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Dr. Lane LECTURE #5. 1. 267A: Cell Cycle 5. Dr. Timothy F. Lane Jonsson Comprehensive Cancer Center, Department of Biological Chemistry Office: 549 BSRB email: [email protected] Actin. Tubulin. DNA. Syncitial Divisions in Drosophila embryo. From Bill Sullivan UCSC.

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Dr. Timothy F. Lane Jonsson Comprehensive Cancer Center, Department of Biological Chemistry

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Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Dr. Lane LECTURE #5

1

267A:Cell Cycle 5

Dr. Timothy F. Lane Jonsson Comprehensive Cancer Center,

Department of Biological Chemistry

Office: 549 BSRB

email: [email protected]

Actin

Tubulin

DNA

Syncitial Divisions in Drosophila

embryo. From Bill Sullivan UCSC

These notes are posted on the www page!

http://bio.research.ucsc.edu/people/sullivan/images.html


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Last time:

We talked in even greater length about the regulation of the cell cycle by cyclins and cdks:

i. We focused on how regulatory signals are terminated so that the cell can

enter subsequent stages, or a new cell cycle.

ii. Focusing on M, we identified a cycB degrading activity called APC.

iii. We found that securins were also degraded by APC, but much earlier.

iv. that turnover could be very precisely programmed by different

E3 ligases (CDH1 for cycB and cdc20 for securins).

v. We identified sequences within the targets (cycB and cdc20) that provided

specific interaction surfaces for binding of APCCDH1 or APCCDC20)

vi. We found that APCCDH1 function continued into G1, and that G1 cyclins

were required to terminate APCCDH1 function.

We have now discussed 4 levels of regulation for cyclins and cdks, including:

i. Transcriptional control of synthesis (pRB represses cycE)

ii. Phosphorylation (wee1 etc) / dephosphorylation (cdc25)

iii. binding to CKIs (Far1, Sic, p15)

iv. turnover by APC.


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Goals:

The context will be Mitosis.

How M-phase is regulated/ended!

Examine how cyclins are turned over in the context of M


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Mitosis:

WHAT DIRECTS APC ACTIVITY:

The APCcdc20 complex peaks in activity in M,

and is responsible for Pds1p/securin proteolysis and anaphase onset.

APCCdh1 cyclosome activity peaks in Anaphase/G1,

and is responsible for promoting Clb2p proteolysis and consequent G1 entry

from M

Pds1/Cut2/securin

Nasmyth, 1999 TIBS 24:98


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

CDH1

separase

LAST TIME: Cell cycle dependent protein degradation during Mitosis

APCcdc20 complex responsible for Pds1p/securin proteolysis and anaphase onset.

APCCdh1 complex responsible for cycB proteolysis and G1 entry.

G1

S

APC

Cyclin B

G2

M

CDH1

o

o

o

o

o

o

Cdc 20

securin

APC


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

THE G1 CYCLINS ARE PREPARED FOR PROTEASOME DEGRADATION

BY A DISTINCT UBIQUITINATION MECHANISM –THE SCF COMPLEX

Mitogen signal for

G0 cells

Cyclin

D1

cdk4

R.P.

Cyclin

E

Go

G1

Cyclin B

M

cdk2

cdk2

Cyclin

A

G2

cdk2

S


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

THE G1 CYCLINS ARE PREPARED FOR PROTEASOME DEGRADATION

BY A DISTINCT UBIQUITINATION MECHANISM –THE SCF COMPLEX

Cdc34 mutants arrest at the G1/S transition,

Cdc34p encodes a ubiquitination enzyme1.

Cln2

cdc28

Go

G1

M

G2

S

1Goebl et al 1988 Science 241:1331


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

THE G1 CYCLINS ARE PREPARED FOR PROTEASOME DEGRADATION

BY A DISTINCT UBIQUITINATION MECHANISM –THE SCF COMPLEX

Cdc34 mutants arrest at the G1/S transition,

Cdc34p encodes a ubiquitination enzyme1.

