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Cell 2001 Feb 9;104(3):453-64

The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting.

Heintzen C, Loros JJ, Dunlap JC. Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.

vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock-associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase-response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.

Genetics 2001 Mar;157(3):1057-65

Analysis of Expressed Sequence Tags From Two Starvation, Time-of-Day-Specific Libraries of Neurospora crassa Reveals Novel Clock-Controlled Genes.

Zhu H, Nowrousian M, Kupfer D, Colot HV, Berrocal-Tito G, Lai H, Bell-Pedersen D, Roe BA, Loros JJ, Dunlap JC. Department of Chemistry and Biochemistry, Advanced Center for Genome Technology, University of Oklahoma, Norman, Oklahoma 73019.

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 13,000 cDNA clones from two time-of-day-specific libraries (morning and evening library) generating approximately 20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For approximately 50% of the sequences (710 of 1431), significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function) were detected. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST sequences. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their expression patterns over the course of the day. Nevertheless, we were able to identify four new clock-controlled genes. On the basis of these data we predict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.

EMBO J 2001 Jan 15;20(1-2):109-17

WC-2 mediates WC-1-FRQ interaction within the PAS protein-linked circadian feedback loop of Neurospora.

Denault DL, Loros JJ, Dunlap JC. Departments of Biochemistry and Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.

Eukaryotic circadian clocks comprise feedback loops where PAS domain-containing transcriptional activators drive gene expression of negative elements. In NEUROSPORA:, clock models posit a White Collar complex (WCC) containing WC-1 and WC-2 that activates expression of the central clock gene frequency (frq); FRQ protein is hypothesized to feed back to block the activity of the WCC. We have characterized the WC-2 protein and its role in this complex: WC-2 is an abundant constitutive nuclear protein, in contrast to rhythmically expressed FRQ and WC-1. WC-2 interacts with WC-1 and FRQ but, significantly, WC-1 and FRQ do not interact in the absence of WC-2. By quantifying the relative numbers of WC-2, FRQ and WC-1 proteins and complexes in cell extracts, both the numbers and types of complexes at different circadian times were estimated, yielding results consistent with the model. Constitutive and abundant WC-2 appears to provide a scaffold allowing for the interaction of two limiting and rhythmically out-of-phase proteins, FRQ and WC-1, and this temporal and physical relationship may be responsible for rhythmic expression of frq.

Cell 1997 Dec 26;91(7):1043-53

Light-induced resetting of a mammalian circadian clock is associated with rapid induction of the mPer1 transcript.

Shigeyoshi Y, Taguchi K, Yamamoto S, Takekida S, Yan L, Tei H, Moriya T, Shibata S, Loros JJ, Dunlap JC, Okamura H
Department of Anatomy and Brain Science, Kobe University School of Medicine,
Japan.

To understand how light might entrain a mammalian circadian clock, we examined
the effects of light on mPer1, a sequence homolog of Drosophila per, that
exhibits robust rhythmic expression in the SCN. mPer1 is rapidly induced by
short duration exposure to light at levels sufficient to reset the clock, and
dose-response curves reveal that mPer1 induction shows both reciprocity and a
strong correlation with phase shifting of the overt rhythm. Thus, in both the
phasing of dark expression and the response to light mPer1 is most similar to
the Neurospora clock gene frq. Within the SCN there appears to be localization
of the induction phenomenon, consistent with the localization of both
light-sensitive and light-insensitive oscillators in this circadian center.

PMID: 9428526, UI: 98088802

Cell 1997 May 2;89(3):477-86

Thermally regulated translational control of FRQ mediates aspects of temperature responses in the neurospora circadian clock.

Liu Y, Garceau NY, Loros JJ, Dunlap JC

Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

Two forms of FRQ, a central component of the Neurospora circadian clock, arise
through alternative in-frame initiation of translation. Either form alone
suffices for a functional clock at some temperatures, but both are always
necessary for robust rhythmicity. Temperature regulates the ratio of FRQ forms
by favoring different initiation codons at different temperatures; when either
initiation codon is eliminated, the temperature range permissive for
rhythmicity is demonstrably reduced. This temperature-influenced choice of
translation-initiation site represents a novel adaptive mechanism that extends
the physiological temperature range over which clocks function. Additionally, a
temperature-dependent threshold level of FRQ is required to establish the
feedback loop comprising the oscillator. These data may explain how temperature
limits permissive for rhythmicity are established, thus providing a molecular
understanding for a basic characteristic of circadian clocks.

PMID: 9150147, UI: 97294391

Cell 1995 Jun 30;81(7):1003-12

Light-induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript.

Crosthwaite SK, Loros JJ, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

To understand how light entrains circadian clocks, we examined the effects of
light on a gene known to encode a state variable of a circadian oscillator, the
frequency (frq) gene. frq is rapidly induced by short pulses of visible light;
clock resetting is correlated with frq induction and is blocked by drugs that
block the synthesis of protein or translatable RNA. The speed and magnitude of
frq induction suggest that this may be the initial clock-specific event in
light resetting. Light induction overcomes frq negative autoregulation so that
frq expression can remain high in constant light. These data explain how a
simple unidirectional signal (light and the induction of frq) may be turned
into a bidirectional clock response (time of day-specific advances and delays).
This light entrainment model is easily generalized and may be the common
mechanism by which the intracellular feedback cycles that comprise circadian
clocks are brought into synchrony with external cycles in the real world.

PMID: 7600569, UI: 95323956

Plant Physiol 1993 Aug;102(4):1299-305

The interplay of light and the circadian clock. Independent dual regulation of clock-controlled gene ccg-2(eas).

Arpaia G, Loros JJ, Dunlap JC, Morelli G, Macino G
Dipartimento di Biopatologia Umana, Universita di Roma La Sapienza, Italy.

Ambient light is the major agent mediating entrainment of circadian rhythms and
is also a major factor influencing development and morphogenesis. We show that
in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene
under the control of the circadian clock and allelic to the developmental gene
easy wettable (eas), is regulated by light in wild-type strains. Light elicits
a direct and important physiological effect on ccg-2(eas) expression as
demonstrated using several mutant Neurospora strains. In white collar mutants
(wc-1 and wc-2) that are "blind" to blue light, ccg-2(eas) mRNA shows no
variation following illumination with saturating light. By contrast, ccg-2(eas)
mRNA is photoinduced in clock-null strains such as frequency (bd;frq). The
results in the clock mutants show that an intact circadian oscillator is not
required for light induction of ccg-2(eas). Thus, ccg-2(eas) is subject to a
dual regulation that involves separable regulation by light and circadian
rhythm.

PMID: 8278550, UI: 94105347

Science 1998 Aug 7;281(5378):825-9

How temperature changes reset a circadian oscillator.

Liu Y, Merrow M, Loros JJ, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

Circadian rhythms control many physiological activities. The environmental
entrainment of rhythms involves the immediate responses of clock components.
Levels of the clock protein FRQ were measured in Neurospora at various
temperatures; at higher temperatures, the amount of FRQ oscillated around
higher levels. Absolute FRQ amounts thus identified different times at
different temperatures, so temperature shifts corresponded to shifts in clock
time without immediate synthesis or turnover of components. Moderate
temperature changes could dominate light-to-dark shifts in the influence of
circadian timing. Temperature regulation of clock components could explain
temperature resetting of rhythms and how single transitions can initiate
rhythmicity from characteristic circadian phases.

PMID: 9694654, UI: 98360068