Estructura de la cromatina y la

regulacin de la expresin gnica

Mara-Paz Ramos, Ph.D.

Cells for Cells Consorcio Regenero S.A.
22.09.2015

 At the simplest level, chromatin is

a double-stranded helical
structure of DNA
DNA is complexed with histones
to form nucleosomes
Each nucleosome consists in eight
histone proteins around which the
DNA wraps 1.65 times
A chromatosome consists of a
nucleosome plus the H1 histone
The nucleosomes fold up to
produce a 30-nm fiber that forms
loops averaging 300 nm in length
The 300-nm fibers are compressed
and folded to produce a 250-nmwide fiber
Tight coiling of the 250-nm fiber
produces the chromatid of a
chromosome

Nucleosome core particle

Cada histona contiene una cola Nterminal, la cual puede sufrir
diversas formas de modificaciones
covalentes, y un motivo en comn,
conocido como histone fold domain
El plegamiento de histonas primero
dimeriza entre s para formar los
dmeros H3-H4 y H2A-H2B. Al
combinar un segundo dmero H3-H4,
dicho tetrmero combina con el
dmero H2A-H2B formando el
octamero final.

Digestin con Nucleasa Micrococal

(MNase)

Cuatro histonas componen el nucleosoma

MNase
digestion

Denaturacin
por Urea 8M

Histonas: protenas nucleares

con rico contenido en Arg y
Lys que forman parte de la
cromatina junto al DNA
Nomenclatura Nucleosmica:
H2A, H2B, H3, H4
No nucleosmica: H1
Peso: 12-16 kDa
H1= 23 kDa
Modificaciones
Acetilaciones, Metilaciones
Fosforilacin
Ubiquitinizacin en regin
CT de H2A
Sumoilizacin

Estructura del nucleosoma durante

la transcripcin

La estructura de la cromatina es
dinmica

Complejos que remodelan cromatina

Variantes de histonas
Modificaciones post-transcripcionales de histonas
Modificaciones covalentes del DNA

Complejos que remodelan la

Cromatina

Complejos
dependientes
de ATP

Histonas cannicas vs variates de

histonas

Histonas cannicas vs variates de

histonas

Ensamblaje de la cromatina
independiente de la replicacin

Variante de histona H3.3

Conceptos Generales
La cromatina es un complejo nucleoproteico. Est
conformada por DNA, histonas y protenas no histnicas.
La cromatina es (i) peridica (ii) compacta pero tambin
(iii) dinmica.
El nucleosoma es la principal unidad repetitiva de la
cromatina.
La cromatina est generalmente reprimida para la unin a
DNA y la transcripcin (modelo de acceso al DNA).
La transcripcin est asociada a profundos cambios en la
estructura de la cromatina
La estructura de la cromatina puede ser regulada por
remodelamiento de los nucleosomas dependientes de ATP
o por modificaciones covalentes de las histonas o

Estrategias para modificar la

estructura de la cromatina

EPIGENTICA
El estudio de cambios en la
funcin gnica que se heredan
a travs de la mitosis y/o
meiosis y que no pueden ser
explicados por cambios en la
secuencia del DNA
Riggs et al., Epigenetic
Mechanisms of Gene Regulation,
1996)