Recombinant cdc34 protein has endogenous

ubiquitin-conjugating activity with histone 2A1

Cln2

cdc28

Go

G1

M

G2

S

1Goebl et al 1988 Science 241:1331


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Ub

Ub

Ub

Ub

THE G1 CYCLINS ARE PREPARED FOR PROTEASOME DEGRADATION

BY A DISTINCT UBIQUITINATION MECHANISM –THE SCF COMPLEX

Cln2

(recomb)

Cdc34 mutants arrest at the G1/S transition,

Cdc34p encodes a ubiquitination enzyme1.

p

cdc28

p

cln2

Recombinant cdc34 protein has endogenous

ubiquitin-conjugating activity with histone 2A1

Extract from cyclin depleted, G1 arrested S.c.

+

Cdc34p ubiquitinates Cln2

and leads to Cln2 destruction2.

The Cln2 in the complex is phosphorylated!

In these in vitro reactions, an additional

modified form of Cln2-P is observed.

This Cln2-P modified form can be

precipitated with anti-ubiquitin.

cdc34

Ub

Ub

p

Ub

p

cln2

The ubiquitination occurs in extracts of wt.

cells, but not extracts of cdc34 t.s. cells.

Moreover, ubiquitination did not occur in cdc28 t.s.

extracts2.

1Goebl et al 1988 Science 241:1331

2Deshaies et al 1995 EMBO J. 14:303


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Cln3

Beta -galactosidase

THE G1 CYCLINS ARE PREPARED FOR PROTEASOME DEGRADATION

BY A DISTINCT UBIQUITINATION MECHANISM –THE SCF COMPLEX

Cln3 is phosphorylated by Cdc28/p34,

and is then ubiquitinated by a

Cdc34-dependent step, leading to

Cln3 degradation.

As with Cln2, Cln3 is not degraded in

cdc28 t.s. mutants!

Expt.

Create a Cln3/gal fusion protein

Expressed in wt or cdc34 mutant yeast.

The proteins were labeled for five minutes

with radioactive amino acids, then the cells

were chased with cold amino acids.

At the times shown, Cln3-gal was

immunoprecipitated and subjected to

electrophoresis and autoradiography

Wild-type yeast strain

cdc34 mutant yeast strain

“[35S]-Met” amino acid pulse

cold amino acid chase

i.p. Electrophoresis

Yaglom et al, 1995 Mol. Cell. Biol. 15:731


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

THE G1 CYCLINS ARE PREPARED FOR PROTEASOME DEGRADATION

BY A DISTINCT UBIQUITINATION MECHANISM –THE SCF COMPLEX

Cln3 is phosphorylated by Cdc28/p34,

and is then ubiquitinated by a

Cdc34-dependent step, leading to

Cln3 degradation.

As with Cln2, Cln3 is not degraded in

cdc28 t.s. mutants!

Expt.

Create a Cln3/gal fusion protein

Expressed in wt or cdc34 mutant yeast.

The proteins were labeled for five minutes

with radioactive amino acids, then the cells

were chased with cold amino acids.

At the times shown, Cln3-gal was

immunoprecipitated and subjected to

electrophoresis and autoradiography

Cdc34

Cln3-beta gal remaining

w.t.

RESULT: Cln3 is very stable in cells

lacking the cdc34 ubiquitin ligase

15

30

45

60

Chase time (min)

Yaglom et al, 1995 Mol. Cell. Biol. 15:731


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

p

p

CKIs are PREPARED FOR PROTEASOME DEGRADATION

BY A CDC34 DEPENDENT PROCESS

SIC1 degradation in G1 is necessary for entry into S phase

Recall from Lecture #3:

To enter S, p40/Sic1 protein is phosphorylated and then degraded,

releasing the inhibition of the CDK activity.

cdc28

cdc28

SIC 1

SIC 1

p

p

Clb2

Clb2

G1

S

Histone

Histone

Histone

Histone

1Mendenhall, 1993 Science 259:216

2Schwob et al, 1994, Cell 79: 233


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

cdc34

P

P

P

P

P

P

P

CKIs are PREPARED FOR PROTEASOME DEGRADATION

BY A CDC34 DEPENDENT PROCESS

  • The transition from G1 to S should involve a rapid initiation of DNA synthesis

  • But must occur only after a series of events in G1 has taken place.

  • Can not be dependent on small errors in the level of phosphorylation of Sic1.

