Stockholm: 7 December, 2012.

The egg and the nucleus: a battle for supremacy

J. B. Gurdon Cambridge, England.

Content
Background
Attack by the egg

Defense by the nucleus Prospects

Background

The original question

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

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

Michail Fischberg

1-nucleolus

2-nucleolus

Intestinal Tract of Feeding T adpole

(GFP-marked))

Partial blastula

Nuclear Transplant Embryo Graft GFP

Muscle derived from intesti GFP-muscle derived from intestine nuclei

by nuclear transfer and grafts

Efficiency of nuclear reprogramming by nuclear transfer to eggs

Switch between celltypes: e.g. intestinal epithelium to muscle and nerve. First nuclear transfers....15% Serial nuclear transfers.....7% Grafts from nuclear transfer embryos.8% Total: 30%

Wilmut, Campbell et al 1996 and 1997.

Nuclear transfer success decreases as donor cells differentiate 40 30 Mammalian nuclear transfers reaching birth Xenopus nuclear transplants reaching feeding tadpole stage

% of total nuclear 20 transfers

10

Blastula Blastula Blastocystt

Differentiation

Adult

Stage of donor nuclei

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

Donor of skin

Egg

Donor of eggs

Cloning
Embryo

Embryonic stem cells Increase cell number Add factors

Stem cell creation

Differentiation

Beating heart muscle

Epigenetic memory

Embryos derived from muscle nuclei remember their origin even in their nerve and endoderm cells
High expression of Neurect muscle oderm genes

56%
Muscle cell Nuclear Blastula Endoderm

52%
transfer

Donor tadpole

Transcription

No transcription
Nature Cell Biol. 2006

H3.3 is required for epigenetic memory.

Elimination by H3.3 mutated from K4 to E4.
High expression of Neurect muscle oderm genes

3%
Muscle cell

Donor tadpole

H3.3E4 mutant Nuclear mRNA transfer

5%
Blastula Endoderm

Transcription
K4, methylatable lysine. E4, gutamine

No transcription

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

Single nuclear transfer to eggs in second meiotic metaphase

SSomatic UV
cell nucleus

Blastula

Frog

New gene expression New cell types

DNA replication Postzygotic only transcription Replication errors in Transplanted somatic nuclei

Incomplete DNA replication damages somatic nuclei transplanted to eggs

Multiple nuclei transferred to growing oocytes in first meiotic prophase

Germinal vesicle
Oocyte [Egg progenitor]

Intense transcription
(no DNA replication)

New gene expression

Days culture

4
No new cell type

Oocyte formation prepares the egg for develpment

G Transcription

100 days
Germ cell Oocyte

Chromatin decondensation Intense gene transcription DNA demethylation

Egg

Blastula Lineage selection

Xenopus oocyte and germinal vesicle

GV

1 mm

The oocyte germinal vesicle contents contribute to post-fertilization development

Germinal vesicle

Matur -ation

Oocyte
First meiotic prophase

Egg
Second meiotic metaphase

Embryo
Mid blastula

Mammalian stem cell genes are rapidly activated in mammalian nuclei transplanted to Xenopus oocytes Nuclei of differentiated cells are reprogrammed slowly.
Mouse/human somatic cell nuclei High

Sox2 Oct4 Nanog

Low

HeLa Mouse thymus

Days

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

Mouse sperm

HFertilized mouse egg

Female pronucleus Male pronucleus

Mammalian cultured cell nuclei : Just after injection

1-2 days after nuclear transfer

Histone replacement in transplanted nuclei.

