epigenetic regulation, stem cells and...
TRANSCRIPT
Epigenetic Regulation,Stem Cells and Cancer
Rudolf Jaenisch
Whitehead Institute and Dept. of Biology, MIT,Cambridge, MA
Levinson and Sweatt, 2005
Development and Differentiation
GeneticGenetic
o ro r
epigenetic changes?epigenetic changes?
Genetics vs. Epigenetics
Maintenance of DNA methylation and histone modification
Felsenfeld, 2007
Allis, Jenuwein, Reinberg, 2007
Histone Modifications
One Genome, many Epigenomes
Allis, Jenuwein, Reinberg, 2007
Three Definitions ofEpigenetics
1. Transmission of information through meiosis or mitosisthat is not based on DNA sequence
2. A mechanism for stable maintenance of geneexpression states that involves physically “marking”the DNA or its associated proteins
3. Mitotically or meiotically heritable changes in geneexpression that are not coded in the DNA itself
Relevant for development and cancer
Epigenetic Regulation,a mechanism that
allows the genome tointegrate
– intrinsic with
– environmental signals
Epigenetics andDisease Relevance
Diet / environment alters epigeneticstate of genome and may affectincidence of long latency / late stagediseases such as:
– Cancer
– Neuro-degenerative diseases
Gene expression affected by diet?
Pseudoagouti Allele: Mice with Variable Coat Color
Agouti gene (A): brownish coat of wild mice
• •
AIAP allele: insertion of IAP retroelement coat variegates between yellow and wild type
color depends on methylation of IAP
Expression (phenotype)
A allele (wt):
AIAP allele, methylated
Skin
AIAP allele, un-methylated
Skin
Ubiquitous: yellow, obese,
tumors
IAP
} normal****IAP
Turner, 2007
Environmental and epigenetic changes:short-term and long-term outcomes
Epigenetics, Environmental Stimuli
and Disease: A few questions
How does diet affect long latency
diseases?• Diet strongly affects cancer incidence
-- Mechanisms?
• How about neurodegenerative diseasessuch as AD, Parkinson’s?
-- Not a “clonal” disease: how to study?
Nuclear Cloning, Stem Cells andEpigenetic Reprogramming
Relevance for
transplantation therapy
Medical Challenges and thePotential of Stem Cells
• Increasing population age:
– Alzheimer, Parkinson, heart failure….
Potential solution:Regeneration, tissue repair
Problem: Suitable donor cells
Tissue Repair by CellTransplantation:
Historical Perspective• Bone marrow transplantation
– Established medical treatment since the 70s to treatleukemia
– Problem: finding suitable donors, immune rejection
• These problems are more serious fortransplantation repair of other tissues
What is the goal stem cell research?
To provide matched cells for“customized” tissue repair
Stem Cells:A developmental hierarchy
B
T
Plts
WBC
RBC
Blood
Pluripotent All cell typesIn vitro
differentiation
Muscle
Bone
Fat
Mesenchymal-Connective
EmbryonicStem Cells
Liver/PancreasSkin, Testes, Gut
Neural
Neurons
Oligoglia
Astroglia
Zygote(TOTIPOTENTIAL)
+
Blastocyst
?
?
Embryonic
Adult
G. Daley
Surani and Reik, 2006
Sources of Pluripotent Cells
Endoderm
Hepatocytes
Ectoderm Gametes/Germ cells
Mesoderm
Neuralstemcells
Hematopoieticstem cells
Mesenchymalstem cells
BoneCartilageMuscle
Fat
Blood
Intestinalstemcells
Pancreaticprogenitor
cells
Hepaticprogenitor
cells
Intestine
Pancreas EggSperm
NeuronsOligodentrocytes
Astrocytes
Skin
Skinstemcells Tissue
precursors/tissue stem
cells
SomaticCells
Multipotentprogenitors
Embryonic Stem CellsPluripotent
ES Cells: A Model System for Development
Embryonicstem cells
Tissue specificprecursor cells
Fetal/adultstem cells
Transplantation
Potential of Embryonic Stem Cells
Differentiatedsomatic cells
Tissueengineering
self-renewalpluripotency
?
