“Epigenetica en Biomedicina: Del Conocimiento al Desarrollo Biotecnológico y Farmacéutico”
Dr. Manel Esteller [email protected]
Director Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL)
ICREA Research Professor Professor of Genetics, University of Barcelona
WE ARE MORE THAN OUR GENES !
DOLLY inrecentyearsepigeneticalterationshavecometopr ominenceincancerresearchinparticularhypermethy lationofcpgislandslocatedinthepromoterregionso ftumorsuppressorgenesisnowfirmlyestablishedasa nimportantmechanismforgeneinactivationincancer oneofthemostremarkableachievementsinthefieldha sbeentheidentificationofthemethylcpgbindingdom ainfamilyofproteinswhichprovidemechanisticlink sbetweenspecificpatternsofdnamethylationandhis tonemodificationsalthoughmanyofthecurrentdatai ndicatethatmethylcpgbindingproteinsplayakeyrol einmaintainingatranscriptionallyinactivestateo fmethylatedgenesmbd4isalsoknowntobeinvolvedine xcisionrepairoftgmismatchesthelatterisamembero fthisfamilyofproteinsandappearstoplayaroleinre ducingmutationsat5methylcytosinethisreviewexam inesthecontributionofmethylcpgbindingproteinsi ntheepigeneticpathwayofcancer
The Epigenetic Dice:
-DNA Methylation
-Histone Modifications
-ncRNAs
-….
Cover by Sanchez-Mut and Esteller, Dev Cell, November 15th 2010 Epigenetics Parameters
Nature, 429, 457-63, 2004
Imprint of famine seen in genes of Second World War babies 60 years on
Towards the Human Epigenome
A B 2,00 2,00 2,00
1,60 1,60 1,60
1,20 1,20 1,20
0,80 0,80 0,80
ESD 5mC
ESD Ac H4 ESD Ac H3
0,40 0,40 0,40
0,00 0,00 0,00 0 15 30 45 60 75 0 15 30 45 60 75 0 15 30 45 60 75 Age (years) Age (years) Age (years) C D
50-year-old 3-year-old twins 50-year-old twins
Francis Collins, director of the National Human Genome Research Institute, said: "Here's something where Mendel, Watson and Crick all seem to have missed some crucial goodies" Discordant Twins for Breast Cancer
Heyn et al., Carcinogenesis 2012 Lupus eritematoso sistémico
Autoimmune disease affecting many tissues and organs
Autoantibodies against nuclear antigens, specially against DNA, histones and nucleosomes % methylation Twin A Average0 8 2 _ 0 1 A SLEVG _ B eTwinta 0.20 0.40 0.60 0.80 Diabetes Diabetes Type 2, Targettissue: Fat Systemic Systemic LupusErythematosus, Target tissue:Lymphocytes 0 1 0 Epigenetics, Epigenetics, Discordant Twinsand Disease 0.20 082_01 AVG_Beta vs AVG_Beta 082_02 AVG_Beta 082_01 Average Average Healthy Twin 082_02 AVG_Beta 0.40 0.60
0.80 twins 20 1 - yo
Average SLE Twin 0 3 0 _ 0 1 A V G _ B e ta 0.20 0.40 0.60 0.80 0 1 0
Average Average Healthy Twin 0.20 030_01 AVG_Beta vs AVG_Beta 030_02 AVG_Beta 030_01 030_02 AVG_Beta 0.40 0.60 TD2 -
twins (397
0.80 - 1 398)
Average0 6 1 _ 0 SLE1 AVG _TwinB e ta 0.20 0.40 0.60 0.80 % methylation Twin A 0 1
0
0.20 061_01 AVG_Beta vs AVG_Beta 061_02 AVG_Beta 061_01 % Average Healthy Twin methylation
061_02 AVG_Beta 0.40 Twin B 0.60
TD2
0.80 - twins (529
1 - 530)
DNA methylation contributes to natural human variation
au
100 A African-American (AF) B 100 100 Caucasian-American (CA) 99
Han Chinese-American (AS)
Height 40 60 80 100 120 100 80 60 40
C AF AS CA
PCA1
1 0 1 2 1 1 0
-
2 2 - -2 -1 0 1 2 PCA2 AF CA AS
D
Ancestral allele allele (%) Ancestral 0 20 40 60 80 100 80 60 40 20 0 Heyn et al., Genome Research 2013
