The egg and the nucleus: a battle for supremacy

J. B. Gurdon

Cambridge, England.

Stockholm: 7 December, 2012.

Attack by the egg

Background

Content

Prospects

Defense by the nucleus

The original question Do all cells in the body have the same sets of genes?

Background

Briggs and King, 1952. Proc. Nat. Acd. Sci., USA

Michail Fischberg

1-nucleolus 2-nucleolus

Intestinal Tract of Feeding Tadpole

(GFP-marked))

Partial blastula

Nuclear Transplant Embryo Graft

GFP

Muscle derived from intesti by nuclear transfer and grafts

GFP-muscle derived from intestine nuclei

Efficiency of nuclear reprogramming by nuclear transfer to eggs

Switch between cell-types: e.g. intestinal epithelium to muscle and nerve.

First nuclear transfers……………..……..…15%

Serial nuclear transfers…………..………..….7%

Grafts from nuclear transfer embryos……….8%

30% Total:

Wilmut, Campbell et al 1996 and 1997.

40

30

20

10

% of total nuclear transfers

Stage of donor nuclei

Mammalian nuclear transfers

Xenopus nuclear transplants reaching feeding tadpole stage

Nuclear transfer success decreases as donor cells differentiate

reaching birth

Blastocystt Blastula Blastula Adult Differentiation

Derivation of functional heart from adult monkey skin (Byrne et al., 2008)

Donor of skin

Embryo

Embryonic stem cells

Egg

+

Add factors

Skin cell nucleus

Cloning

Donor of eggs

Stem cell creation

Increase cell number

Differentiation

Beating heart muscle

Epigenetic memory

muscle

Donor tadpole

Muscle cell

Nuclear Blastula

Neurect -oderm

Endoderm

Embryos derived from muscle nuclei remember their origin even in their nerve and endoderm cells

No transcription

High expression

of

56%

52%

genes

Transcription

transfer

Nature Cell Biol. 2006

muscle

Donor tadpole

Muscle cell

Nuclear Blastula

Neurect -oderm

Endoderm

H3.3 is required for epigenetic memory.

No transcription

High expression

of

3%

5%

genes

Transcription

transfer

Elimination by H3.3 mutated from K4 to E4.

H3.3-E4 mutant mRNA

K4, methylatable lysine.

E4, gutamine

EPIGENETIC MEMORY

Can be explained if:

1. H3.3 promotes continuing transcription of active genes, and if

2. Egg cytoplasm reverses gene transcription with a 50% efficiency.

First meiotic prophase oocytes

to analyse the mechanism of nuclear reprogramming

UV

DNA replication

transcription only

Blastula SSomatic cell nucleus

New gene expression New cell types

Frog

Replication errors in Transplanted somatic nuclei

Postzygotic

Single nuclear transfer to eggs in second meiotic metaphase

Incomplete DNA replication

damages somatic nuclei

transplanted to eggs

Days culture O 4

Intense transcription

(no DNA replication)

New gene expression

Germinal vesicle

[Egg progenitor]

Multiple nuclei transferred to growing oocytes in first meiotic prophase

Oocyte

No new cell type

100 days

Oocyte

Egg

Blastula

Oocyte formation prepares the egg for develpment

Germ cell

Transcription G

Lineage selection

Chromatin decondensation Intense gene transcription DNA demethylation

Xenopus oocyte and germinal vesicle

GV

1 mm

.

.

Oocyte Egg Embryo

First meiotic prophase

Second meiotic metaphase

Mid blastula

The oocyte germinal vesicle contents

Matur -ation

contribute to post-fertilization development

Germinal vesicle

Mammalian stem cell genes are rapidly activated in mammalian nuclei transplanted to Xenopus oocytes

1 2 3 4

nuclei

Mouse/human somatic cell

Days

High

Low

thymus Mouse

HeLa Oct4 Nanog

Nuclei of differentiated cells are reprogrammed slowly.

Sox2

Oocyte transcription assay

Living oocyte transcription assay 1. Multiple somatic nuclei in one oocyte.

2. Linear accumulation of new transcripts.

3. Multiple initiations of transcription per gene per day.

4. Oocyte injections show resistance

Transcriptional activation:

attack by egg cytoplasm

Male

Female pronucleus

pronucleus

HFertilized mouse egg Mouse sperm

Mammalian cultured cell nuclei

Just after injection

:

1-2 days after nuclear transfer

Histone replacement in transplanted nuclei.

