1
Walther Flemming: ‘Zellsubstanz, Kern und Zelltheilung.’ Leipzig, 1882
Cohesin, Chromosomesand Cancer
David Hansemann (1890):Über asymmetrische Zelltheilung in Epithel Krebsen und deren biologische BedeutungVirchow’s Arch. Pathol. Anat. 119, 299-326
'Besonders wichtig erscheint die asymmetrische Theilung vom Standpunkte desPanmerismus, […], dass die biologischen Eigenschaften einer Zelle an bestimmtegeformte Elemente der Zelle gebunden sind,…'
'Es ist also wohl möglich, dass stellenweise eine Verzögerung der Längsspaltungeingetreten war.'
Aneuploidy in a human colon cancer
human karyotype
colon cancer
2
Lengauer et al. (1997)
Centromere 7Centromere 18
HT29HCT116
Genetic instability in colorectal cancer
• 85% of colocrectal cancers display chromosomal instability(as do most other solid tumours)
• a high degree of aneuploidy is taken as poor prognosis- evolution in new environment- resistance against treatment
• chromosomal instabilitiy can be seen in the earliest detectablelesions (2 mm adenomas)
Chromosomal instability: Facts
Does chromosomal instability cause cancer?
3
Effect of taxol in chemotherapy might depend on the checkpoint status
4
Saccharomycescerevisiae:mutations in>136 genes causechromosomeinstabilityP. Hieter (1990)
5
Tetraploidy as a route to aneuploidy (I)
a virus-induced cell fusion
b endoreplication
c cytokinesis failure
Tetraploidy as a route to aneuploidy (II)
a tetraploidy checkpoint (?)
b assymetric division on multipolar spindle
c regeneration of stable 2N karyotype
d stable propagation as tetraploids
Tetraploidy as a route to aneuploidy (III)
6
How important is tetraploidy as route to aneuploidy?
spontaneous non-disjunction in immortalized human keratinocytes (N/TERT-1)
leads to cytokinesis regression and binucleate formation
Shi and King, 2005
tetraploid p53- mouse mammary epithelial cells (transient cytochalasin B
treatment), but not diploid controls, gave rise to tumours in nude mice
Pellman, 2005
Tetraploidy as a route to aneuploidy (IV)
7
The cohesin cycle
Scc2/4
8
Milutinovich and Koshland 2003
Campbell and Cohen-Fix 2002
Haering and Nasmyth, 2003
How does cohesin hold together sister chromatids?
A topological interaction between cohesin and DNA
circular minichromosome
loss of the cohesin-DNA interaction by:
1 cleavage of Scc1 (separase)
2 engineered cleavage of Smc3 (TEV protease)
3 restriction of the minichromosome
Ivanov and Nasmyth (2005) Cell 122, 849-860
9
The Smc 'heads' are ABC ATPases that dimerize upon ATP binding:
ATP BindingWalker A: GxxGxGKS/TWalker B: hhDExDATP HydrolysisC-motif: LSGG
P. furiosus SMC:(Hopfner, 2004)
ATP Binding ATP HydrolysisDNA
ATP Binding ATP HydrolysisDNA
Scc2/4
10
50 nm
Atomic force microscopy reveals a head-hinge interaction
Cohesin Smc1/3 dimer:
opening ofheads or hinge?
Replisome
Replisome
+
Replisome
2. Cohesin re-assembly after fork passage
1. Replication fork sliding through the cohesin ring
a)
b)
And what happens during DNA replication?
Identification of replication products for segregation in mitosis
Biochemistry is easy with soluble proteins,but what does cohesin look like bound to chromosomes?
11
DNase digest
M=S N0(6 Rs)/(1-v2 ).
Siegel and Monty, 1966
12
why does a stable Scc1 cleavage productcause chromosome loss?
13
Sister chromatid cohesion factors in congenital disorders
human Scc2 (NIPBL): Cornelia de Lange syndrome
human Eco1 (ESCO2): Roberts syndrome
Hierarchical folding models of a chromosome
‘scaffold’?
A proteinaceous chromosome scaffold?
• salt extraction ofnon-histone proteins• visualisation ofchromosome scaffoldby electron microscopy
U. Laemmli
scaffold after chemicaltreatment, is there a stablescaffold in live cells?
14
Poirier and Marko, 2002
Newt chromosome between micro-pipettes
10 μm
+ D
Nase
Mitotic chromosomes are chromatin networkswithout a mechanically contiguous protein scaffold
Studying protein dynamics on chromosomes byFluorescense Recovery After Photobleaching (FRAP)
Topo
1. Topoisomerase II fused to Green Fluorescent Protein (GFP)2. Photobleaching of a defined region within the nucleus3. Does Topo II-GFP from the surrounding replenish fluorescense in the bleached area?
Answer, Yes: Topo II is not stably bound to chromosomes, but is dynamic and mobile
Christensen et al. 2002
Nse2
Sister chromatid cohesionDNA repair (sister recombination)Chromatin domain borderChromosome condensation
Haering, Nasmyth 2003
Eukaryotic Prokaryotic
15
Nse2
Haering, Nasmyth 2003
Eukaryotic Prokaryotic
Chromosome condensationTranscriptional silencingDNA repairReplication checkpoint signalling
Condensin and mitotic chromosome condensation
Condensin ( Ba: Barren (Brn1))
Maeshima and Laemmli, 2003
Nse2
Haering, Nasmyth 2003
Eukaryotic Prokaryotic
DNA repair( , UV; sister recombination?)