DNA methylation and natural human variation: pathways and diseases DNA methylation and natural human variation: pathways and diseases
Taste Skin Pigmentation DNA methylation and natural human variation: pathways and diseases
Taste Skin Pigmentation
Diabetes Infection (HBV, HIV, Measles Parkinson’s Virus, E. coli)
DNA methylation plasticity of human adipose-derived stem cells in lineage commitment
A OSTEOGENIC MYOGENIC PTPRS - 293 + 92 Differentiation Suppression of Differentiation Suppression of medium the inductor medium the inductor
Adult Stem Cells_1
Adult Stem Cells_2
In vitro myocytes_1 PTPRS B - 293 + 92 C In vitro 800 myocytes_2
600 )
expression 400 SAA1
- 159 + 162 Cells 200
myocytes
PTPRS vs GAPDH) vsPTPRS
Relative
(
tem S Regressed 0
1 2 3
from ( Adult Stem Cells_1 SAA1 Adult - 159 + 162 Stem Cells_2
) 150
In vitro Osteocytes_1
Cells 100
expression osteocytes
In vitro tem S
Osteocytes_2 Regressed 50
from
(
SAA1 vs GAPDH)vsSAA1
( Relative 0 1 2 3 PIWIL2 PIWIL2 - 152 + 160
- 152 + 160 )
Adult 150
Stem Cells_1 Cells
100 osteocytes
Adult tem S
Stem Cells_2 Regressed expression
from 50
(
)
PIWIL2 vs GAPDH) vs PIWIL2 Relative
In vitro ( myocytes_1 0
1 2 3 4 5 Cells
In vitro myocytes
osteocytes_2
tem
S
Regressed
from (
Berdasco et al., American Journal of Pathology 2012 Stem Cells and Sirtuins Blue: DAPI Green: alpha-actin Embryonic Stem Cells (H-181) Differentiation upon siRNA of SIRT1 (mesodem)
Differentiation upon Sirtinol treatment (24h) Differentiation upon bFGF withdrawal
Epigenetic Marks The Epigenetic Marks: DNA Methylation
Esteller, Nat Rev Genet 2007 Esteller, New England Journal of Medicine, 2008 Heyn and Esteller, Nature Review Genetics, 2012 Roriguez-Paredes and Esteller, Nature Medicine, 2011
THE HISTONE CODE
H3, N-terminus H4, N-terminus
Me Ac P Ac Me Ac Ac Ac Ac
activation 4 9 10 14 18 23 27 36 5 8 12 16 20
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPH- SGRGKGGKGLGKGGAKRHRKVLRDN-
Me Ac silencing The Dark Genome Melo and Esteller, FEBS Letters, 2010 Melo and Esteller, FEBS Letters, 2010
Nature Genetics, 2009
Cancer Cell, 2010
Science, 2009 Esteller, Nature Reviews Genetics 2011 Rodriguez-Paredes and Esteller, Nature Medicine, 2011 Preclinical Clinical Epigenetic drugs stage stage DNA Demethylating Agents
Histone Deacetylase Inhibitors
Histone Methyltransferase Inhibitors
Histone Demethylase Inhibitors
Histone Kinase Inhibitors
Histone Acetyltransferase Inhibitors
Bromodomain Inhibitors
Sirtuin Inhibitors
microRNA Related Compounds
Rodriguez-Paredes and Esteller, Cancer Discovery 2011 Outside Cancer Rett Syndrome
Rett Syndrome (RS) is a neurodevelopmental disorder believed to affect 1 in 10,000 females..
Germline mutations in MECP2 (Methyl-CpG Binding Protein-2) found in 65% of Rett patients. MECP2 is located in Xq28.
Girls with Rett Syndrome appear to develop normally until 6 to 18 months of age. They then enter a period of regression, losing speech and hand skills they had acquired. Most girls develop seizures, repetitive hand movements, irregular breathing and motor-control problems. A slowing of the rate of head growth also becomes apparent. Most researchers agree that RS is a developmental disorder rather than a progressive, degenerative disorder. The girls can live to adulthood, but most never regain the ability to use their hands or to speak.