  • In fact, six out of nine phosphorylation sites on Sic1 must be phosphorylated before

  • ubiquitination/degradation becomes effective.

  • This results in a delayed, but sharp initiation of Sic1 degradation,

  • activation of cdc2/S cyclin protein kinase activity, and the G1/S transition.

cdc28

Cln2

SIC 1

SIC 1

DEGRADATION

G1

S

Nash et al, 2001 Nature 414:514-521


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

cdc34

cdc4

ub

CDC34 DEPENDENT UBIQUITINATION REQUIRES THE SCF complex

Several other gene products are required for the G1/S transition

When mutated – some produce phenotypes like cdc34 (i.e., arrest at G1/S).

These include: skp1, cdc53 and cdc4.

The SCF complex is required to

recognize pSic1 protein and catalyze

its ubiquitination.

Skowyra et al expressed recombinant

forms of all these proteins in an insect cell system

and used co-immunoprecipitation procedures

to demonstrate that four proteins

– Cdc34, Cdc4, Cdc53, and Skp1 –

could form a macromolecular complex1.

Feldman et al demonstrated that this complex,

in the presence of ubiquitin and the E1 and E2 enzymes,

can ubiquitinate phosphorylated Sic12.

The cdc4 protein recognizes the multi-phosphorylated

Sic1 protein, and causes its binding to the ubiquitinating complex

Sic 1

p

p

Skp1

Cdc53

1Skowyra et al 1997 Cell 91:209

2Feldman et al 1997, Cell 91: 221


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

cdc34

cdc4

ub

ub

ub

ub

ub

ub

G1

Cyclin

S-phase

Cyclin

S-phase

Cyclin

cdc28

cdc28

cdc28

Sic 1

Sic 1

p

p

Inactive kinase

Skp1

Cdc53

S-phase

Cyclin

proteasome

S-phase

Cyclin

cdc28

cdc28

Sic 1

p

p

active kinase


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

p

p

p

p

p

p

p

p

cyclin

CDC34 DEPENDENT UBIQUITINATION targets multiple targets

p

cdk

cdk

WEE1

cyclin

Phosp.

Thr 161

cyclin

WEE1

Dephosp.

Thr 161

p

cdc25

p

cdk

cdc25

cyclin

ACTIVE CDK

Degrade

cyclin

CKI

CKI

cdk

cdk

p

cyclin


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

cdc34

cdc4

ub

Structure of SCF

cdc4 associates with Skp1 through

a motif on Cdc4 that is called an F-box.

The name SCF complex

(for Skp1, Cdc53, F-box protein).

F-box

cdc4

Sic 1

p

p

Skp1

Cdc53

Bai et al 1996, Cell 86:263

Skowra et al 1997, Cell 91:209


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Structure of SCF

cdc4 associates with Skp1 through

a motif on Cdc4 that is called an F-box.

The name SCF complex

(for Skp1, Cdc53, F-box protein).

Bai et al identified a number of proteins that contain

F-box domains. They suggested that different

F box domain proteins might select

alternative targets for SCF.

Skowra et al then demonstrated

that this is the case:

F-box

cdc4

F box-2

F box-3

Bai et al 1996, Cell 86:263

Skowra et al 1997, Cell 91:209


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

cdc34

ub

ub

ub

ub

ub

ub

Structure of SCF

cdc4 associates with Skp1 through

a motif on Cdc4 that is called an F-box.

The name SCF complex

(for Skp1, Cdc53, F-box protein).

Cln2

Cln2

p

p

Bai et al identified a number of proteins that contain

F-box domains. They suggested that different

F box domain proteins might select

alternative targets for SCF.

GRR1

Skp1

Cdc53

Skowra et al then demonstrated

that this is the case:

F-box protein, Grr1, associates with SCF components

Skp1, Cdc53, and Cdc34.

SCFGrr1not bind to p-Sic1, but does bind to p-Cln2 & p-Cln3

It appears that the SCF complex –

interacts with a variety of F-box proteins,

via the Skp1 recognition site.

The F-box containing subunits target the SCF

ubiquitination activity to different substrates.