Cultured cell nuclei in oocyte GV

H1o histone-GFP replaced by

Somatic

oocyte B4 histone-RFP

Jerome Jullien

Donor H3.3 Oocyte H3.3 Merge

0%

82%

99%

5

10

15

. Histone H3.3 is incorporated into translanted nuclei

Uptake of linker histone and pol II correlates with reprogramming

Linker histone B4 (oocyte origin)

1 hour 24 hours 48 hours

Polymerase II (unphosphorylated)

Polymerase II (serine 5 phosph.)

DAPI

H2B-cherry

YFP-RPB1 (RNA polymerase)

(core histone)

GFP-TBP (TATA-binding protein

of somatic cells)

TBP2 -cherry (TATA-binding protein

of oocytes)

Uptake and loss of chromosomal proteins 0 hour 24 hours

Loss

Loss

Loss

Gain

Gain

Time sequence of polymerase II components binding to genes

Hours since nuclear injection 24 48

% o

f nuc

lei

boun

d

100

50

75

25

0

Histone B4 Pol II ser5P Pol II ser2P

Component bound

2

4

6

Abso

lute

tran

scrip

t lev

el

norm

alize

d to

24

hr sa

mpl

e

0 24 48 Hours after nuclear injection

,

, , with a-amanitin

, normal

Transcriptional reprogramming depends on polymerase II of oocyte origin

inject a-am resistant donor nuclei

(which kills ooc pol II)

Linker histone B4

Polymerase II Ser2

Transcription

Globin TSS Sox 2 TSS High

Low High

Low

High

Low

Reprogramming is selective at the level of polymerase II

Hours after nuclear transfer at 17 C o 48 36 24 12

High

Low

Tran

scrip

tion

3 6

Time course of transcriptional activation of somatic cell nuclei by oocytes

40

Resistance to reprogramming:

defence by the nucleus

Repressed Xi in female mammals

MEF Xi

3

Xi

0

EPI

Xi

0

Xi

3

EPI

100%

50%

Oct

4 tran

script

s rel

ativ

e to

active

X o

n da

y 3

Days

Epiblast-Xi, but not MEF-Xi, genes are strongly reactivated in injected oocytes

MEF

MacroH2A helps to explain resistance to reprogramming

MacroH2A is knocked down by inhibitory RNA,

and induces Oct4 and Sox2 in MEF-Xi cells.

Conclusion

macroH2A marks embryonic

differentiation and acts as an

epigenetic resistance to nuclear

reprogramming

Selective gene transcription 48 hours after nuclear transfer to Xenopus oocytes

High

Low

Tra

nscr

ipt

accu

mul

atio

n

Hours since injection 0 48

3368 (21%)

1176 (7%)

3805 (23%)

7890 (49%)

Numbers (%) of genes

Resistance is gene and cell-type specific

Chromatin modification

Nil U16

H3K27me3 H3K9me2/3

Histone H3 Histone H3 Histone H3

Histone modifications in nuclei can be changed after transfer to oocytes

H2AUb

Inject mRNs on day 1. Transplant nuclei on day 2. Reisolate transplanted nuclei on day 3 for Western analysis

Nil K6b Nil K4D

K6b, H3K27me3, H3K27 demethylase.

K4D, H3K9 demethylase.

U116, H2A deubiquitinase.

mRNAs:

DAPI H3K9Me3

No overexpression

Histone modifiers overexpressed in the oocyte efficiently modify transplanted nuclei chromatin

Anti-HA

Overexpressed HA-histone demethylase

methylation

KDM4D Control

H2A- Cherry

GFP- HP1alpha

Merge

Loss of HP1 alpha binding to transplanted chromatin after lysine demethylase overexpression

H2A- Cherry

GFP- HP1gamma

Merge

KDM4D Control

0

2

4

6

8

10

12

14

16

T0 RFP T48 KDM4D T48 USPs T48

Prok 2

Overexpression of histone 2A deubiquitinase removes resistance

Tra

scri

pt le

vel

mRNAs Injected

Chromatin depletion

10

4

5

6

7

8

9

10

10

10

10

10

Log

tra

nscr

ipt co

nten

t

, Oct4 mRNA , luciferase mRNA

.

RNA is removed, replaced, and quantitated by RT-PCR

RNA +RNase +RNase +RNase inhib. +More RNA

Oct

4 an

d lu

cife

rase

43

RNA RNA

1 2 3 4 1 2 3 4 Days

Tra

nscr

ipts

per

gen

e

Normal RNA _

Oct4. Pluripotency. Thymus nuclei

560

1 2 3 4 1 2 3 4 Days

Normal RNA _

Thymus nuclei

1000

Resistance of nuclei transplanted to oocytes RNA depletion from donor nuclei does not affect rate or extent of reprogramming

Sox2. Pluripotency.