umped into the generates a diversity of cell types with and fundamental questions remain to
These include disparate, yet stable, profiles of gene be answered, many of which center on
ocesses, such expression and distinct cellular func- the propagation of epigenetic informaze (an interac- tions. Thus, cellular differentiation may tion through cellular division and differs in which one be considered an epigenetic phenom- entiation. We highlight some of these
hanges in the enon, largely governed by changes in questions as challenges to the emergffect variega- what Waddington described as the ing field. We also refer readers to the
phila (in which epigenetic landscape rather than review articles appearing in this special
nvironment of alterations in genetic inheritance (Wad- issue, as well as a new textbook enti(PTGS) and transcriptional
expression); dington, 1957; Figure 1). More (TGS)
spe- RNA
tled interference
Epigenetics (Allis et al., 2007; see
cific
Book Review by Y. Shi, page
(RNAi)-related
pathways,
respectively, in almost 639
all of this issue).
ci in
eukaryotes. These RNAs
teroften act in concert with
now
Mechanisms
various components of Epigenetic
the
at Work
cells chromatin and DNA
ommethylation
machinery
to
Much
of todays epigenetic
sms
achieve stable silencing.
research is converging on
pheAlthough we might not conthe study of covalent and
ntly
sider PTGS-inducing RNAs
(e.g., microRNAs, siRNAs,
noncovalent modifications
n of
etc.) to be epigenetic in
of DNA and histone proteins
ngs
nature, TGS-evoking RNAs
and the mechanisms by
onal
(e.g., repeat-associated siRwhich such modifications
NAs, Xist RNA, and small
ces,
RNAs
in
S.
pombe)
are
more
influence overall chromatin
and
clearly epigenetic, as they
structure. Chromatin, the
edican induce long-term silenccomplex of DNA and its intil of
ing effects that can be inherited through cell division
mately associated proteins,
ars
(Bernstein and Allis, 2005).
provides an attractive canCell.
Figure 1. Waddingtons Classical Epigenetic Landscape
well
In 1957, Conrad Waddington proposed the conceptEpigenetic
of an epigenetic
Pathways didate for shaping the fealandscape to represent the process of cellular decision-making
durIntersect
tures of a cells epigenetic
cs?
ing development. At various points in this dynamicIt visual
metaphor,
is
becoming
clear
that
landscape (see Review by
a of
the cell (represented by a ball) can take specific permitted
significant trajectocrosstalk exists
B.E. Bernstein et al., page
heir
ries, leading to different outcomes or cell fates. Figure
reprinted
from
between
different
epigenetic
Waddington, 1957.
e in
pathways. In S. pombe, 669
for of this issue). Diverse

El cambio de la teora de
Waddington

Cell

example, biochemical purification of the RNAi machin128,revealed

February
23, 2007
ery
an interaction

Figure 2. A Current View of the Epigenetic Molecular

Machinery
2007
Elsevier Inc. 635

during repli
understand
little about n
epigenetic in
Key ques
chromatin-b
ance surpr
unanswered
are histone
inherited th
tion, and if
this occur (s
Groth et al.,
issue)? How
porated hi
from parent
there an ac
for templatin
fications du
cation? Is te
histone mod
a consequen
to adjacent
as in posit
egation? Or
parental mo
new histone
sary? Are h
fications eno
modification
cellular ide
nisms shoul
plating of ne

Goldberg et al. (2007) Cell

Known factors that regulate epigenetic phenomena are shown directing

Epigentica

Las modificaciones epigenticas son

traducidas en funciones biolgicas a travs
de la interaccin con distintas protenas
H3K27m3

Promoter

Gene body
DNMT

K4

H3K4m3

Promoter

Gene body

methylated CpG site

WRITERS

unmethylated CpG site

Las modificaciones epigenticas son

traducidas en funciones biolgicas a travs
de la interaccin con distintas protenas
H3K27m3

Promoter

Gene body
MeCP2

K4

H3K4m3

Promoter

Gene body

methylated CpG site

READERS

unmethylated CpG site

Diversos dominios reconocen las

acetilaciones y metilaciones de las
histonas

Bromodominio

Cromodominio

Tudor Domain

Dedos PHD

Las modificaciones epigenticas son

traducidas en funciones biolgicas a travs
de la interaccin con distintas protenas
H3K27m3

Promoter

Gene body

K4

H3K4m3

Promoter

Gene body

methylated CpG site

ERASERS

unmethylated CpG site

s H3K4me2 or H3K4me1 (refs 17, 18). We iden

H3K4me2 enrichment
in ES cellsisfrom 1 kb to . 1
DNA methylation
inversely correlated with H3K4me3

Mean methylation
level of H C Ps (%)

a
100
50
0

ion
s (%)