Cln2

p

p

Bai et al 1996, Cell 86:263

Skowra et al 1997, Cell 91:209


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Structure of SCF: Selecting multiple targets:

SCFGRR1 targets G1 cyclins for proteosomal turnover:

SCFCDC4 targets Sic1 for proteosomal turnover:


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

p

p

Structure of SCF: Selecting multiple targets:

Wee1 is also degraded by an SCF Cdc34 mechanism.

Recall that wee1 (a kinase that phosphorylates p34cdc2) in S. pombe

must be inactivated for cells to move from G2 into M,

wee1 protein is phosphorylated and degraded.

wee1

ATP

Cdc2

p34

Cdc2

p34

cdc 13

cdc 13

ATP

HISTONE H1

H1-P

Michael and Newport 1998, Science 282: 1886


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

p

p

Structure of SCF: Selecting multiple targets:

Wee1 is also degraded by an SCF Cdc34 mechanism.

Recall that wee1 (a kinase that phosphorylates p34cdc2) in S. pombe

must be inactivated for cells to move from G2 into M,

wee1 protein is phosphorylated and degraded.

Expt: Using an oocyte extract system,

it was demonstrated that a dominant-negative cdc34 mutant

can block wee1 degradation.

Thus, in addition to ubiquitinating G2 phase cyclins and CKIs,

an SCF complex regulates entry into mitosis by initiating wee1 protein degradation.

What F-box protein targets wee1 kinase for ubiquitination?

wee1

wee1

DEGRADATION

cdc34dn

Michael and Newport 1998, Science 282: 1886


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

cdc34

ub

What F-box protein targets Wee1 for destruction?

Ayad et al used a biochemical search to

find new substrates for APCCDH1.

They in vitro translated pools of cDNAs,

mixed them with:

[mitotic extracts + CDH1]

vs [mitotic extracts without CDH1],

looked for proteins degraded in the presence of CDH1.

RESULT:

One of the substrates was called Tome-1.

Wee1

p

p

???

Skp1

Cdc53

Ayad et al 2003, Cell 113:101-113


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

G2/M transition

What F-box protein targets Wee1 for destruction?

As expected for a APCCDH1 substrate,

Tome-1 is degraded in a cell cycle dependent

manner similar to cyclin B (A)

The Tome-1 sequence contains

what looks like an F-box.

Tome-1 co-IPs with Skp-1 and Cul-1.

If Tome-1 is reduced by using a siRNA

- wee1 degradation is inhibited.

The experiment was done by pulse labeling

cells with [35S]cys after incubation with siRNA,

then doing IP’s at various times after labeling.

Non-mitotic extracts can be stimulated to enter M,

and activate CDK activity on histones.

If Tome-1 is depleted from extracts with anti-Tome-1,

and the extracts are then stimulated.

Tome-1 depleted extracts enter mitosis later.

G1/S synchronized cells.

100

Tome-1 siRNA

Percent wee1

50

Control RNA

50

150

Time (min)

60

40

H1 kinase activity

Tome-1 depleted extract

20

Time (min)

50

100

Ayad et al 2003, Cell 113:101-113


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

cdc34

ub

ub

ub

ub

ub

ub

What F-box protein targets Wee1 for destruction?

wee1

Tome-1 is named for trigger of mitotic entry.

Tome-1 helps to regulate the transition

from G2 into M by acting as an F-box protein

leading to the degradation of wee1,

shifting the wee1/cdc25 balance in the

direction of cdc25.

This causes dephosphorylation of cdc2

and activation of the cdc2/cyclin B kinase

activity required for mitosis.

However, Tome-1 is degraded by an

APC-dependent process, so that wee1

can be built back up in the next cycle,

preventing premature mitosis.

TF: Tome-1 connects the APC and SCF

protein degradation cycles.

From the yeast data base, there are

over 20 “F-box” containing proteins

that are candidates for SCF-regulatory

molecules that control cell cycle transitions

of various types, by promoting ubiquitination

and degradation Koepp et al 1999 Cell 97:431

wee1

p

p

wee1

p

p

Tome-1

Skp1

Cdc53

wee1

p

p


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

p

p

p

p

p

p

p

p

cyclin

cyclin

p

cdk

cdk

WEE1

cyclin

Phosp.

