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.
Science 2000 Jul 7;289(5476):107-10
Interconnected feedback loops in the Neurospora circadian system.
Lee K, Loros JJ, Dunlap JC. Department of Genetics, Dartmouth Medical School, Hanover, NH 03755-3844, USA.
In Neurospora crassa, white collar 1 (WC-1), a transcriptional activator and positive clock element, is rhythmically expressed from a nonrhythmic steady-state pool of wc-1 transcript, consistent with posttranscriptional regulation of rhythmicity. Mutations in frq influence both the level and periodicity of WC-1 expression, and driven FRQ expression not only depresses its own endogenous levels, but positively regulates WC-1 synthesis with a lag of about 8 hours, a delay similar to that seen in the wild-type clock. FRQ thus plays dual roles in the Neurospora clock and thereby, with WC-1, forms a second feedback loop that would promote robustness and stability in this circadian system. The existence also of interlocked loops in Drosophila melanogaster and mouse clocks suggests that such interlocked loops may be a conserved aspect of circadian timing systems.
Genetics (2001) 157: 1057-1065
Analysis of expressed sequence tags from two starvation, time of day-specific
libraries of Neurospora crassa reveals novel clock-controlled genes
Hua Zhu*1,4, Minou Nowrousian*2,4, Doris Kupfer*1,
Hildur V. Colot*2,
Gloria Berrocal-Tito*2, Lai H*1, Deborah Bell-Pedersen*3,
Bruce A. Roe*1,
Jennifer J. Loros*2, Jay C. Dunlap*2
*1Advanced Center for Genome Technology, Department of
Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019
*2Departments of Genetics and Biochemistry, Dartmouth Medical School,
Hanover, NH 03755
*3Department of Biology, Biological Sciences, Texas A&M University,
College Station, TX 77843
*4These authors contributed equally to this work.
In an effort to determine genes which 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 ESTs and 986 ESTs
present in multiple cDNA clones. For about 50 % of the sequences
(710 of 1,431), significant matches to sequences in the NCBI
database (of known or unknown function) were detected. About
50 % of the ESTs (721 of 1,431) showed no similarity to previously
identified genes. You can view the contigs or single clones and their
putative homologues here. You can also search the EST sequence data
using BLAST.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. Based upon 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.
J Biol Chem 1998 Jan 2;273(1):446-52
Glyceraldehyde-3-phosphate dehydrogenase is regulated on
a daily basis by the circadian clock.
Shinohara ML, Loros JJ, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH
03755, USA.
Circadian clocks function to govern a wide range of rhythmic activities in
organisms. An integral part of rhythmicity is the daily control of target genes
by the clock. Here we describe the sequence and analysis of a novel
clock-controlled gene, ccg-7, showing similarity to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), a glycolytic enzyme widely used as a constitutive
control in a variety of systems. That ccg-7 encodes GAPDH was confirmed by
demonstrating that in vitro synthesized CCG-7 possesses GAPDH activity. Rhythms
in both ccg-7 mRNA accumulation and CCG-7 (GAPDH) activity are observed in a
clock wild-type strain where the peak in GAPDH activity lags several hours
behind the peak in ccg-7 mRNA accumulation in the late night. Together with our
previous observation that ccg-7 mRNA is not developmentally regulated, we show
that ccg-7 is not induced by environmental stresses such as glucose or nitrogen
deprivation (which also trigger development), heat shock, or osmotic stress.
Thus, the finding that GAPDH is clock-regulated points to a specific role for
the circadian clock in controlling aspects of general metabolism and provides
evidence for circadian regulation of a gene found in most living organisms.
PMID: 9417102, UI: 98079082
Proc Natl Acad Sci U S A 1996 Nov 12;93(23):13096-101
Circadian clock-controlled genes isolated from Neurospora
crassa are late night- to early morning-specific.
Bell-Pedersen D, Shinohara ML, Loros JJ, Dunlap JC
Department of Biochemistry, Darmouth Medical School, Hanover, NH
03755, USA.
An endogenous circadian biological clock controls the temporal aspects of life
in most organisms, including rhythmic control of genes involved in clock output
pathways. In the fungus Neurospora crassa, one pathway known to be under
control of the clock is asexual spore (conidia) development. To understand more
fully the processes that are regulated by the N. crassa circadian clock,
systematic screens were carried out for genes that oscillate at the
transcriptional level. Time-of-day-specific cDNA libraries were generated and
used in differential screens to identify six new clock-controlled genes (ccgs).
Transcripts specific for each of the ccgs preferentially accumulate during the
late night to early morning, although they vary with respect to steady-state
mRNA levels and amplitude of the rhythm. Sequencing of the ends of the new ccg
cDNAs revealed that ccg-12 is identical to N. crassa cmt encoding copper
metallothionein, providing the suggestion that not all clock-regulated genes in
N. crassa are specifically involved in the development of conidia. This was
supported by finding that half of the new ccgs, including cmt(ccg-12), are not
transcriptionally induced by developmental or light signals. These data suggest
a major role for the clock in the regulation of biological processes distinct
from development.
PMID: 8917550, UI: 97075124
Mol Cell Biol 1996 Feb;16(2):513-21
Distinct cis-acting elements mediate clock, light, and
developmental regulation of the Neurospora crassa eas (ccg-2)
gene.
Bell-Pedersen D, Dunlap JC, Loros JJ
Department of Biochemistry, Dartmouth Medical School, Hanover, NH
03755, USA.
