Transcription factor loading: please take my place!

Interactions of transcription factors with chromatin are highly dynamic. Now Voss et al. (2011) demonstrate that two transcription factors with identical DNA-binding specificities do not compete for occupancy at a given DNA element, but instead, one factor can even facilitate the binding of another. This assisted loading probably involves chromatin-remodeling machines.

SIRT1 regulates circadian clock gene expression through PER2 deacetylation.

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. Here, we show that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry.

Circadian Dbp transcription relies on highly dynamic BMAL1-CLOCK interaction with E boxes and requires the proteasome.

The transcription factors BMAL1 and CLOCK drive the circadian transcription of clock and clock-controlled genes, such as Dbp. To investigate the kinetics of BMAL1 binding to target genes in real time, we generated a cell line harboring tandem arrays of Dbp repeats and monitored the binding of a fluorescent BMAL1 fusion protein to these arrays by time-lapse microscopy. BMAL1 occupancy at the Dbp locus was highly circadian and strictly dependent on CLOCK. Moreover, BMAL1-CLOCK associations with Dbp were extremely unstable and displayed stochastic, proteasome-dependent fluctuations. Proteasome inhibition prolonged the residence time of BMAL1-CLOCK but resulted in an immediate attenuation of Dbp transcription. In cells harboring a single Dbp-luciferase reporter gene copy, this silencing was shown to be caused by a decrease in both the frequencies and sizes of transcriptional bursts. Thus, BMAL1 and CLOCK may act as Kamikaze activators, in that they are rapidly degraded once bound to Dbp chromatin.

Simulated body temperature rhythms reveal the phase-shifting behavior and plasticity of mammalian circadian oscillators.

The circadian pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus maintains phase coherence in peripheral cells through metabolic, neuronal, and humoral signaling pathways. Here, we investigated the role of daily body temperature fluctuations as possible systemic cues in the resetting of peripheral oscillators. Using precise temperature devices in conjunction with real-time monitoring of the bioluminescence produced by circadian luciferase reporter genes, we showed that simulated body temperature cycles of mice and even humans, with daily temperature differences of only 3°C and 1°C, respectively, could gradually synchronize circadian gene expression in cultured fibroblasts. The time required for establishing the new steady-state phase depended on the reporter gene, but after a few days, the expression of each gene oscillated with a precise phase relative to that of the temperature cycles. Smooth temperature oscillations with a very small amplitude could synchronize fibroblast clocks over a wide temperature range, and such temperature rhythms were also capable of entraining gene expression cycles to periods significantly longer or shorter than 24 h. As revealed by genetic loss-of-function experiments, heat-shock factor 1 (HSF1), but not HSF2, was required for the efficient synchronization of fibroblast oscillators to simulated body temperature cycles.

Flexible phase adjustment of circadian albumin D site-binding protein (DBP) gene expression by CRYPTOCHROME1.

The albumin D site-binding protein (DBP) governs circadian transcription of a number of hepatic detoxification and metabolic enzymes prior to the activity phase and subsequent food intake of mice. However, the behavior of mice is drastically affected by the photoperiod. Therefore, continuous adjustment of the phase of circadian Dbp expression is required in the liver. Here we describe a direct impact of CRYPTOCHROME1 (CRY1) on the phase of Dbp expression. Dbp and the nuclear receptor Rev-Erbalpha are circadian target genes of BMAL1 and CLOCK. Surprisingly, dynamic CRY1 binding to the Dbp promoter region delayed BMAL1 and CLOCK-mediated transcription of Dbp compared with Rev-Erbalpha. Extended presence of CRY1 in the nucleus enabled continuous uncoupling of the phase of Dbp from Rev-Erbalpha expression upon change from short to longer photoperiods. CRY1 thus maintained the peak of DBP accumulation close to the activity phase. In contrast, Rev-Erbalpha expression was phase-locked to the circadian oscillator and shaped by accumulation of its own gene product. Our data indicate that fine-tuning of circadian transcription in the liver is even more sophisticated than expected.

Airflow and Air Velocity Measurements While Playing Wind Instruments, with Respect to Risk Assessment of a SARS-CoV-2 Infection.