Thr 161

cyclin

WEE1

Dephosp.

Thr 161

p

cdc25

p

cdk

cdc25

ACTIVE CDK

Degrade

cyclin

CKI

CKI

CKI

cdk

cdk

p

cyclin


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

SCF

SCF

SCF

CDH1

Grr1

Cln2

separase

cdc4

Sic1

CDH1

APC

G1

S

Tome-1

APC

Cyclin B

G2

M

wee1

CDH1

o

o

o

Tome-1

o

o

o

Cdc 20

securin

APC


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

The SCF complex

The APC/cyclosome


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

1)Replication of the DNA must occur once,

for each gene in a cell cycle, and

G2 phase cells are fused to S phase cells – the G2 phase cell DNA does not replicate,

despite the presence of a factor that can cause G1 phase cell DNA to replicate.

Is there a factor that “licenses ” DNA replication for G1 phase cells?

a factor that is removed after replication in S phase?

If so, the DNA must be re-licensed before the next S phase.

DNA Replication Licensing


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

DNA Replication Licensing

DNA replication occurs at origins of replication (ORC).

The origin replication complex, or ORC,

binds to replication origins.

The ORC is “always” associated with

origins of replication.


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

DNA Replication Licensing

“Licensing” occurs as the cells complete mitosis:

Cdc18p, Cdt1p, and the MCM proteins

(the mini-chromosome maintenance oligomer)

are loaded onto the ORC,

licensing it for replication.


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

DNA Replication Licensing

Then the MCM is added

Unlicensed origin


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

DNA Replication Licensing

The licensed replication complex is activated

for DNA replication at the G1/S transition.

In yeast, this activation requires

Cdc28/clb5 or Cdc28/clb6 –“the S phase cyclins”.

In mammalian cells, this activation requires

Cdk2-cyclinE. The activation of the licensed

replication complex by the CDK results in

additional proteins binding to the origin. The

ORC is left at the origin, while the assembled,

activated replication machinery proceeds down

the DNA. The functional target(s) of the CDK required

to activate a licensed origin are not yet known.

A cdk/S phase cyclin reaction(s) is required to initiate DNA synthesis


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

DNA Replication Licensing

After DNA replication is initiated, Cdc6/18 is then destroyed by an SCFcdc4 ubiquitination and

proteasome degradation.

Unlicensed origin


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

High throughput screens:

Do the temperature sensitive screens of Hartwell and Nurse identify all cell cycle genes?

Stevenson et al used over-expression of yeast genomic fragments and cDNA libraries, in which

the genes are over-expressed from the GAL1 promoter, to look for genes that would cause cell

cycle arrest in S. cerevisiae when the cells are shifted to galactose. Using high-throughput

screening, 150,000 colonies were screened,

179 genes/ORFs were identified that modulate the cell cycle.

Cells were analyzed by flow cytometry after 6-8 hours in the presence of galactose.

Strains in the left column are shifted toward a 1C amount of DNA relative to cells containing a

control plasmid, cells in the right column are shifted toward a 2C content of DNA.

43% of the known genes cloned had previously been associated with cell cycle.

19 new ORFs were identified that modulated cell cycle.

Seven of these ORFs cause accumulation in M phase, when examined microscopically.

Stevenson et al 2001,PNAS 98: 3946


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Control morphology studies and flow cytometry patterns for S. Cerevisiae

Strains in the left column are shifted toward a 1C amount of DNA relative to cells containing a

control plasmid, cells in the right column are shifted toward a 2C content of DNA.

43% of the known genes cloned had previously been associated with cell cycle.

19 new ORFs were identified that modulated cell cycle.

Seven of these ORFs cause accumulation in M phase, when examined microscopically.

Stevenson et al 2001,PNAS 98: 3946


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Control flow cytometry patterns for S. Cerevisiae

150 thousand colonies were screened. 179 colonies were isolated in which over expression of gal-driven genomic fragments or cDNAs caused cell cycle arrest.

Stevenson et al 2001,PNAS 98: 3946


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Strains in the left column are shifted toward

a 1C amount of DNA relative to cells

containing a control plasmid.