1

2

3

4

5

6

7

8

9

10 400 mM NaCl

75 mM NaCl

, Oct4

, c-Jun

R. Halley-Stott

Protein depletion in somatic nuclei removes memory and enhances transcription

P Protein removal from nuclei by salt and Triton

Nil 75mM 200mM 400mM NaCl

S

ox2

tran

scrip

ts

C2C12 ES

HMG 10 10 3 1 EED 10 10 8 0 BMi1 10 10 5 0

HP! 10 10 9 0 Brg1 10 10 6 1

H2A 10 10 10 10

chromosomal proteins

Resistance to reprogramming is maintained at high salt concentrations

Levels of

Pol II 10 10 9 0 after salt treatment

The battle for supremacy

The egg The nucleus

Designed to transform sperm to an embryo

active nucleus

Designed to maintain the same pattern of gene expression

Tries to do the same for somatic nuclei

Tries to resist any change

Prospects

To defeat resistance and win

efficient cell replacement

Gurdon lab

Charles Bradshaw (bioinformatics)

Funding:

Acknowledgements

Jerome Jullien

Rick Halley-Stott

Vincent Pasque

Kei

Miyamoto

Nigel Garrett

Patrick Narbonne

Past colleagues

Ronald Laskey Donald Brown

Laurence Korn Eduardo De Robertis

Marvin Wickens Alan Colman

Ann Clarke

Valerie Moar

Christpher Graham John Knowland

Present colleagues

James Byrne

Jerome Jullien Kei Miyamoto

Rick Halley-Stott

Vincent Pasque

Marta Teperek

Eva Hoermanseder

Stan Wang

Celia Delahay

ACKNOWLEDGEMENTS

MEDICAL RESEARCH COUNCIL WELLCOME TRUST

CANCER RESEARCH CAMPAIGN

Stina Simonsson Carolina Astrand

Doug Melton

END

Sperm cell Embryonic cell Specialised cell

Images from Dr Kei Miyamoto Marta Teperek

99% 35% 1%

% of normal development after nuclear transfer (to a feeding tadpole)

A sperm nucleus is specially designed to yield normal development

Conclusions

2. 2. Stable comitment can be reversed by nuclear transfer to eggs.

3. Nuclei from diferentiated cells show a strong resistance to reprogramming.

4. Resistance is strongly cell-type and gene specific.

5. Resistance depends on histone modifications and on other stable chromosomal components.

.

1. Some cells (endoderm) undergo a very early stable commitment to their lineage pathway.

Nuclear reprogramming

Vincent Pasque (Xi) Richard Halley-Stott (Trn)

Kei Miyamoto Polym. Actin)

Acknowledgements

Jerome Jullien (B4)

39

Kazutaka Murata (Histone mods) Marta Teperek (Sperm)

Other Laboratories G. Crabtree (Stanford)

G. Almouzni (Paris) K. Ohsumi (Nagoya)

K.Shinkai (Kyoto)

Welcome Trust

Welcome Trust

Medical Research Council

Medical Reseacrh Councill

.

Single nuclear transfer to unfertilized eggs

Skin cells

nucleus in egg

Cloned animal

Somatic

Skin

Skin

Oocyte

Multiple somatic nuclei Germinal vesicle FRAP

GV isolation

Fluorescence recovery after photobleaching

Germinal vesicle with

injected nuclei

To determine the exchange rate of a defined protein in transplanted nuclei

1 hour 6 hours 24 hours 48 hours

Time after nuclear transfer

Histone B4

Pol II total

Pol II Ser 2

DAPI

Increase in polymerase II after nuclear transfer

0 24 48 72 Hrs

0 24 48 72 Hrs

0 24 48 72 Hrs

0 24 48 72 Hrs

0 24 48 72 Hrs

2

4 6

2

4 6

2

4 6

2

4 6

2

4 6

H3K4Me2

H3K4Me3 ,

,

B-globin promoter

Oct4 coding region

Sall4 promoter

Sox2RR2 reg. region

Sox2 promoter

Histones in gene control regions are methylated – Chip analysis

Nuclei from retinoic acid treated ES cells

Epigenetics and chromatin, 2010.

MacroH2A helps to explain resistance to reprogramming

MacroH2A is high on MEF-X:i resists reprogramming. but absent from EPI-Xi: is reprogrammed.

