Transcript ppt - Chair of Computational Biology

V2: Feedback loops control the mammalian circadian core
clock
Gallego et al.
Nat.Rev.Mol.Cell.Biol.
8, 140 (2007)
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.
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 protein1 (PDP1) activates the transcription of Clk.
Gallego et al. Nat.Rev.Mol.Cell.Biol. 8, 140 (2007)
WS 2010 – lecture 2
Cellular Programs
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)
WS 2010 – lecture 2
Cellular Programs
3
Casein kinase I (CKI) has many roles in the
mammalian circadian clock
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.
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)
WS 2010 – lecture 2
Cellular Programs
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
periodicity of gene expression.
Sancar,
Nat. Struct. Mol. Biol. 15, 23 (2008)
WS 2010 – lecture 2
Cellular Programs
5
What is epigenetics?
Epigenetics refers to alternate phenotypic states that are not based in
differences in genotype, and are potentially reversible, but are generally stably
maintained during cell division.
Examples: imprinting, twins, cancer vs. normal cells, differentiation, ...
The narrow interpretation of this concept is that of stable differential states of gene
expression.
A much more expanded view of epigenetics has recently emerged in which multiple
mechanisms interact to collectively establish
- alternate states of chromatin structure (open – packed/condensed),
- histone modifications,
- associated protein (e.g. histone) composition,
- transcriptional activity, and
- in mammals, cytosine-5 DNA methylation at CpG dinucleotides.
Laird, Hum Mol Gen 14, R65 (2005)
Basic principles of epigenetics:
DNA methylation and histone modfications
The human genome contains
23 000 genes that must be
expressed in specific cells at
precise times.
Cells manage gene expression
by wrapping DNA around
clusters (octamers) of globular
histone proteins to form
nucleosomes.
These nucleosomes of DNA
and histones are organized into
chromatin, the building block of
a chromosome.
Rodenhiser, Mann,
CMAJ 174, 341 (2006)
Bock, Lengauer, Bioinformatics 24, 1 (2008)
Epigenetic modifications
Strands of DNA are wrapped around histone octamers, forming nucleosomes.
These nucleosomes are organized into chromatin, the building block of a
chromosome.
Rodenhiser, Mann,
CMAJ 174, 341 (2006)
Reversible and site-specific histone modifications occur at multiple sites at the
unstructured histone tails through acetylation, methylation and phosphorylation.
DNA methylation occurs at 5-position of cytosine residues within CpG pairs in a
reaction catalyzed by DNA methyltransferases (DNMTs).
Together, these modifications provide a unique epigenetic signature that regulates
chromatin organization and gene expression.
Cytosine methylation
3-6 % of all cytosines are methylated in human DNA.
Mammalian genomes contain much fewer (only 20-25 %) of the CpG dinucleotide than is
expected by the G+C content. This is typically explained in the following way:
As most CpGs serve as targets of DNA methyltransferases, they are usually methylated.
5-Methylcytosine, whose occurrence is almost completely restricted to CpG dinucleotides, can
easily deaminate to thymine.
If this mutation is not repaired, the affected CpG is permanently converted to TpG (or CpA if
the transition occurs on the reverse DNA strand).
Hence, methylCpGs represent mutational hot spots in the genome.
If such mutations occur in the germ line, they become heritable.
A constant loss of CpGs over thousands of generations can explain the scarcity of this special
dinucleotide in the genomes of human and mouse.
Esteller, Nat. Rev. Gen. 8, 286 (2007)
effects in chromatin organization affect gene expression
Schematic of the reversible changes in chromatin organization that influence
gene expression:
genes are expressed (switched on) when the chromatin is open (active), and they
are inactivated (switched off) when the chromatin is condensed (silent).
White circles = unmethylated cytosines;
red circles = methylated cytosines.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
Basic principles of epigenetics:
DNA methylation and histone modfications
Changes to the structure of chromatin influence gene expression:
genes are inactivated (switched off) when the chromatin is condensed (silent),
and they are expressed (switched on) when chromatin is open (active).
These dynamic chromatin states are controlled by reversible epigenetic patterns of
DNA methylation and histone modifications.
Interestingly, repetitive genomic sequences are heavily methylated, which means
transcriptionally silenced.
Enzymes involved in this process include
- DNA methyltransferases (DNMTs),
- histone deacetylases (HDACs),
- histone acetylases,
- histone methyltransferases and the
- methyl-binding domain protein MECP2.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
DNA methylation
Typically, unmethylated clusters of CpG pairs are located in tissue-specific genes
and in essential housekeeping genes, which are involved in routine maintenance
roles and are expressed in most tissues.
These clusters, or CpG islands, are targets for proteins that bind to unmethylated
CpGs and initiate gene transcription.
In contrast, methylated CpGs are generally associated with silent DNA, can block
methylation-sensitive proteins and can be easily mutated.
The loss of normal DNA methylation patterns is the best understood epigenetic
cause of disease.
In animal experiments, the removal of genes that encode DNMTs is lethal; in
humans, overexpression of these enzymes has been linked to a variety of cancers.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
Esteller, Nat. Rev. Gen. 8, 286 (2007)
Esteller, Nat. Rev. Gen. 8, 286 (2007)
New: 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
periodicity of gene expression.
Sancar,
Nat. Struct. Mol. Biol. 15, 23 (2008)
WS 2010 – lecture 2
Cellular Programs
15
Intro of Arabidopsis thaliana
Arabidopsis thaliana is a small flowering plant that
is widely used as a model organism in plant
biology.
Arabidopsis is a member of the mustard
(Brassicaceae) family, which includes
cultivated species such as cabbage and radish.
Arabidopsis is not of major agronomic significance,
but it offers important advantages for basic
research in genetics and molecular biology.
TAIR
Some useful statistics for Arabidopsis thaliana
– Its small genome (114.5 Mb/125 Mb total) has been
sequenced in the year 2000.
– Extensive genetic and physical maps of all 5 chromosomes.
– A rapid life cycle (about 6 weeks from germination to mature
seed).
– Prolific seed production and easy cultivation in restricted
space.
– Efficient transformation methods utilizing Agrobacterium
tumefaciens.
– A large number of mutant lines and genomic resources many
of which are available from Stock Centers.
– Multinational research community of academic, government
and industry laboratories.
TAIR
Such advantages have made Arabidopsis a model organism for studies of the
cellular and molecular biology of flowering plants.
TAIR collects and makes available the information arising from these efforts.
Arabidopsis thaliana chromosomes
red: Sequenced portions,
light blue: telomeric and centromeric
regions,
black: heterochromatic knobs,
magenta: rDNA repeat regions
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 DAPI-stained
and small nucleolar RNAs (`RNAs')
chromophores
Nature 408, 796 (2000)
Arabidopsis thaliana genome sequence
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.
Nature 408, 796 (2000)
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
altered activity states”.
Zhang, Science 320, 489 (2008)
The epigenetic landscape of A. thaliana
The relative abundance of genes,
repeats, cytosine methylation and
siRNAs is shown for the length of A.
thaliana chromosome 1, which is ~30
Mb long.
diagram of
chromosome.
euchromatic arms,
pericentromeric heterochromatin;
centromeric core.
Henderson & Jacobson, Nature 447, 418 (2007)
Motiv density along Arabidopsis chromosomes
Distribution of genes, repetitive sequences,
DNA methylation, siRNAs, H3K27me3, and
low nucleosome density (LND) regions.
Left: chromosomal distributions on chr 1.
The x axis shows chromosomal position.
Zhang, Science 320, 489 (2008)