Stevenson et al 2001,PNAS 98: 3946


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Strains in the right column are shifted toward

a 2C amount of DNA relative to cells

containing a control plasmid.

Stevenson et al 2001,PNAS 98: 3946


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

High throughput screens:

Spellman et al. synchronized yeast cells three ways

(with mating factor, using the cdc15 ts mutant, and using an elutriation rotor).

They extracted RNA at various times after release from the cell cycle blocks, and then

performed microarray analyses of mRNA levels for the whole yeast genome,

versus RNA from exponentially growing cells.

They first used “phase analysis,” in which they plotted the peak time of expression for these genes.

They found 800 cell cycle regulated genes;

300 in G1, 71 in S, 121 in G2, 195 in M and 113 “M/G1” genes.

They compared the upstream promoter sequences of the genes, and found increased presence

of appropriate transcription factor response elements.

They then used “cluster analysis”, which “sorts through all the data to find the pairs of genes

that behave most similarly in each experiment, and then progressively adds other genes to the

initial pairs to form clusters of apparently co-regulated genes.”

RESULT: Nine clusters were identified (3 in G1, 2 in S, 1 in M, and 3 in M/G1).

About half the 800 cell cycle regulated genes are in these nine clusters.

Spellman et al. 1998, Mol Biol Cell. 9: 3273


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

Analysis of the 5’ regions of the genes in a cluster shows that such genes share common promoter

elements.

For example, within the G1 group of genes is the “CLB2 cluster” of 76 genes .

CRCGAAA

ACGCGN

52% of the G1 “CLB2 cluster” genes have this sequence in the promoter; only 13% of all yeast genes have this sequence.

58% of the G1 “CLB2 cluster” genes have this sequence in the promoter; only 6% of all yeast genes have this sequence.

Spellman et al. 1998, Mol Biol Cell. 9: 3273


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

76 genes are in the G1 “CLB2” cluster

SBF

MBF

Swi4

Swi6

Mbp1

Swi6

CRCGAAA

ACGCGN

52% of the G1 “CLB2 cluster” genes have this sequence in the promoter; only 13% of all yeast genes have this sequence.

58% of the G1 “CLB2 cluster” genes have this sequence in the promoter; only 6% of all yeast genes have this sequence.

Spellman et al. 1998, Mol Biol Cell. 9: 3273


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

All the data from Spellman et al, 400,000 data points, are freely available on the web

for investigation and manipulation by any informatics aficionados.

SBF

MBF

Swi4

Swi6

Mbp1

Swi6

CRCGAAA

ACGCGN

However, all is not necessarily well…..

Shedden and Cooper [Nuc. Acids. Res. 30:2920 (2002)] have reviewed this paper and

conclude

1) “…when the degree of cyclicity for genes in different experiments are compared,

a large degree of non-reproducibility is found.”

2) “Specific genes can show a wide range of cyclical behavior between different experiments;

a gene with high cyclicity in one experiment can show essentially no cyclicity in another

experiment.”

Spellman et al. 1998, Mol Biol Cell. 9: 3273


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

Cell cycle gene profiling has also been extended to mammalian cells.

Cho et al synchronized fibroblasts by a double thymidine block and analyzed mRNA profiles

every two hours by microarray, for cyclically expressed genes.

Twelve “patterns” were identified, which included 731 transcripts. An attempt to classify these

patterns into groups of genes involved in related functions is described.

Whitfield et al synchronized Hela cells by (1) double thymidine block, (2) high thymidine followed

by nocodazole or (3) mitotic collection and pooling on ice. Cells were released, RNA was isolated

at various times, and microarray analysis was performed.

They identified ~850 cell cycle regulated genes.

Extensive temporal correlations and functional classifications are provided.

Cho et al 2001, Nature Genetics 27: 48

Whitfield et al 2002, Mol. Biol. Cell 13: 1977


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

BUT THERE ARE REAL PROBLEMS:

Whitfield et al compared the ~700 genes from the Cho et al paper with 595 genes

in their own study.

Only 96 genes were identified as cell cycle regulated in both studies!!

They comment “We have no ready explanation of this difference in results, except to note

that there are differences in the cell lineage, the microarray technology, and in the analysis

methods….