ES
NPC
ES
NPC
ES
NPC
K4me3 K4me3 K4me3 K4me2 K4me3 None
n = 6,147
n = 191
n = 184

ES
N
Bivalent B
n = 11

All sampled sequence (excluding H C Ps)

100

Meissner et al. (2008) Nature

mote
W
Epithelial
modi
OPEN
DNA
doi:10.1038/nature14248
Brain
low D
Muscle
DNA
Heart
Sm. musc.
medi
Digestive
and E
were
Other
genes
ENCODE
Data
2012
proxi
1,2 states
Chrom.
Roadmap Epigenomics Consortium{, Anshul Kundaje1,2,3*, Wouter Meuleman
*, Jason Ernst1,2,4*, Misha Bilenky5*,
RefSeq genes
sion l
1,2
5
1,2
1,2
1,2
2,6
RNA-seq
Angela Yen , Alireza Heravi-Moussavi , Pouya Kheradpour , Zhizhuo Zhang
, Jianrong Wang , Michael J. Ziller ,
b H3K36me3
genes
7
8
9
1,2
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H4K20me1
Viren Amin , John W. Whitaker , Matthew D. Schultz , Lucas D. Ward , Abhishek
Sarkar , Gerald Quon ,
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H3K79me1
Richard S. Sandstrom10, Matthew L. Eaton1,2, Yi-Chieh Wu1,2, Andreas R. Pfenning
,
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Wang
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Melina
Claussnitzer
,
H3K9me1
2
DNase
Yaping Liu1,2, Cristian Coarfa7, R. Alan Harris7, Noam Shoresh2, Charles B. Epstein
, Elizabeta Gjoneska2,12, Danny Leung8,13,
Ch
DGF
15Input
Wei Xie8,13, R. David Hawkins8,13, Ryan Lister9, Chibo Hong14, Philippe Gascard
, Andrew J. Mungall5, Richard Moore5,
H3K4me3
ion
th
16
10
FigureKaul
2H3K9ac
|16Data sets available
for each1,2,17
reference
epigenome. List of 12
g hHansen
Tam5, Theresa K.e
Canfield10, R.
, Rajinder
,
a b
c d Eric Chuah5, Angela
f2 Scott
i jk
H3K56ac, Peter J. Sabo , Mukul S. Bansal
TxFln
18
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1,2
20
Annaick Carles , Jesse R. Dixon , Kai-How Farh , Soheil Feizi , Rosa
KarlicH2AK9ac
, Ah-Ram
Kim ,111
Ashwinikumar
Kulkarni Epigenomics
,
epigenomes
including
by the
Roadmap
program (E001
H2BK5ac
10
7
2,23
DNA
Daofeng Li21, Rebecca Lowdon21, GiNell Elliott21, Tim R. Mercer22, Shane
J.
Neph
,
Vitor
Onuchic
,
Paz
Polak
,
H3K4me2
E113)
See Supplementary Table 1(Fig.
for
H3K18ac16 by ENCODE (E114E129).
8,13
20
1,2
10 and
1,2
Nisha
Rajagopal
,
Pradipta
Ray
,
Richard
C.
Sallari
,
Kyle
T.
Siebenthall
,
Nicholas
A.
Sinnott-Armstrong
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H3K4me1
Cell type/
H3K27ac
21,42
10
24,25
21
21names
26 scores. ad, Tissue
11
list
of
and
quality
and
cell
types
grouped
by
H4K5ac
Michael Stevens
, Robert E. Thurman , Jie Wu
, Bo Zhang , Xin Zhou , Arthur
E. Beaudet , Laurie A. Boyer ,
tion,ty
tissue
H4K8ac
2,23,27
28
29
30 H3K4ac
5,31,32
33
Philip
L.
De
Jager
,
Peggy
J.
Farnham
,
Susan
J.
Fisher
,
David
Haussler
,
Steven
J.
M.
Jones
,
Wei
Li
,
of
biological
material
(a),
anatomical
location
(b),
reference
epigenome
H3K14ac
group
EID Epigenome name 5,32
enrich
H3K23ac 35,41
Marra , Michael T. McManus34, Shamil Sunyaev2,23,27
A. Thomson
, Thea D. Tlsty15, Li-Huei Tsai2,12,
H2AK5ac
fetal lungA.
fibroblasts
21 , James
E017 IMR90 Marco
IMR90
identifier