MacroH2A is knocked down by inhibitory RNA, and induces Oct4 and Sox2 in MEF-Xi cells

muscle

Donor tadpole

Muscle cell

Nuclear Blastula

Neurect -oderm

Endoderm

Epigenetic memory

No transcription

High expression

of

56%

52%

genes

Transcription

transfer

Nature Cell Biol. 2006

The resistance of MEF Xi nuclei to reprogramming

by oocytes is not explained by

DNA methylation or by

histone H3K27 me

Nature Cell Biol.2007

Nature Cell Biol.2007

WAVE-1 is required for zygotic genome activation and embryonic development

Oocyte

Egg

Embryo development

+ Antisense Wave-1 _

Nucleus

Nucleus

Arrest as gastrulae

Meiotic maturation

(Wiskott-Aldrich syndrome)

(no antisense)

(with antisense)

Histone modifiers overexpression in the oocyte:

- H3K9 demethylase KDM4D efficiently removes H3K9Me2/3 from transplanted nuclei and leads to loss of HP1 alpha

-H2A deubiquitinases (USP16&21) reduce ubiquitinated H2A

level in transplanted nuclei

10

4

5

6

7

8

9

10

10

10

10

10

Log

RN

A c

onte

nt

, endogenous G3PDH

, endogenous Xist

RNA can be depleted from donor nuclei by RNase.

Permeabilized cells, containing RNA, are treated with RNase,

+RNase

of tre

ated

cel

ls

43

then assayed for residual RNA.

No RNase

Rick Halley Stott

Past colleagues

Ronald Laskey

Donald Brown

Laurence Korn

Eduardo De Robertis

Marvin Wickens

Alan Colman

Ann Jewkes

Valerie Speight

Christpher Graham

John Knowland

Present colleagues

James Byrne Jerome Jullien

Kei Miyamoto

Rick Halley-Stott

Vincent Pasque

Marta Teperek

Eva Hoermanseder

Stan Wang

Celia

ACKNOWLEDGEMENTS

MEDICAL RESEARCH COUNCIL WELLCOME TRUST

CANCER RESEARCH CAMPAIGN

Nil 75mM 200mM 400mM NaCl

S

ox2

tran

script

s

C2C12 ES

HMG 10 10 3 1

EED 10 10 8 0

BMi1 10 10 5 0

HP! 10 10 9 0

Brg1 10 10 6 1

H2A 10 10 10 10

chromosomal

proteins

Resistance to reprogramming is maintained at high salt concentrations

Levels of

Pol II 10 10 9 0 after salt

treatment

Newt chromosome II.

Amphibian lampbrush chromosomes

Oogenesis and development in the mouse

Meiotic

Mouse 17

days days 10

Germ cell

divisions

Mature oocyte Zygote

Muscular response

Oogenesis Development

Major events in nuclear reprogramming

Chromatin decondensation

New (pluripotency) gene expression

DNA demethylation

DNA replication, cell proliferation

Repression of unwanted genes

(lineage selection)

Amphibia

Oocyte in meiotic prophase

Eggs and embryos

DNA demethylation

Stem cell genes are rapidly activated in mammalian nuclei transplanted to Xenopus oocytes

1 2 3 4

nuclei

Mouse/human somatic cell

Days

High

Low

Thymus

Mouse

ES nuclei

Oct4 Nanog

Nuclei of most differentiated cells resist reprogramming.

Sox2

nuclei Differentiated

(resistant)

Resistance to reprogramming is pronounced when comparing different donor cell-types. [by up to 50X]

Time following nuclear transplantation (hrs)

480 400 320 240

160 80

0

35

28

24

12

4

3 24 48 72 96 3 24 48 72 96

Sox2 Oct4

C2C12 MEF C2C12

MEF

Histone variant macroH2A

• macro domain = 2/3 of macroH2A

• vertebrate-specific variant

• ‘hallmark’ of vertebrate heterochromatin

0

25

50

ES 48h MEF 48h

Prok2 (RIP)

0

1

2

3

4

ES 48h MEF 48h

Ooep (RIP)

0

50

100

150

ES 48h MEF 48h

Gadd45 a (RIP)

0 0,5

1 1,5

2 2,5

ES 48h MEF 48h

GAPDH (RIP)

Examples of genes with restricted expression in MEF nuclei after transplantation to Xenopus oocytes

0 24 48 72 Hrs

0 24 48 72 Hrs

0 24 48 72 Hrs

0 24 48 72 Hrs

0 24 48 72 Hrs

2

4 6

2

4 6

2

4 6

2

4 6

2

4 6

H3K4Me2

H3K4Me3 ,

,

B-globin promoter

Oct4 coding region

Sall4 promoter

Sox2RR2 reg. region

Sox2 promoter

Histones in gene control regions are methylated – Chip analysis

Nuclei from retinoic acid treated ES cells

Epigenetics and chromatin, 2010.

