V2: Feedback loops control the mammalian circadian core clock

1. 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. Cellular Programs

2. 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 Programs

3. 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 3

4. 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) Cellular Programs

5. 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) Cellular Programs 5

6. 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)

7. 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)

8. 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.

9. 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)

10. 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)

11. 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)

12. 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)

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

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

15. 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

16. 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

17. 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.

18. 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 and small nucleolar RNAs (`RNAs') DAPI-stained chromophores Nature 408, 796 (2000)

19. 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)

20. 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)

21. The epigenetic landscape of A. thaliana diagram of chromosome. euchromatic arms, pericentromeric heterochromatin; centromeric core. 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. Henderson & Jacobson, Nature 447, 418 (2007)

22. 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)