Due to airborne transmission of the coronavirus, the question arose as to how high the risk of spreading infectious particles can be while playing a wind instrument. To examine this question and to help clarify the possible risk, we analyzed 14 wind instruments, first qualitatively by making airflows visible while playing, and second quantitatively by measuring air velocity at three distances (1, 1.5, 2 m) in the direction of the instruments' bells. Measurements took place with wind instrumentalists of the Bamberg Symphony in their concert hall. Our findings highlight that while playing, no airflows escaping from any of the wind instruments-from the bell with brass instruments or from the mouthpiece, keyholes or bell with woodwinds-were measurable beyond a distance of 1.5 m, regardless of volume, pitch or what was played. With that, air velocity while playing corresponded to the usual value of 1 m/s in hall-like rooms. For air-jet woodwinds, alto flute and piccolo, significant air movements were seen close to the mouthpiece, which escaped directly into the room.

Diverse odor-conditioned memories require uniquely timed dorsal paired medial neuron output.

Amnesiac mutant flies have an olfactory memory defect. The amn gene encodes a homolog of vertebrate pituitary adenylate cyclase-activating peptide (PACAP), and it is strongly expressed in dorsal paired medial (DPM) neurons. DPM neurons ramify throughout the mushroom bodies in the adult fly brain, and they are required for stable memory. Here, we show that DPM neuron output is only required during the consolidation phase for middle-term odor memory and is dispensable during acquisition and recall. However, we found that DPM neuron output is required during acquisition of a benzaldehyde odor memory. We show that flies sense benzaldehyde by the classical olfactory and a noncanonical route. These results suggest that DPM neurons are required to consolidate memory and are differently involved in memory of a volatile that requires multisensory integration.

Properties, entrainment, and physiological functions of mammalian peripheral oscillators.

In mammals, the circadian timing system is composed of multiple oscillators that are organized in a hierarchical manner. The central pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, is believed to orchestrate countless subsidiary clocks in the periphery. These peripheral oscillators are cell-autonomous, self-sustained, resilient to cell division, and virtually insensitive to large fluctuations in general transcription rates. However, they are probably not coupled within an organ, and daily zeitgeber signals emanating from the SCN appear to be required to ensure phase coherence within and between tissues. Peripheral clocks are implicated in a variety of biochemical pathways, and recent results tightly link circadian rhythms to several aspects of metabolism. Thus, the expression of many key enzymes conducting rate-limiting steps in various metabolic pathways is regulated in a circadian fashion by core clock components or clock-controlled transcription factors. Genetic loss-of-function studies have now established a role for mammalian circadian clock components in energy homeostasis and xenobiotic detoxification, and the latter manifests itself in the daytime-dependent modulation of drug efficacy and toxicity.

REV-ERBs: more than the sum of the individual parts.

REV-ERB nuclear receptors have been believed to stabilize the circadian clock machinery through an accessory but dispensable feedback loop. Recent work now challenges this assumption by demonstrating that REV-ERBs are essential core clock components, in addition to serving as pivotal regulators of rhythmic metabolism.

Cold-inducible RNA-binding protein modulates circadian gene expression posttranscriptionally.

In mammalian tissues, circadian gene expression can be driven by local oscillators or systemic signals controlled by the master pacemaker in the suprachiasmatic nucleus. We show that simulated body temperature cycles, but not peripheral oscillators, controlled the rhythmic expression of cold-inducible RNA-binding protein (CIRP) in cultured fibroblasts. In turn, loss-of-function experiments indicated that CIRP was required for high-amplitude circadian gene expression. The transcriptome-wide identification of CIRP-bound RNAs by a biotin-streptavidin-based cross-linking and immunoprecipitation (CLIP) procedure revealed several transcripts encoding circadian oscillator proteins, including CLOCK. Moreover, CLOCK accumulation was strongly reduced in CIRP-depleted fibroblasts. Because ectopic expression of CLOCK improved circadian gene expression in these cells, we surmise that CIRP confers robustness to circadian oscillators through regulation of CLOCK expression.