GROWTH IN PUBLICATIONS ABOUT STEM CELLS
0
200
400
600
800
1000
1200
1400
1980
1982
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
*
Year
Pu
bM
ed
Pu
blicati
on
s p
er
year
EMBRYONIC
STEM CELLS
ADULT STEM
CELLS
HUMAN
ES CELLS
*ProjectedYear
Publications on stem cellsover the last 20 years
HumanES CellsIsolated
MouseES CellsIsolated
The interest in stem cells has grown exponentially
G. Daley
Therapeutic Limitationsof Embryonic Stem Cells
• ES cells are derived from donatedembryos:
This causes immune rejection
One potential solution:
– Nuclear cloning to create“customized” ES cells
Nuclear transfer
oocyteDonor cell
NuclearTransfer
Reproductive cloning
Blastocyst
NT to create patient-specific ES cells
Inner Cell Mass
Customized patient-specificES cell
G. Daley
Nuclear Cloning is very
Inefficient
• Most clones die soon after implantation
Question:
Survival of NT clones dependenton differentiation state of donor
cell type?
Loss of Nuclear Potency withIncreasing Age of Donor
An old question:
Is the genome of
terminally differentiated
cells reprogrammable by
nuclear cloning?
Nuclear Cloning of TerminallyDifferentiated Cells
Genetic vs. epigenetic changes
A. Monoclonal mice:• B / T cells: visualization of Ig and TCR genomic
rearrangements(Hochedlinger and Jaenisch, 2002)
B. CNS: Cloning of postmitotic matureneurons• Does brain development / neuronal functions involve
epigenetic as well as genetic alterations?(Eggan et al, 2004; Li et al, 2004)
C. Cancer:• Can the malignant state be reversed?
Distinction between epigenetic (= reversible) andgenetic (= irreversible) changes in tumor(Hochedlinger et al, 2004; Blelloch et al, 2004)
State of Donor Cell Differentiationand Efficiency of NT:
Higher Survival of ES Cell Clones
Donor Cells Survival to adults (from cloned blastocysts)
Somatic cells 1-3 %Fibroblasts, Sertoli, cumulus cells
Terminally differentiated cells < .001 %
B, T cells, neurons, cancer
ES cellsES cells 15-25 % 15-25 %ES nuclei are easier to reprogram
Lesson from NuclearCloning:
Differentiation state of donor cellsaffects reprogramming efficiency
Likely due to differences in
epigenetic state
Gene Expression andPhenotype of Cloned Animals
1. Widespread faulty gene expression• 4-5% of all genes• 30-50% of imprinted genes
2. "Normal" appearing clones often develop seriousabnormalities with age
Faulty reprogramming may preclude thegeneration of normal cloned individuals
However, offspring of clones are always normal(The problem are not mutations but an abnormal epigenetic state)
Degree of Abnormalities in Clones:A continuum with few defined stages
Degree of abnormalityHigher Lower
Dead Survivors
Su
rviv
al
Long term Survivors:
Are they really"normal"?
Age of clones
BirthImplantation
• Even clones that survive to birth have oftenserious abnormalities and die later
• Widespread epigenetic dysregulation
“Normal” clones may be theexception
Lessons from animal cloning:
Cloning of Humans
Testifying in the
United StatesCongress:
Charlatans, Clowns and Publicity:Consequences for Legislation ?
Embryonicstem cells
“Customized”embryonicstem cells
Sexuallyproduced
embryo
Asexuallyproduced
embryo
Therapeutic Applications of Embryonic Stem Cells
“Customized” ES cells from cloned blastocysts: patient’s own cells
ES cells from IFV embryos: different from patient, immune rejections
Cell Cell 2002, 109: 17-272002, 109: 17-27
ClonedES Cells
Rag2-/-
Gene Correction
CorrectedES Cells
Egg Tail Tip CellβThalassemiaSickle cell anemiaFanconi’s anemiaLeukemia
G. Daley
I. Nuclear transferand ES cellderivation
II. Derivation of bone marrow
cells and
transplantationinto “patient”
Conclusion
In principle,therapeutic SCNT will work in humans
to generate “customized” cell fortreatment of
– Parkinsons
– Diabetes
– Blood diseases…..
BUT…..