Cultured cell nuclei in oocyte GV

Somatic H1o histone-GFP replaced by oocyte B4 histone-RFP

Jerome Jullien

Histone H3.3 is incorporated into translanted nuclei

Donor H3.3 Oocyte H3.3 Merge

0%

10

82%

15

99%

Uptake of linker histone and pol II correlates with reprogramming

1 hour 24 hours 48 hours

Linker histone B4 (oocyte origin) Polymerase II (unphosphorylated) Polymerase II (serine 5 phosph.) DAPI

Uptake and loss of chromosomal proteins

0 hour 24 hours

(RNA polymerase)

YFP-RPB1

Loss Gain Loss Loss

H2B-cherry

(core histone)

(TATA-binding protein of somatic cells) (TATA-binding protein of oocytes)

GFP-TBP

TBP2 -cherry

Gain

Time sequence of polymerase II components binding to genes

Component bound

% of nuclei bound

100 75 50 25

Histone B4 Pol II ser5P Pol II ser2P

24 48 Hours since nuclear injection

Transcriptional reprogramming depends on polymerase II of oocyte origin

Absolute transcript level normalized to 24 hr sample

6 4 2

, ,

, normal , with a-amanitin

(which kills ooc pol II) inject a-am resistant donor nuclei

24 Hours after nuclear injection

48

Reprogramming is selective at the level of polymerase II Globin TSS Sox 2 TSS

High Linker histone B4 Low High Polymerase II Ser2 Low High Transcription Low

Time course of transcriptional activation of somatic cell nuclei by oocytes

Transcription

High

Low 3 6 12 24 36 o Hours after nuclear transfer at 17 C 48 40

Resistance to reprogramming:

defence by the nucleus

Repressed Xi in female mammals

EpiblastXi, but not MEFXi, genes are strongly reactivated in injected oocytes 100% Oct4 transcripts relative to active X on day 3

50%

Days

EPI Xi 0

EPI Xi 3

MEF Xi 0

MEF Xi 3

MacroH2A is knocked down by inhibitory RNA, and induces Oct4 and Sox2 in MEFXi cells.

MacroH2A helps to explain resistance to reprogramming

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 Transcript accumulation
Numbers (%) of genes

3368 (21%) 1176 (7%)

3805 (23%) 7890 (49%) 0 Hours since injection 48

Low

Resistance is gene and celltype specific

Chromatin modification

Histone modifications in nuclei can be changed after transfer to oocytes

Inject mRNs on day 1. Transplant nuclei on day 2. Reisolate transplanted nuclei on day 3 for Western analysis mRNAs: Nil K6b
H3K27me3 Histone H3

Nil K4D
H3K9me2/3 Histone H3

Nil U16
H2AUb Histone H3

K6b, H3K27me3, H3K27 demethylase. K4D, H3K9 demethylase. U116, H2A deubiquitinase.

Histone modifiers overexpressed in the oocyte efficiently modify transplanted nuclei chromatin
DAPI H3K9Me3 AntiHA

methylation

No overexpression

Overexpressed HAhistone demethylase

Loss of HP1 alpha binding to transplanted chromatin after lysine demethylase overexpression
Control KDM4D Control KDM4D