(EID,
c)
and
abbreviated
name
(d).
PB,
peripheral
blood.
ENC
H4K91ac
typica
8
36
20,37
38
2,39,40
E002 ES-WA7
cellsWang , Robert A. Waterland , Michael Q. Zhang
Wei
, Lisa H. Chadwick H2BK120ac
,H2BK12ac
Bradley E. Bernstein
1,
21
E008 H9 cells
14
9
5,18
2,6
7shown separately.
8,13
TssA
H2BK15ac
2012
reference
epigenomes
are
eg,
Normalized
stran
Joseph
F.
Costello
1,
Joseph
R.
Ecker
1,
Martin
Hirst
1,
Alexander
Meissner
1,
Aleksandar
Milosavljevic
1,
Bing
Ren
1,
E001 ES-I3 cells
H2BK20ac
H3K27me3
E015 HUES6John
cells A. Stamatoyannopoulos101, Ting Wang211 & Manolis Kellis1,21
37
states
ES cell
for
the
core
set
of
five
histone
cross-correlation
quality
scores
(NSC)
H3K9me3
E014 HUES48 cells
WGBS
E016 HUES64 cells
and h
Hi-C
marks (e), additional acetylation marks (f) and DNase-seq (g). h, Methy
E003 H1 cells
20
E024 ES-UCSF4 cells
s
t
s
c by
a
data
WGBS (red), RRBSd (blue) and mCRF
(green). A total of 104bindi
E020 iPS-20b cells
H3K4me1
obl
fibr
Data
g
E019 iPS-18 cells
n
lu
E018 iPS-15b cells
The reference human genome sequence set the stage for studies of genetic
variation data
and itssets
association
with
90 disease,reference epigenomes. i, Ge
methylation
available
inhuman
95IMRdistinct
iPSC
surem
E021 iPS DF 6.9 cells
n
i
s
butcells
epigenomic studies lack a similar reference. To address this need,expression
the NIH Roadmap
Consortium
generated
set
E022 iPS DF 19.11
data Epigenomics
using RNA-seq
(yel
ata (brown) and microarray expressionmatin
d
13
3
neuronal
progenitor
cultured
cells
E007 H1 derived
the largest collection so far of human epigenomes for primary cells and tissues.
Here we describe the3 integrative analysis
H3K4me3
1
E009 H9 derived neuronal progenitor cultured cells
j,
A
total
of
26
epigenomes
contain
184
additional
histone
modification
m
highl
ofneuron
111 reference
human epigenomes generated as part of the1 programme, profiled for histone modification patterns, DNA
E010 H9 derived
cultured cells
+ mesoderm
E013 HUES64 derived
CD56
accessibility, DNA methylation and RNA expression. We establish global
maps
of regulatory elements,
define(purple)
regulatorywere used for training acces
k, Sixty
highest-quality
epigenomes
the c
HUES64 derived CD56+ ectoderm
ES-deriv. E012
E011 HUES64 derived
modules
of+ coordinated
activity, and their likely activators and repressors.
Westate
showmodel,
that diseaseand
trait-associated
CD184
endoderm
meth
chromatin
which
was
then
applied
to
the
full
set
of
epigeno
E004 H1 BMP4 derived mesendoderm
11
DNase
variants are enriched in tissue-specific epigenomic
E005 H1 BMP4genetic
derived trophoblast
15 marks, revealing biologically relevant cell types for diverse
matin
(purple
orange).
E006 H1 derived
mesenchymal
stem
cellsproviding a resource for interpreting the
13 molecular
human
traits,
and
basisand
of human
disease. Our results demonstrate
E062 Primary mononuclear cells (from PB)
Gi
roleblood
of epigenomic
information for understanding gene regulation, cellular differentiation and human disease.