Gene activation in somatic nuclei transplanted to oocytes is selective

Expresssed in MEFs, But NOT in transplanted MEF nuclei,

Number %

NOT expressed in MEFs, BUT in transplanted MEF nuclei

Expressed in MEFs and in transplanted MEF nuclei ,

Repressed 7113 41

Activated 1176 9

No change 3308 29

+/-mRNA injection

Anti-H3K9Me2/3

Anti-H3

Anti-H2AUb

Histone modifiers overexpressed in the oocyte efficiently modify transplanted nuclear chromatin

Somatic nuclei transplantation

24h 24h

Collect GV

Wash unbound protein

WB analysis

Day -1 Day 0 Day +1

Nil

With enzyme, day-1

Nuclei

“ “ “

Antibody

Chromatin modifiers that alter the epigenetic state of transplanted nuclei

Acknowledgements

39

Welcome Trust

Welcome Trust Medical Research Council

Vincent Pasque (Xi) Belgium Richard Halley-Stott (Resistance) S. Africa

Kei Miyamoto (Polymerized actin) Japan

Jerome Jullien (Histone B4, H3.3) France

Kazutaka Murata (Histone mods) Japan

Marta Teperek (Sperm progenitors) Poland

Stan Wang USA

Tadpole cloned from a muscle cell Tadpole from fertilized egg

derived from transplanted muscle cell nucleus

Normal eye

Lens

Retinal layers

Pigmented iris

Normal eye

by cloning from a muscle cell

DNA replication is retarded in Amphibian somatic cell nuclear transfers

30 60 90 120 150

Mins after fertilization or nuclear transfer

replic-

DNA

ation

comp-

lete

100%

0%

Nuclear transfers

with chromosomal damage

Fertilized

eggs

oooo

Mitosis

DNA DNA

Sox

2 tr

ansc

ription

2

1

rela

tive

to

contr

ol a

t 24

hou

rs

B4 histone is required for gene activation in oocytes

, Sox2 transcripts , c-jun transcripts

B4 Dom. Neg. 24 hrs

B4 antibody

24 hrs Control

24 hrs 0 hrs

Sox Jun Sox Jun Sox Jun Sox Jun

Jerome Jullien

5

4

3

2

1 Indu

ced

tran

scrip

t lev

el

Transcription is enhanced by actin polymerization

K. Miyamoto. Gen.Devel.2011.

Oct

4/GA

PDH

A model of nuclear actin function

Transcriptional activation is much enhanced by WAVE-1

Toca-1

WAVE-1

Rac1

N-WASP Enhances reprogramming seen by pluripotency gene transcription in oocytes

No effect on Reprogramming

Actin polymerization

(Wiskott-Aldrich syndrome)

WAVE-1 is required for zygotic genome activation and embryonic development

Oocyte

Egg

Embryo development

+ Antisense Wave-1 _

Nucleus

Nucleus

Arrest as gastrulae

Meiotic maturation

(Wiskott-Aldrich syndrome)

(no antisense)

(with antisense)

Genes with restricted expression in MEF or ES nuclei after transplantation to Xenopus oocytes

(41%) 2123 2864 2048 NOT

expressed in ES after NT.

NOT expressed in MEF nuclei after NT.

Expressed in ES nuclei after NT.

Expressed in MEF nuclei after NT.

Expressed in MEF and ES

(40%) (31%)

(29%)

Histone modifications in nuclei can be changed after transfer to oocytes

Nil K6b Nil U16

H3K27me3 H3K9me3/2 H2AUb

Histone H3 Histone H3 Histone H3

Nil K4D Overexpressed mRNAs

0 hour: mRNA injections. 24 hours: nuclear injections. 48 hours:reisolation of injected nuclei and Western analysis.

K6b, H3K27 demethylase. K4D, H3K9 demethylase. U16, H2A deubiquitinase. Western blots to show loss of histone modifications 48 hours after mRNA injection.

Overexpression of H2A deubiquitinases removes restriction

0

5

10

15

T0 RFP T48 KDM4D T48 USPs T48

prok2

0

0,5

1

1,5

T0 RFP T48 KDM4D T48 USPs T48

ooep

0

0,2

0,4

0,6

0,8

1

1,2

1,4

T0 RFP T48 KDM4D T48 USPs T48

sox2

0

0,5

1

1,5

2

T0 RFP T48 KDM4D T48 USPs T48

Jun

0

5

10

15

T0 RFP T48 KDM4D T48 USPs T48

prok2

Transcriptional reprogramming

Eggs and oocytes have a very high content of histone H3.3.

Histone H3.3 prolongs transcription of somatic nuclei in oocytes.

CONCLUSIONS