H2ACherry

H2ACherry

GFPHP1alpha

GFPHP1gamma

Merge

Merge

Overexpression of histone 2A deubiquitinase removes resistance

Prok 2
Trascript level
16 14 12 10 8 6 4 2 0 T0 RFP T48 KDM4D T48 USPs T48

Injected

mRNAs

Chromatin depletion

RNA is removed, replaced, and quantitated by RTPCR

, Oct4 mRNA , luciferase mRNA 9 8

10

Oct4 and luciferase

Log transcript content

10

10 7 10 6

10 5 10 4 RNA RNA + RNase RNA + RNase + RNase inhib. + More RNA

43

Resistance of nuclei transplanted to oocytes

RNA depletion from donor nuclei does not affect rate or extent of reprogramming

Oct4. Pluripotency. Thymus nuclei Normal Transcripts per gene RNA _

Sox2. Pluripotency. Thymus nuclei Normal RNA _

560

1000

12 3 4

12 3 4 Days

12 3 4

12 3 4 Days

Protein depletion in somatic nuclei removes memory and enhances transcription

10 9 8 7 6 5 4 3 2 1 75 mM NaCl , Oct4 , cJun 400 mM NaCl

R. HalleyStott

Protein removal from nuclei by salt and Triton

Resistance to reprogramming is maintained at high salt concentrations

Sox2 transcripts

ES

C2C12
HMG EED BMi1 HP! Brg1 Pol II H2A

Nil
10 10 10 10 10 10 10

75mM 200mM 400mM

10 10 10 10 10 10 10 3 8 5 9 6 9 10 1 0 0 0 1 0 10

NaCl

Levels of chromosomal proteins after salt treatment

The battle for supremacy The egg

Designed to transform sperm to an embryo active nucleus Tries to do the same for somatic nuclei

The nucleus
Designed to maintain the same pattern of gene expression

Tries to resist any change

Prospects

To defeat resistance and win efficient cell replacement

Acknowledgements
Gurdon lab

Charles Bradshaw (bioinformatics)

Jerome Rick HalleyStott Jullien Nigel Vincent Garrett Pasque Patrick Narbonne

K Miyamoto e i

Funding:

Past colleagues Donald Brown Ronald Laskey Doug Melton Eduardo De Robertis Laurence Korn Marvin Wickens Alan Colman Christpher Graham John Knowland Ann Clarke Valerie Moar James Byrne Stina Simonsson Carolina Astrand

ACKNOWLEDGEMENTS
Present colleagues Jerome Jullien Kei Miyamoto Rick Halley-Stott Vincent Pasque Marta Teperek Eva Hoermanseder Stan Wang Celia Delahay MEDICAL RESEARCH COUNCIL WELLCOME TRUST CANCER RESEARCH CAMPAIGN

END

A sperm nucleus is specially designed to yield normal development

Sperm cell Embryonic cell Specialised cell

99%

35%

1%

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

Images from Dr Kei Miyamoto Marta T eperek

Conclusions
1. Some cells (endoderm) undergo a very early stable commitment to their lineage pathway.
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 celltype and gene specific. 5. Resistance depends on histone modifications and on . other stable chromosomal components.

Acknowledgements

Nuclear reprogramming Jerome Jullien (B4) Kei Miyamoto Polym. Actin)

Vincent Pasque (Xi)

Richard HalleyStott (Trn) Kazutaka Murata (Histone mods) Marta Teperek (Sperm) Welcome Trust
Wel com e Trus

Other Laboratories G. Crabtree (Stanford) K. Ohsumi (Nagoya) Medical G. Almouzni (Paris) Research Council K.Shinkai (Kyoto)

Medical Reseacrh Councill

39

Single nuclear transfer to unfertilized eggs

Skin nucleus in egg Cloned animal

Skin

Skin cells

Somatic

Fluorescence recovery after photobleaching

T o determine the exchange rate of a defined protein in transplanted nuclei

Germinal vesicle

Multiple somatic nuclei

FRAP

GV isolation

Oocyte

Germinal vesicle with injected nuclei

Increase in polymerase II after nuclear transfer

Time after nuclear transfer 1 hour Histone B4 6 hours 24 hours 48 hours

Pol II total

Pol II Ser 2

DAPI

Histones in gene control regions are methylated Chip analysis

Nuclei from retinoic acid treated ES cells
6 4 2 0 6 4 2 Sox2 promoter 6 4 2 Oct4 coding region

24

48

72 Hrs

24

48

72 Hrs

Sox2RR2 reg. region 6 4 2

Bglobin promoter

0 6 4 2

24

48

72 Hrs

24

48

72 Hrs

Sall4 promoter , H3K4Me2 , H3K4Me3 Epigenetics and chromatin, 2010.

24

48

72 Hrs

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

Epigenetic memory
High expression of Neurect muscle oderm genes

56%
Muscle cell Nuclear Blastula Endoderm

52%
transfer

Donor tadpole

Transcription

No transcription
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

WAVE1 is required for zygotic genome activation and embryonic development

(WiskottAldrich syndrome)

Meiotic maturation Egg Oocyte

Nucleus Nucleus

Embryo development (no antisense) Arrest as gastrulae (with antisense)

Antisense + _ Wave1

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

RNA can be depleted from donor nuclei by RNase.

