Cellular Programs V2: Feedback loops control the mammalian circadian core clock The mammalian circadian rhythms core clock is a transcription–translation negative- feedback loop with a delay between transcription and the negative feedback. It is initiated by a heterodimeric transcription factor that consists of CLOCK and BMAL1. CLOCK and BMAL1 drive expression of their own negative regulators, the period proteins PER1 and PER2 and the cryptochromes CRY1 and CRY2. Over the course of the day, the PER and CRY proteins accumulate and multimerize in the cytoplasm, where they are phosphorylated by casein kinase Iε (CKIε) and glycogen synthase kinase-3 (GSK3). They then translocate to the nucleus in a phosphorylation-regulated manner where they interact with the CLOCK–BMAL1 complex to repress their own activator. At the end of the circadian cycle, the PER and CRY proteins are degraded in a CKI- dependent manner, which releases the repression of the transcription and allows the next cycle to start. An additional stabilizing feedback loop, which involves the activator Rora and the inhibitor Rev-Erbα, controls BMAL1 expression and reinforces the oscillations. RRE, R-response element. Gallego et al. Nat.Rev.Mol.Cell. Biol. 8, 140 (2007) WS 2010 – lecture 2
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V2: Feedback loops control the mammalian circadian core clock
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Cellular Programs
V2: Feedback loops control the mammalian circadian core clock
The mammalian circadian rhythms core clock is a transcription–translation negative-feedback loop with a delay between transcription and the negative feedback. It is initiated by a heterodimeric transcription factor that consists of CLOCK and BMAL1. CLOCK and BMAL1 drive expression of their own negative regulators, the period proteins PER1 and PER2 and the cryptochromes CRY1 and CRY2. Over the course of the day, the PER and CRY proteins accumulate and multimerize in the cytoplasm, where they are phosphorylated by casein kinase Iε (CKIε) and glycogen synthase kinase-3 (GSK3). They then translocate to the nucleus in a phosphorylation-regulated manner where they interact with the CLOCK–BMAL1 complex to repress their own activator. At the end of the circadian cycle, the PER and CRY proteins are degraded in a CKI-dependent manner, which releases the repression of the transcription and allows the next cycle to start. An additional stabilizing feedback loop, which involves the activator Rora and the inhibitor Rev-Erbα, controls BMAL1 expression and reinforces the oscillations. RRE, R-response element.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
WS 2010 – lecture 2
Cellular Programs
Circadian clock in D. melanogaster
(1) Clock (CLK) and cycle (CYC) activate the
transcription of the circadian genes in D.
melanogaster.
(2) Period (PER) and timeless (TIM) form
heterodimers in the cytoplasm where they
are phosphorylated by double-time (DBT)
and shaggy (SGG).
(3) PER and TIM then translocate to the
nucleus where PER inhibits the
transcriptional activity of the CLK–CYC
complex.
(4) Similarly to the mammalian clock, a
number of kinases regulate PER and TIM.
(5) In the stabilizing loop, the protein vrille
(VRI) inhibits, whereas PAR-domain protein-
1 (PDP1) activates the transcription of Clk.Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
WS 2010 – lecture 2
Cellular ProgramsWS 2010 – lecture 23
Why add phosphorylation to the clock?
Why are post-transcriptional modifications of crucial importance?
Transcription–translation feedback cycles generally operate on a timescale of up
to a few hours. If, following synthesis, the repressor proteins PER and CRY
translocated to the nucleus to repress CLOCK and BMAL1, the whole cycle
would take just a few hours rather than one day.
To maintain the daily oscillations of clock proteins, a significant delay between
the activation and repression of transcription is required. This is ensured by
regulation through post-translational modifications.
Reversible phosphorylation regulates important processes such as nuclear entry,
formation of protein complexes and protein degradation. Each of these can
individually contribute to introduce the delay that keeps the period at ~24 hours.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
Cellular Programs
Casein kinase I (CKI) has many roles in the mammalian circadian clock
b Phosphorylation of PER proteins increases over the course of the circadian day, peaking when the
repression of the positive transcription factors CLOCK and BMAL1 is maximal. There are several
phosphorylation sites for CKI on PER proteins.
c The phosphorylation of PER proteins regulates protein stability. Phosphorylation of 1-2 distinct sites
on PER1 and PER2 target these proteins for ubiquitin-mediated degradation by the proteasome.
Degradation of PER proteins can reset the clock
.
d PER and CRY proteins are not the only substrates of CKI in the clock. CKIε-mediated
phosphorylation of the circadian regulator BMAL1 increases its transcriptional activity.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
Casein kinase I (CKI)
a regulates the nuclear localization of the
circadian repression protein period (here
PER1). In some cell types, CKI activity
promotes the cytoplasmic accumulation of
PER1, whereas in others it mediates the
nuclear translocation of PER1.
WS 2010 – lecture 2
Cellular ProgramsWS 2010 – lecture 25
Dual roles of CLOCK acetyltransferase activity
CLOCK acetylates (Ac) histones H3 and H4 in nucleosomes (green) to confer
‘open’ chromatin structure and enable CLOCK-BMAL1 to bind to the E-boxes in
cognate promoters and turn on transcription.
CLOCK also acetylates BMAL1, making it a target for binding of the CRY
repressor, concomitant with deacetylation of histones by histone deacetylases
(HDAC). These dual effects of acetylation by CLOCK contribute to circadian
Gene density (`Genes') ranges from 38 per 100 kb to 1 gene per 100 kb;
expressed sequence tag matches (`ESTs') ranges from more than 200 per 100 kb to 1 per 100 kb.
Transposable element densities (`TEs') ranged from 33 per 100 kb to 1 per 100 kb.
Black and green ticks marks: Mitochondrial and chloroplast insertions (`MT/CP').
black and red ticks marks: Transfer RNAs and small nucleolar RNAs (`RNAs')
Nature 408, 796 (2000)
DAPI-stainedchromophores
Arabidopsis thaliana genome sequence
Nature 408, 796 (2000)
The proportion of Arabidopsis proteins having related counterparts in eukaryotic genomes varies by a factor of 2 to 3 depending on the functional category. Only 8 ± 23% of Arabidopsis proteins involved in transcription have related genes in other eukaryotic genomes, reflecting the independent evolution of many plant transcription factors. In contrast, 48 ± 60% of genes involved in protein synthesis have counterparts in the other eukaryotic genomes, reflecting highly conserved gene functions. The relatively high proportion of matches between Arabidopsis and bacterial proteins in the categories `metabolism' and `energy' reflects both the acquisition of bacterial genes from the ancestor of the plastid and high conservation of sequences across all species. Finally, a comparison between unicellular and multicellular eukaryotes indicates that Arabidopsis genes involved in cellular communication and signal transduction have more counterparts in multicellular eukaryotes than in yeast, reflecting the need for sets of genes for communication in multicellular organisms.
Plant epigenetics
The genomes of several plants have been sequenced, and those of many
others are under way.
But genetic information alone cannot fully address the fundamental question of
how genes are differentially expressed during cell differentiation and plant
development, as the DNA sequences in all cells in a plant are essentially the
same.
Several important mechanisms regulate transcription by affecting the
structural properties of the chromatin:- DNA cytosine methylation, - covalent modifications of histones, and - certain aspects of RNA interference (RNAi),
They are referred to as “epigenetic” because they direct “the structural
adaptation of chromosomal regions so as to register, signal or perpetuate