E034 Primary Tthe
cellscentral
from primary
(from PB)
E045 Primary T cells effector/memory enriched (PB)
cer re
RNA-seq
E033 Primary T cells from cord blood
genome-wide
strand cross-correlation37 (Fig. 2eg); inter-repl
E044 Primary T regulatory cells (from PB)
based
Blood &
E043 PrimaryWhile
T helper
cells
(from
PB)
the primary sequence of the human genand cells. We used a diversity of assays, including
and
M
E039 Primary T helper naive cells (from PB)
correlation;
multidimensional
scaling
of
data
sets
from
different
is largely
preserved
in all human cell types,
chromatin immunoprecipitation
(ChIP)9,10,16,17,
T cell
E041 Primaryome
T helper
cells PMA-I
stimulated
EPIGENOME ROADMAP
Ge
nom
than
7,18
E042 Primarythe
T helper
17
cells
PMA-I
stimulated
e-wI (DNase)
epigenomic
landscape of each cell can vary
DNA digestion
by DNase
, bisulfite
WGBS
duction
centres
(Supplementary
Fig. 1);
correlation across pairs of1
A Nature special issue
ide
E040 Primary T helper
memory cells (from PB)
me
1,2,19,20
ylatio
asu immunopreciE037 Primaryconsiderably,
T helper memory
cells (from PB)
contributing
to
distinct
gene
exprestreatment
,
methylated
DNA
rem
nature.com/epigenomeroadmap
ent
E048 Primary T CD8+ memory cells (from PB)
sets (Extended
Fig.
1e); consistency
between assays carried
o
21
s fo
r allrestricmatin
programs
and
biological
functions14. EpipitationData
(MeDIP)
, methylation-sensitive
E038 Primarysion
T helper
naive cells
(from
PB)
ma
rks
22
8
E047 Primarygenomic
cells (from PB)
T CD8+ naive
information,
such as covalent histone modifications, DNA tion enzyme
digestion
(MRE)
,
and
RNA
profiling
,
each
followed
by
multiple mapping centres (Supplementary Table 2); read mapping
Inter
E029 Primary monocytes (from PB)
andblood
DNA methylation can be interrogated in each cell and massivelyFigure
parallel3short-read
sequencing
(-seq). The
resulting
dataand
setsmarks. a, Chromatin active
38,39
B cells from cord
E031 Primaryaccessibility
|
Epigenomic
information
across
tissues
lity
for
bisulfite-treated
reads
;
and
agreement
with
imputed
d
haematopoietic
stem
cells (HSCs)
E035 Primarytissue
type using
high-throughput
molecular assays2,58. The resulting were assembled into publicly accessible websites and databases, which
E051 Primary HSCs G-CSF-mobilized male
state annotations across 127 reference epigenomes (rows, Fig. 2) in a ,3.5-Mb interm
HSC &
a broadly data
useful resource
for the
scientific and
biomedicalor
combeen instrumental
for annotating cis-regulatory elements serve as
HSCshave
G-CSF-mobilized
female
E050 Primarymaps
Outlier
sets were
flagged,
removed
replaced, and lower-cove
region
chromosome
9. Promoters
are reference
primarilyepigeconstitutive (red vertical
E036 Primary HSCs short term culture
sion,
B cell
weon
report
the integrative
analysis of 111
other
non-exonic
genomic features with characteristic epigenomic munity. Here
B cells
(from
PB)
E032 Primaryand
Chromatin state annotations in 127

