V2: Feedback loops control the mammalian circadian core clock

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)


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.


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.


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.



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)

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

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.


Strands of DNA are wrapped around histone octamers, forming nucleosomes.

These nucleosomes are organized into chromatin, the building block of a

chromosome.

Cytosine methylation

3-6 % of all cytosines are methylated in human DNA.

Esteller, Nat. Rev. Gen. 8, 286 (2007)

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.

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.





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)

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 chromosomesred: 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 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

altered activity states”.Zhang, Science 320, 489 (2008)

The epigenetic landscape of A. thaliana

Henderson & Jacobson, Nature 447, 418 (2007)

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.

Motiv density along Arabidopsis chromosomes

Zhang, Science 320, 489 (2008)

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.

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V2: Feedback loops control the mammalian circadian core clock