The Neurospora crassa eas (ccg-2) gene, which encodes a fungal hydrophobin, is
transcriptionally regulated by the circadian clock. In addition, eas (ccg-2) is
positively regulated by light and transcripts accumulate during asexual
development. To sort out the basis of this complex regulation, deletion
analyses of the eas (ccg-2) promoter were carried out to localize the
cis-acting elements mediating clock, light, and developmental control. The
primary sequence determinants of a positive activating clock element (ACE) were
found to reside in a 45-bp region, just upstream from the TATA box. Using a
novel unregulated promoter/reporter system developed for this study, we show
that a 68-bp sequence encompassing the ACE is sufficient to confer clock
regulation on the eas (ccg-2) gene. Electrophoretic mobility shift assays using
the ACE reveal factors present in N. crassa protein extracts that recognize and
bind specifically to DNA containing this element. Separate regions of the eas
(ccg-2) promoter involved in light induction and developmental control are
identified and shown not to be required for clock-regulated expression of eas
(ccg-2). The distinct nature of the ACE validates its use as a tool for the
identification of upstream regulatory factors involved in clock control of gene
expression.
PMID: 8552078, UI: 96140417
Mol Gen Genet 1995 Apr 20;247(2):157-63
Light induction of the clock-controlled gene ccg-1 is not
transduced through the circadian clock in Neurospora crassa.
Arpaia G, Loros JJ, Dunlap JC, Morelli G, Macino G
Dipartimento di Biopatologia Umana, Policlinico Umberto I,
Universita di Roma
La Sapienza, Italy.
Ambient light and the circadian clock have been shown to be capable of acting
either independently or in an interrelated fashion to regulate the expression
of conidiation in the ascomycete fungus Neurospora crassa. Recently several
molecular correlates of the circadian clock have been identified in the form of
the morning-specific clock-controlled genes ccg-1 and ccg-2. In this paper we
report studies on the regulation of ccg-1, an abundantly expressed gene
displaying complex regulation. Consistent with an emerging consensus for
clock-controlled genes and conidiation genes in Neurospora, we report that
ccg-1 expression is induced by light, and show that this induction is
independent of the direct effects of light on the circadian clock. Although
circadian regulation of the gene is lost in strains lacking a functional clock,
expression of ccg-1 is still not constitutive, but rather fluctuates in concert
with changes in developmental potential seen in such strains. Light induction
of ccg-1 requires the products of the Neurospora wc-1 and wc-2 genes, but
surprisingly the requirement for wc-2 is suppressed in conditional mutants of
cot-1, a gene that encodes, a cAMP-dependent protein kinase. These data provide
insight into a complex regulatory web, involving at least circadian clock
control, light control, metabolic control, and very probably developmental
regulation, that governs the expression of ccg-1.
PMID: 7753024, UI: 95272524
Genes Dev 1992 Dec;6(12A):2382-94
The Neurospora circadian clock-controlled gene, ccg-2, is
allelic to eas and encodes a fungal hydrophobin required for
formation of the conidial rodlet layer.
Bell-Pedersen D, Dunlap JC, Loros JJ
Department of Biochemistry, Dartmouth Medical School, Hanover, NH
03755, USA.
The Neurospora crassa clock-controlled gene (ccg-2) is transcriptionally
activated by the circadian clock in a time-of-day-specific manner. Transcript
and sequence analyses of ccg-2 reveal that the predicted Ccg-2 polypeptide
bears significant similarity to a class of low-molecular-weight, cysteine-rich,
hydrophobic proteins (hydrophobins), first identified in Schizophyllum, and
including the product of the developmentally regulated Aspergillus gene,
rodletless, required for spore surface rodlets. Allelism between ccg-2 and
easily wettable (eas) (one of the first developmental genetic loci identified
in Neurospora) was predicted on the basis of this similarity, their close
genetic linkage, and previous findings demonstrating that eas mutants lack
rodlets. In this study allelism is confirmed experimentally by showing that (1)
transformation of an eas mutant strain with ccg-2 DNA results in phenotypic
complementation, including restoration of surface rodlets, (2) inactivation of
the ccg-2 gene, by RIP, results in an eas phenotype including loss of rodlet
fascicles, and (3) the original eas strain has dramatically reduced levels of
ccg-2 mRNA. Thus, the clock-controlled ccg-2 gene encodes an integral component
of fungal asexual spores important for spore dispersal. The dramatic reduction
of ccg-2 expression in the eas mutant has no apparent effect on the normal
operation of the circadian clock, confirming that there is no feedback of this
clock output on the oscillator itself. These data, in conjunction with the
previous observation that ccg-2 is light induced, serve to focus attention on
the dual interacting role of light and the circadian clock in the regulation of
fungal spore development.
PMID: 1459460, UI: 93093472
Mol Cell Biol 1991 Jan;11(1):558-63
Neurospora crassa clock-controlled genes are regulated at
the level of transcription.
Loros JJ, Dunlap JC
Department of Biochemistry, Dartmouth Medical School, Hanover, NH
03755, USA.
Although an extensive number of biological processes are under the daily
control of the circadian biological clock, little is known about how the clock
maintains its regulatory networks within a cell. An important aspect of this
temporal control is the daily control of gene expression. Previously we
identified two morning-specific genes that are regulated by the clock through
daily control of gene expression (J. Loros, S. Denome, and J.C. Dunlap, Science
243:385-388, 1989). We have now introduced a method for transcriptional
analysis in Neurospora crassa and used this nuclear run-on procedure to show
that regulation of mRNA abundance for these two morning-specific genes occurs
at the level of transcription. This transcriptional regulation by the circadian
clock provides a basis for isolating circadian rhythm mutants.
PMID: 1824715, UI: 91094873