Sanger Institute:

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Sanger Institute Jürg Bähler
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Schizosaccharomyces pombe (Fission Yeast)

• unicellular eukaryote (fungus)

• genome 14 Mb, 5,000 genes

• easy to handle / genetics,
• relatively simple organism

• evolutionary distant to
• S. cerevisiae (budding yeast)

• no beauty but what a beast!

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Now which one is the higher eukaryote?!
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• Synchronization during cell cycle
• Centrifugal synchronization elutriation
• (selective synchronization)
• cdc25 block-release conditional muant
• (whole-culture synchronization)

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Expression profiling 4 major waves of gene
expression
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Transcriptional regulation of clusters 1 and 2
Cluster 1
Cluster 2
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Identification of regulatory promoter motifs
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Probably the best cell-cycle data in the world
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Core cell cycle-regulated genes
S. pombe (high amplitude)
S. cerevisiae (Cho Spellman)
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95
(4)
2981 orthologs in total
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Core cell cycle-regulated genes
Mitosis and cell division
CDC5, IPL1, KIN3 plo1, ark1, fin1 Polo, Aurora,
and NimA kinases CDC20 slp1 Activator of
APC SPO12 wis3 Putative cell-cycle
regulator KAR3, KIP1 klp5, klp6, klp8 Kinesin
microtubule motor MOB1, DBF2 mob1, sid2 Proteins
involved in MEN/SIN MYO1 myo3 Myosin II heavy
chain BUD4 mid2 Protein involved in
cytokinesis ACE2 ace2 Transcription
factor HOF1 imp2 Protein involved in cell
division DSE4 eng1 Glucanase for cell
separation CHS2 chs2 Protein involved in
septum formation TOS7 mac1 Putative role in
cell separation
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Core cell cycle-regulated genes
DNA replication
POL1, POL2 pol1 and cdc20 DNA polymerases a and
e RFA1 ssb1 Single-stranded DNA-binding
protein CDC6 cdc18 Regulator of DNA replication
initiation MRC1 mrc1 DNA replication checkpoint
protein RNR1 cdc22 Ribonucleotide
reductase SMC3, MCD1 psm3 and rad21 Cohesins HTZ1
pht1 Histone variant 8 histone genes 9 histone
genes Histones H2A, H2B, H3, and H4
Others mik1 SWE1 Kinase inhibiting
cyclin-dependent kinase cig2 CLB1-CLB6 B-type
cyclins msh6 MSH6 Mismatch-repair
protein rhp51 RAD51 DNA repair protein
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Modification of cell cycle-regulation in response
to environmental or genetic perturbations
Changes in genes expression during entry into
quiescent phase. Left gene expression levels.
Right Grow curve and glucose concentration in
the media.
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Regulation of Gene Expression at Multiple Levels
DNA DNA-bd prot.
Chromatin
transcription rate
mRNA RNA-bd prot.
Transcripts
decay rate
translation rate
Post-transcriptional control
Proteins
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Global data on different layers of gene
expression control
mRNAs
Expression profiling (steady-state mRNA levels)
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Our DIAMONDS team Sofia Aligianni (Postdoc)
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ChIP-chip cell cycle transcription factor complex

• MBF (E2F analogue) / Forkhead positive and
  negative roles
• Combinatorial and dynamic binding of different
  subunits
• as a function of cell cycle
• - Changes during meiotic cell cycle?

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Affymetrix tiling chipsBrian Wilhelm, Samuel
Marguerat, Chris Penkett, Stephen Watt

• 20 bp resolution each direction, entire S. pombe
  genome in both directions 2.5 million probes
• All transcripts, non-coding RNAs, antisense
  transcripts
• Transcript lengths and splicing
• High-resolution ChIP-chip

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• Conclusions
• Main discrepancies not in data themselves
• but in interpretation/analysis of data
• Integrated data better than any single data set
• Available data do not allow to reliably predict
  more than
• 400-500 periodic genes
• For S. pombe there are now more cell-cycle
  microarray
• data available than for any other organism

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What type and formats of data are of
interest? ArrayExpress http//www.sanger.ac.uk
/PostGenomics/S_pombe/ CBS http//www.cbs.dtu.d
k/cellcycle/
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Determine ribosome numbers associated with
transcriptsTranslational Profiling
Fractionate polysomes, cDNA labelling,
hybridization to microarrays
Resolve ribosome-bound mRNAs by density gradient
centrifugation
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Measurement of mRNA stability
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Determination of mRNA stability
Transcript levels at different times after
transcriptional shut-down with phenanthroline
Samuel Marguerat
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Perturbations of cell growth/proliferation

• Gene deletions/overexpressions (TFs,
  chromatin/transl. regulators, RBPs, non-coding
  RNAs, conserved/unknown prot. that are
  periodically expressed)
• Promoter swaps and constitutive expression for
  selected key regulators
• Fusion proteins (SET domain of methyltransferase
  with TF DNA-bd domain)

• Starvation/G0 experiments -N (G1 arrest), -G (G2
  arrest) and refeeding
• Rapamycin treatment and TOR pathway mutants
  (growth control at multiple levels, ribosome
  synthesis, differences to budding yeast TOR
  kinases)

• Uncouple growth from division cdc2 mutant,
  growth arrest with nutrients
• Diploid and tetrapolid vs haploid cells
  coordination growth/division

• Long-term adaptation/chemostat Ace2/MBF act in
  parallel -gt extend G1 (nitrogen source or cyclin
  mutant) re-wiring of regulatory network?
• Also promising constructs from above / 2n vs 1n
  / Hsp90 mutant