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

frank.uhlmann@cancer.org.uk