Permeabilized cells, containing RNA, are treated with RNase, then assayed for residual RNA.
10 9 10 Log RNA content of treated cells 8 , endogenous G3PDH , endogenous Xist

10 7 10 6

10 5 10 4 No RNase + RNase 43 Rick Halley Stott

Past colleagues Christpher Graham John Knowland James Byrne

ACKNOWLEDGEMENTS Present colleagues

Jerome Jullien Kei Miyamoto Rick Halley-Stott Vincent Pasque Marta Teperek Eva Hoermanseder Stan Wang Celia MEDICAL RESEARCH COUNCIL WELLCOME TRUST CANCER RESEARCH CAMPAIGN

Ronald Laskey Donald Brown Laurence Korn Eduardo De Robertis Marvin Wickens Alan Colman Ann Jewkes Valerie Speight

Resistance to reprogramming is maintained at high salt concentrations

Sox2 transcripts

ES C2C12

Nil
10 10 10 10 10 10 10

75mM 200mM 400mM

10 10 10 10 10 10 10 3 8 5 9 6 9 10 1 0 0 0 1 0 10

NaCl

Levels of chromosomal proteins after salt treatment

HMG EED BMi1 HP! Brg1 Pol II H2A

Newt chromosome II.

Amphibian lampbrush chromosomes

Oogenesis and development in the mouse

Oogenesis

Development

Mouse

Germ cell 17 days

Meiotic divisions Mature oocyte Zygote 10 days

Muscular response

Major events in nuclear reprogramming

Amphibia
Oocyte in meiotic prophase Eggs and embryos

Chromatin decondensation New (pluripotency) gene expression DNA demethylation DNA demethylation DNA replication, cell proliferation Repression of unwanted genes (lineage selection)

Stem cell genes are rapidly activated in mammalian nuclei transplanted to Xenopus oocytes Nuclei of most differentiated cells resist reprogramming.
Mouse/human somatic cell nuclei High

Sox2 Oct4 Nanog

Low

Differentiated ES nuclei

Mouse Thymus nuclei (resistant)

3 4 Days

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

Sox2
480 400 320 240 160 80 0 3 48 24 72 96
MEF C2C12

Oct4
35 28 24 12 4 3 48 24 72 96
C2C12 MEF

Time following nuclear transplantation (hrs)

Histone variant macroH2A

macro domain = 2/3 of macroH2A vertebratespecific variant hallmark of vertebrate heterochromatin

Examples of genes with restricted expression in MEF nuclei after transplantation to Xenopus oocytes
Gadd45 a GAPDH (RIP)
2,5 2 1,5 1 0,5 0 ES 48h MEF 48h 150 100 50 0 ES 48h MEF 48h

(RIP)

Ooep (RIP)
4 3 2 1 0 ES 48h MEF 48h 0 25 50

Prok2 (RIP)

ES 48h

MEF 48h

Histones in gene control regions are methylated Chip analysis

Nuclei from retinoic acid treated ES cells
6 4 2 0 6 4 2 Sox2 promoter 6 4 2 Oct4 coding region

24

48

72 Hrs

24

48

72 Hrs

Sox2RR2 reg. region 6 4 2

Bglobin promoter

0 6 4 2

24

48

72 Hrs

24

48

72 Hrs

Sall4 promoter , H3K4Me2 , H3K4Me3 Epigenetics and chromatin, 2010.