16
Nse2
Eukaryotic Prokaryotic
Nucleoid compactionChromosome segregation
(non-essential)
Haering, Nasmyth 2003
Chromatin Immunoprecipitation (ChIP) to analyse chromomal proteins
Protein crosslinking
to DNA
(formaledhyde)
Chromatin
preparation and
fragmentation
(sonication)
Immunoprecipitation
(ChIP)
Data analysis Hybridisation
Crosslink reversal,
DNA amplification,
and
labelling
CEN
Chr VI
Cohesin localisation along yeast Chromosome VI
Scc2/4 Scc2/4
CohesinCohesin loader
17
Scc2/4
ATPATP
ADP + Pi
Cohesin loading onto chromosomes by Scc2/4
Scc2
Scc1
Scc1(16oC
10 min)
Scc1(16oC
30 min)
Scc2/4
ATPATP
Scc2 Scc2 Scc2
Scc1 Scc1 Scc1
ADP + Pi
Cohesin loading onto chromosomes by Scc2/4
Scc2
Scc1
Scc1(16oC
10 min)
Scc1(16oC
30 min)
Scc2/4
ATPATP
Scc2 Scc2 Scc2
Scc1 Scc1 Scc1
ADP + Pi
Cohesin loading onto chromosomes by Scc2/4
18
Scc2
Scc1
Scc1(16oC
10 min)
Scc1(16oC
30 min)
Scc2/4
ATPATP
ADP + Pi
Scc2 Scc2 Scc2
Scc1 Scc1 Scc1
Cohesin loading onto chromosomes by Scc2/4
Scc2/4 Scc2/4
Cohesin
Cohesin loader
Scc2/4 Scc2/4
Cohesin
Cohesin loader
19
Scc2/4
Cohesin
Scc2/4
Cohesin loader
Scc2/4 Scc2/4
Pol II
Pol II
Pol II
Cohesin
Comparison of cohesin and condensin along chromosome V
cohesin (Scc1)
condensin (Smc2)
20
Cohesin
Scc2/4
Condensin
Cohesin/Condensinloader
An alternating pattern of cohesin and condensin structures the chromosome
Wittmann et al. 2001
Rieder et al.
Microtubule dynamics during mitosis
GTP, stable GTP, unstable (regulatory)minus end
plus end
21
The GTP/GDP status at themicrotubule end determinesits stability
GTP GDP
Surveillance of chromosome attachment
• Bipolar attachmentgenerates tension
• A tensionlesskinetochoreproduces await anaphasesignal
22
Screen for mutants that don’t monitor attachment
(Hoyt, Murray)
mad and bub genes mutated in
several human cancers
Mad2
Bub1Bub3
Mad1
Mad3
mad: ‘mitotic arrest defective’
bub: ‘budding un-inhibited by benomyl’
Mad2 localises to kinetochores until bipolar tension is achieved
How
ell e
t al.
2000
J. C
ell B
iol. 150,
123
3-12
50P
tk1
(rat
kan
garo
o ki
dney
epi
thel
ial)
cells
Mad2koff
Mad2
Mad2inactive ?
active ?
Mad2 turns over at anunattached kinetochoreand is converted into aconformation that inhibitsCdc20
23
XMAP 215
XKCM1 (Kinesin)
Loss of cohesionbetween sisterchromatidstriggers separationand movement ofkinetochores toopposite spindlepoles
ANAPHASE A
24
1 A sliding force isgenerated betweenoverlappingpole microtubules
2 Astral microtubulespull at thecentrosomes
1
2
ANAPHASE B
Conly Rieder et al.
Separase
Securin
uu
uu
u
APC
Cdc20
Cdk
Cyclin
uu
uu
u
Anaphase Followed by Mitotic Exit:
Conly Rieder et al.
Separase
Securin
uu
uu
u
APC
Cdc20
Cdk
Cyclin
uu
uu
u
Cdc14phosphatase
25
Conly Rieder et al.
Separase
Securin
uu
uu
u
APC
Cdc20
Cdk
Cyclin
uu
uu
uAPC
Cdh1
Cdc14phosphatase Cdk substrates
Sic1 Cdk inhibitor
(Sic1 transcr. Factor)Swi5
26
Separase
Securin
uu
uu
u
APC
Cdc20
Spo12
Slk19
Cdk
Tem1
Cdc15
Dbf2Mob1
Lte
1Bub2Bfa1
Net1- P
The mitotic exit network (MEN):
Phase 1:Separase-dependent early anaphase release (FEAR) Polo kinase, Slk19, Spo12
Phase 2:Postive feedback loop(activated by Cdc14)
Tem1 GTPase Bfa1/Bub2 GAP Lte1 GEF
Cdc15 kinaseDbf2/Mob1 kinase
Cdc14
phosphorylation of the Cdc14 inhibitor Net1
Polo
other organisms?budding yeast
Cdk
Cyclin
Cdk
Cyclin
G1 M->A G1 M->A
Separase Separase
?
cohesin cleavage cohesin cleavage
exit exit
phosphatases?Cdc14
27
Sorger, Annu. Rev. Cell Dev. Biol. 2003
62
48
kDa
Ask1-HA
cycl meta 15 30 45 60 min
anaphase
Sullivan et al., J. Biol. Chem. 2004
Ask1 dephosphorylation at anaphase onset:
1086420
Separase
0 10 20 30 40Time (min)
TEV1
0 10 20 30 40
TEV2
0 10 20 30 40pole
to p
ole
dist
ance
(μm
)
Anaphase B in the absence of Cdc14 activation
Frank Uhlmann
Cancer Research UK London Research Institute
44 Lincoln’s Inn Fields
London WC2A 3PX
UK