We suspect that the most significant differences may well be in the degree of synchrony

achieved…”

591

GENES

731

GENES

CHO cells

HeLa cells

Only 96 cell cycle regulated genes overlap in these two studies!


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

BUT THERE ARE REAL PROBLEMS:

Whitfield et al compared the ~700 genes from the Cho et al paper with 595 genes

in their own study.

Only 96 genes were identified as cell cycle regulated in both studies!!

They comment “We have no ready explanation of this difference in results, except to note

that there are differences in the cell lineage, the microarray technology, and in the analysis

methods….

We suspect that the most significant differences may well be in the degree of synchrony

achieved…”

Again, all is not well….. Shedden and Cooper have also reviewed the data from the Cho et al

article. They conclude that the cyclic variations observed do not support the proposal that

there are numerous cell-cycle-specifically expressed genes in human cells.”

591

GENES

731

GENES

CHO cells

HeLa cells

Only 96 cell cycle regulated genes

overlap in these two studies!

Shedden and Cooper 2002, PNAS 99: 4379


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

Genomic Analysis of Transcription factor Binding (ChIP on CHIP).

A variety of transcription factors have been shown to bind to the upstream regions of members

of the various “clusters” of cell-cycle regulated genes in S. cerevisiae.

G1 phase of the yeast cell cycle bind either

SBF, a heterodimer of Swi4p and Swi6p, or

MBF, a heterodimer of Mbp1p and Swi6p.

SBF

MBF

Swi4

Swi6

Mbp1

Swi6

CRCGAAA

ACGCGN

Iyer et al 2001, Nature 409: 533


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

ChIP positive genes

All

intergenic

sequences

Sequence

SBF

MBF

CRCGAAA 10% 49% ---

ACGCGN 20% --- 59%

HIGH THROUGHPUT SCREENING, GENOMICS, BIO INFORMATICS

AND PROTEOMICS COME TO CELL CYCLE ANALYSIS

Genomic Analysis of Transcription factor Binding (ChIP on CHIP).

Iyer et al used chromatin immunoprecipitation assays to identify genes that bind SBF and MBF

targets.

They identified about 200 candidate target genes.

49% of the Swi4 ChIP targets contain CRCGAAA, the SBF target;

only 10% of all intergenic regions have this sequence.

59% of the MBF putative targets have the ACGCGN sequence;

only 20% of all intergenic regions have this sequence.

Of particular interest: “Not every promoter bound by SBF or MBF in vivo contained a

recognizable consensus binding site. Moreover, most of the coding sequences and many of

the promoters that contain the consensus sequences show no evidence of binding to these

factors in vivo.”

Using the data of Spellman et al, they found that 66% of the genes identified by ChIP

with Swi4p,Swi6 or Mbp1 are cell cycle regulated, as opposed to 13% of all yeast genes.

Iyer et al 2001, Nature 409: 533


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

(Swi4 or Mbp1)

Anti-Swi4

Anti-Mbp1


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

c

RB

RB

Cyclin

D1

p

CHECKPOINTS AND THE CELL CYCLE

To control for undesired outcomes in cell cycle processes, cells have developed "checkpoints".

Endogenous genes

altered in human

cancer

CDK

4

E2F

E2F

Late G1, Early S

Go, Early G1

What would you expect the phenotype of a cell that makes no RB to be? What would you expect the phenotype of a cell to be if it produces cyclin D1 when it should not?


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

CHECKPOINTS AND THE CELL CYCLE

Endogenous genes

altered in human

cancer

CKI

CDK

CKI

CDK

p

p

cyclin

cyclin

Active CDK

Inactive CDK

What would you expect the phenotype of a cell that makes no CKI to be?


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

p

p

p

p

p

p

p

p

CHECKPOINTS AND THE CELL CYCLE

Growth factor binding to receptor and triggering a mitogenic G0 to G1 response

RAS

GAP

GNEF

GRB2

RAF

MEK

MAPK

MAPK

ATP

Endogenous genes

altered in human cancer


Dr timothy f lane jonsson comprehensive cancer center department of biological chemistry

Three previous M267 exams on cell cycle have been placed on the www site

The exam will be joined with Dr. Lusis. Open Book.


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