total WC2 levels above a threshold.

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.