Problems withTherapeutic SCNT
1. Procedure too inefficient, costlyfor routine treatment
2. Ethical objections to usinghuman eggs for therapy
“Customized” Cells for Tissue Repair:
The key issue
Achieving reprogramming withoutthe need for human eggs
• We need to understand thereprogramming rules
The egg does not accomplish amiracle but a biochemical
reaction
Dedifferentiation and differentiation in the test tube:
A strategy for cell based therapy
Somatic cellsFibroblasts,
Skin…
“Reprogrammed” ES cell
Cells frompatient
“Customized”cells for therapy
Differentiated cells for transplantationNeurons, Muscle, β cells...
Reprogrammingin petri dish
Differentiationin petri dish
granulocytes
Reversibility of the Epigenetic State
Change in epigenetic state
Different strategies togenerate pluripotentfrom somatic cells
• Nuclear transplantation ofsomatic cell nuclei
• Fusion of ES and somatic cells
• Direct conversion of a somaticinto an ”ES” like cellSomatic embryonic epigenetic state
Key question ofReprogramming:
• Why is NT with somatic donornuclei so inefficient?
• What is the molecular circuitrythat distinguishes pluripotentfrom somatic cells?If we understand the key epigenetic switches of
differentiation, we may eventually be able toconvert one cell type into the other(“transdifferentiation, plasticity”)
Death of Clones after Implantation:
Questions
Degree of abnormalityHigher Lower
Survivors
Su
rviv
al
Age of clones
BirthImplantation
• Why do most clones
die after implantation?• Which genes not
correctly reprogrammed?
}Dead
“Pluripotency” GenesOct4, Nanog, Sox2
• Expressed in early embryo, EScells, not in somatic cells
• Regulators of pluripotency andself-renewal
• Following NT their activationappears crucial for– Cloned embryos to survive after
implantation(Nichols, 1999; Boiani, 2002; Bortvin, 2003; Chambers, 2003; Mitsui, 2003)
Oct4, Nanog, Sox2:Key regulators of stem cells
Issues:– Molecular control circuits of
•Self-renewal
•Pluripotency
– Embryonic vs. adult stem cells
•Similar / different regulation?
Embryonic Stem (ES) Cells
Key Properties:
• Self-renewal
• Pluripotent
What controlsES cellidentity?
Important implications for understanding nuclear reprogrammingand directed differentiation of ES cells.
The molecular circuitry ofpluripotency and
self renewal
We have to understand thekey factors that
– Set up the pluripotent state
– Maintain the pluripotent state
– Induce differentiation of stemcells
Genome-wide Location Analysis
Protein:DNA interactions
Your Favorite Gene Here
Oligos chosen to represent this region
Targets 8 kbp upstream and 2 kbp downstream oftranscription start sites for all annotated genes
(~18K) in the human genome (RefSeq, Ensembl,MGC, H-inv)
Oligonucleotide Promoter Array
R. Young
Oct4, Sox2, Nanog Co-Occupy Many Target Genesin Human ES Cells
Transcriptional Regulatory Hierarchy in Human ES Cells
ActivationTranscription FactorsChromatin RegulatorsCell Cycle RegulatorsSignaling Molecules
RepressionDevelopmental
Transcription Factors
• Co-occupy many target genes
• Associate with active genes encoding proliferation factors
• Associate with inactive genes encoding developmental transcription factors
• Contribute to specialized regulatory circuitry that provide important clues into ES cell pluripotency
EScell
Specialized cell
self-renewal Oct4
Nanog Sox2
Insights from Core Regulatory Circuitry in Human Embryonic Stem Cells
Oct4, Sox2, and Nanog:
• Identified in Drosophila as regulators of homeotic (HOX)genes
• Conserved from fly to human• Essential roles in early embryonic development• Maintain repressive gene expression patterns and cellular
identity through epigenetic modification of chromatin
Polycomb Group (PcG) Proteins
EScell
differentiatedcell
Self-renewal,pluripotency Oct4
Nanog
PcG proteins
Regulation of Pluripotency inEmbryonic Stem Cells
Oct4, Nanog and PcGs co-occupy a set of repressed genesthat encodes developmental transcription factors.
This set of target genes must be repressedto maintain pluripotency
ES cell regulatory circuitry
differentiated
cells
self-renewal
Suz12
Pol II
Ethical / Political Problemswith Embryonic Stem Cells
and Therapeutic SCNT:
Scientific solutions?
Any solutions?
The New Yorker
Alternatives to embryonic stemcells?
Do we need to use SCNT togenerate “customized”ES cells for therapy?
or
Such as Adult Stem cells?