+ 103 +
N2
B MPP1 57 ºC 57
Annealing Silencing Silencing NAB
UB -
Human Human Mutation, 2005 B
MeCP2 UB
I
5´-CTTAAAAATCATCTCRCAAAAACT-3´ 5´-YGAATTTGGTTAGGGTATTYGGG-3´ Control R106W MPP1
Primer sequence Forward Reverse 264
- in Rett Syndrome Patients Syndrome Rett in
Gene MPP1 N2 N1 release of MeCP2 of release
Clustering expression analysis and and analysis expression Clustering NoMut3
NoMut1
NoMut4
NoMut2
R255X
R306C
R294X
P391X
T158M
R106W 36 36 38 38 35 35 49 49 36 36 41 41 105 105 108 108 84 84 9090 125 125 125 123 123 126 126 112 112 125 125 120 120 56 56 53 53 77 77 7171
Mouse Model of Rett
1
3 2
7.5gr/10.25cms 1.-Encéfalo 299mg 2.-Mesencéfalo 3.-Cerebelo
1 3 2
8.1gr/11.05cms 310mg Dynamic DNA Methylation Reconfiguration During Brain Development
Lister et al., Science 2013 •DNA Methylation and Schizophrenia DNA Methylation Map of Mouse Brain Identifies Targets of Epigenetic Disruption in Alzheimer’s Disease
Sanchez-Mut et al., Brain 2013 Promoter hypermethylation of the phosphatase DUSP22 mediates PKA-dependent TAU phosphorylation and CREB activation in Alzheimer's disease
Sanchez-Mut et al., Hippocampus 2013 DNA Methylation Profiling of Human Atheroesclerosis
Zaina et al., Circulation: Cardiovascular Genetics 2014 …Epigenomes “The Book of Life” (Almost…) Figure 1 Esteller Epigenetic differences in monozygotic twins and distinct penetrance of disease
Epigenetic differences among species and distinct skills
Epigenetic differences among species and distinct skills
Epigenetic differences in cloned animals and distinct phenotypes
Epigenetic Epigenetic changes in changes development in germinal and growth cells
Epigenetic alterations and human diseases
Esteller, The Lancet, 2008
Heyn and Esteller, Nature Reviews Genetics, 2012 Heyn and Esteller, Nature Reviews Genetics, 2012 A 450,000 CpG Site Prognostic Classifier of 500 Non-Small Cell Lung Cancer Patients A. B. Group 1 Group 2
Adeno Squamous Large 0 0.5 1 1.0 Hazard ratio, 2.30 (95% CI, 1.36-3.89) p: 0.002
0.8
free free survial - 0.6
group2 (n=174) 0.4
0.2 group1 (n=24)
0 Proportion with recurrence with Proportion 0 5 10 15 20 Years after surgery
C. Group 1 Group 2 D. Adeno Squamous Large 0 0.5 1 1.0 Hazard ratio, 2.46 (95% CI, 1.32-4.59) p: 0.005
0.8
free free survial - 0.6 group2 (n=114)
0.4
0.2 group1 (n=33)
0 Proportion with recurrence with Proportion 0 5 10 15 20 Years after surgery Cancer of Unknown Primary
The Distinct DNA Methylomes of Newborns and Centenarians
16,800,000 82% 1.0 Distance correlation to closest CpG site 80% 16,600,000 78%
76% 16,400,000
74% level (%) level 16,200,000 72%
CpG methylation CpG methylation 70% Methylated Methylated CpG sites 16,000,000 68% NB Y26 Y103 NB Y26 Y103 Correlation
0.8
0.1
1 10 100 1000 0.6 Distance
Chromosome 2 1.00 0.9 ** Meth. level Meth. ** 0.90 ** ** 0.8 0.4 0.80 0.7 0.70 0.6 0.8 0.60 **
0.5 0.50 NB NB 0.40 0.4 Y103 ** Y103 0.30 0.3
0.6 0.20 0.2 Mean methylation level methylation Mean 0.10 level methylation Mean 0.1
Chromosome 8 0.00 0 Meth. level Meth. 0.4 Promoter Exonic Intronic Intergenic CG poor CGI promoters promoters
CpG poor CpG island promoters promoters 140; 17% 479; 45%
9000 8000 DMRs
7000 15669; Hypomethylated 6000 87% in Y103 671; 83% 585; 55% Hypermethylated 5000 in Y103 4000 Tissue-specific genes Housekeeping genes 3000 8; 13% DMR numberDMR 2000 611; 34% 1000 0 2261; 13% Promoter Exonic Intronic Intergenic
1200; 66%
56; 87%
Proc Natl Acad Sci USA, 2012 Diseases Associated with Premature Aging: Hutchinson-Gilford Progeria and Werner syndrome
HG/W Nonagenarians Newborn Prediction of Biological Age Using DNA Methylation The Biomedical Iceberg
The Genome
The Epigenome
Manel Esteller, 2014