24

48

72 Hrs

Gene activation in somatic nuclei transplanted to oocytes is selective Number

Expresssed in MEFs, But NOT in transplanted MEF nuclei, NOT expressed in MEFs, BUT in transplanted MEF nuclei Expressed in MEFs and in transplanted MEF nuclei
,

%
41

Repressed

7113

Activated

1176

No change

3308

29

Histone modifiers overexpressed in the oocyte efficiently modify transplanted nuclear chromatin
Somatic nuclei transplantation + /mRNA injection 24h 24h Collect GV Wash unbound protein

WB analysis

Day 1

Day 0

Day + 1

Nil Nucle i With enzyme, day1

Antibody AntiH3 AntiH3K9Me2/3 AntiH2AUb

Chromatin modifiers that alter the epigenetic state of transplanted nuclei

Acknowledgements
Jerome Jullien (Histone B4, H3.3) Kei Miyamoto (Polymerized actin)
Vincent Pasque (Xi)

France Japan
Belgium

Richard HalleyStott (Resistance) Kazutaka Murata (Histone mods) Marta Teperek (Sperm progenitors) Stan Wang
Wel com e Trus

S. Africa Japan Poland USA

Welcome Trust

Medical Research Council

39

T adpole from fertilized egg

T adpole cloned from a muscle cell

Normal eye
Normal eye derived from transplanted muscle cell nucleus by cloning from a muscle cell

Lens Retinal layers

Pigmented iris

DNA replication is retarded in Amphibian somatic cell nuclear transfers

Mitosis

100% DNA replication complete 0%

DNA Fertilized
eggs

DNA

Nuclear transfers with chromosomal damage 30 60 90 oooo 120 150

Mins after fertilization or nuclear transfer

B4 histone is required for gene activation in oocytes

Sox2 transcription relative to control at 24 hours , Sox2 transcripts 2 1 , cjun transcripts

Sox Jun

Sox Jun Control

Sox Jun

Sox Jun

B4 Dom. Neg. B4 antibody

0 hrs

24 hrs

24 hrs

24 hrs Jerome Jullien

Transcription is enhanced by actin polymerization

Induced transcript level

5 Oct4/GAPDH 4 3 2 1

K. Miyamoto. Gen.Devel.2011.

A model of nuclear actin function

Transcriptional activation is much enhanced by WAVE-1

(Wiskott-Aldrich syndrome)

Actin polymerization
Toca-1 Rac1

N-WASP
No effect on Reprogramming

WAVE-1 Enhances reprogramming seen by pluripotency gene transcription in oocytes

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

(Wiskott-Aldrich syndrome)

Meiotic maturation Egg Oocyte

Nucleus Nucleus

Embryo development (no antisense) Arrest as gastrulae (with antisense)

+ _

Antisense Wave-1

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

Expressed in MEF nuclei after NT. NOT expressed in ES after NT.

2864 (41%)
(40%)

2123

(31%) 2048 (29%)

Expressed in ES nuclei after NT. NOT expressed in MEF nuclei after NT.

Expressed in MEF and ES

Histone modifications in nuclei can be changed after transfer to oocytes

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

Nil K6b

Overexpressed mRNAs
Nil K4D H3K27me3 Histone H3 H3K9me3/2 Histone H3

Nil U16 H2AUb Histone H3

K6b, H3K27 demethylase.

U16, H2A deubiquitinase. Western blots to show loss of histone modifications 48 hours after mRNA injection.

K4D, H3K9 demethylase.

Overexpression of H2A deubiquitinases removes restriction sox2

1,4 1,2 1 0,8 0,6 0,4 0,2 15 0 T0 10 5 0 T0 RFP T48 KDM4D T48 USPs T48 2 1,5 1

Jun

prok2
RFP T48 KDM4D T48 USPs T48

0,5 0 T0 RFP T48 KDM4D T48 USPs T48

prok2
1,5 1 0,5 0 T0 RFP T48 KDM4D T48 USPs T48 T0

ooep

15 10 5 0

RFP T48 KDM4D T48 USPs T48

Transcriptional reprogramming

CONCLUSIONS Eggs and oocytes have a very high content of histone H3.3. Histone H3.3 prolongs transcription of somatic nuclei in oocytes.