Promise Challenges
Reproductive
cloning
Clones are
abnormal
CustomizedES cells
Ethics,human eggs
Reprogramming
in test tube
Fused cells
are 4n
SimplicityNo evidence of reprogramming
SpermatogonialStem Cells
(derived from spermatigonia)
No ethical problems
DisturbedImprinting
Different Approaches to obtain “Customized”
Pluripotential Cells from Postnatal Animals
Transduction of Transduction of ““reprogrammingreprogramming””factors into somatic cells andfactors into somatic cells and
selection for activation ofselection for activation ofpluripotency genespluripotency genes
Reprogrammingin test tube
What are the key
epigenetic
effectors?
Dedifferentiation and differentiation in the test tube:
A strategy for cell based therapy
Somatic cellsFibroblasts,
Skin…
“Reprogrammed” ES cell
Cells frompatient
“Customized”cells for therapy
Differentiated cells for transplantationNeurons, Muscle, β cells...
Reprogrammingin petri dish
Differentiationin petri dish
Epigenetics, Cancer and
the Reversibility of theMalignant State
Progressive promoter methylation andselection for silencing in tumors
Jones andBaylin, 2006
Genes that are frequently hypermethylatedand /or mutated in cancer
Only mutatedOnly methylated
Methylated and mutatedJones and Baylin, 2006
Epigenetic Alterationsand Cancer:
• How stable is themalignant state?
• Reversible by nucleartransfer into egg?
The Concept
Normaldevelopment
embryonic cell
epigeneticchanges
differentiated cell
Nuclearcloning
Tumorigenesis
normal cell
malignant cell
geneticchanges
+
epigeneticchanges
? Nuclearcloning ?
Tumor donor cells Eggs
injectedBlastocysts ES cell lines
30 0
Ras-inducible melanoma 600 8 2
23 0
1450Lymphomas,leukemias
p53-/- spontaneousbreast cancer (cell line CK5)
189Solid
tumors
Nuclear Cloning and Cancer
Somatictumors
Total 2515 61(2.5%) 2 (3 %)
Embryonalcarcinomas
METT1,P19, F9
435 32 17(7%) (53%)
Melanoma donor
NT ES cell derivedfrom melanoma
heartkidney intestine
GFP
light
Melanoma Derived ES Cells form Chimeric Pup
Chimeric mouse from Melanoma DonorDerived ES cell
Coat color and tumors in chimeras
Chimera # 1 Chimera # 3
Chimera # 2 Chimera # 4
Chimera # 6
Chimera # 8
Reprogramming of Cancer CellGenome
Malignant tumor cell genome can bereprogrammed by nuclear transfer into pluripotentES cells with the potential to differentiate intomost if not all somatic tissues
Key question:• NT ES cell derived from tumor cell or non-tumor
support cell?
Approach:• CGH analysis of donor cells, NT ES cells and
chimeras (tumors and fibroblasts)
Chr 8
R545 parental R545-1 NT ES
R545
SCID
tumorR545-1NT ES cells
Melanoma
Rhabdo-
myosarcoma
MPNST
Fibroblasts
R5
45
-1 N
T E
Sc
ell
de
rive
d
CGH Analysis of Melanoma Donor andtheir Cloned ES Cell Derivatives
R545
parental
Do
no
r
Reprogramming of CancerCell Genome
Malignant tumor cell genome
– Can be reprogrammed by nucleartransfer into pluripotent ES cells withthe potential to differentiate into most ifnot all somatic tissues
– Tumor phenotype largely determined byepigenetic changes
Pardal, Clarke, Morrison, 2003
Somatic Stem cells / Cancer Stem Cells
Epigenetic silencing, stem cells and cancer
Baylin and Jones, 2007
Epigenetic modifications in cancer are reversible: relevance for therapy
Allis, Jenuwein, Reinberg, 2007
L. BoyerK. PlathA. MeissnerY. YamadaC. BeardT. BrambrinkR. BlellochZ. WangM. WernigL. MedeirosM. RayA. Tajonar
M. GuentherT. LeeM. ColeS. JohnstonR. Jenner B. ChevalierJ. Zucker S. LevineT. VolkertR. YoungD. GiffordM. KybaG. Daley
K. HochedlingerK. Eggan C. Brennan
M. KimL. Chin (Harvard)