ARTICLE

HSC &
B cell
Mesench.

E035
E051
E050
E036
E032
E046
E030
E026
E049
E025
E023
E052
E055
E056
E059
E061
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E058
E028
E027
E054
E053
E112
E093
E071
E074
E068
E069
E072
E067
E073
E070
E082
E081
E063
E100
E108
E107
E089
E090
E083
E104
E095
E105
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E078
E076
E103
E111
E092
E085
E084
E109
E106
E075
E101
E102
E110
E077
E079
E094
E099
E086
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Integrative analysis of 111 reference

human epigenomes
FAM205B

ATP8B5P

SIT1 NPR2

RECK

RNF38

MELK

PAX5

GRHPR

FAM205B

ATP8B5P

SIT1 NPR2

RECK

RNF38

MELK

PAX5

GRHPR FRMPD1

FRMPD1

SHB

ALDH1B1

SHB

ALDH1B1

H3K4me1
H3K4me3
H3K36me3
H3K27me3
H3K9me3
H3K27ac
H3K9ac
DNase-Seq
DNA methyl
Gene expr.
Addtl marks
Chrom. states

IMR90 fetal lung fibroblasts

WGBS

H3K4me1

DNase
H3K4me3

RNA-seq

Chromatin states

127 reference epigenomes

Individual mark data sets across epigenomes

Primary cells

ES cell derived

Primary cultures

Sample type

ARTICLE RESEAR

Modificaciones en el histone tale

Diversas enzimas modifican a las

histonas

Metilasas (HMT)
Demetilasas (KDM)
Lys, Arg

Kinasas
Fosfatasas
Ser, Thr

Acetilasas (HAT)
Deacetilasas (HDAC)
Lys

Acetilacin de las histonas

Histonas acetiladas: modelo

electroesttico

Protena de la Tetrahymena de
55 kDa con actividad HAT
Histonas

Tetrahymena p55: la primera HAT

caracterizada

Tetrahymena p55 = S. cerevisiae Gcn5

Metilacin de histonas

Modificaciones en las histonas

regulan la transcripcin

Activacin de la transcripcin

COMPASS
Setd2

Represin de la transcripcin

Existen mltiples anticuerpos que

reconocen las modificaciones de las
histonas

Inmunoprecipitacin de la
Cromatina (ChIP)

Visualizacin de ChIP-seq a travs

de UCSC Genome Browser
Scale
chr10:

50 kb
62,300,000

30

mm9
62,350,000

siCt
0
30

siKdm5b
0
CpG Islands

131

UCSC Genes (RefSeq, GenBank, tRNAs & Comparative Genomics)

Tet1
Tet1
Tet1

ENCODE
H3K4me3 track
0

Scale
chr13:

30

5 kb
101,550,000

mm9
www.genome.ucsc.edu

101,555,000

Transcripcin y la modificacin de
histonas

RNAP II

MLL3/4

H3K4me3
P
P

Y
PS
ST
SP

P
ST
SP

SY

Y
PS
ST
SP

RNAP II
SETD2

TF I IH

H3K36me3
P
P

CAP

Y
PS
ST
SP

Y
PS

P-TEFb

P
ST
SP

SY

TF I IH

ST
SP

5
CAP

The recruitment of COMPASS is produced by pSer5 during

transcriptional initiation. Through its subunit MLL3/4, H3K4me3 is
catalyzed at TSS. Continuing with transcription elongation, SETD2, the
only enzyme capable of producing H3K36me3 in mammals, gets recruited
to the transcription foci by pSer2, which then recruits the TFIIS
elongation

La hiptesis del cdigo de las

histonas

Posibles mecanismos que explicaran este efecto:

Las modificaciones produciran un cambio en la estructura de
la cromatina hacia un estado abierto o cerrado de sta.
Reclutamiento de protenas regulatorias.

N
O

Metilacin del DNA

O

Cytosine

Uracil

NH2
N
O

C
N

methyl-Cytosine

Substrato: CpG (CHG, CHH) dinucletidos

En mamferos, metilacin es global

pero no
CpG island
O uniforme:
N
Thymine

Catlisis: DNA MetilTransferasas

(DNMTs)
DNMT3A/B de novo metila al DNA en
el contexto CpG
DNMT1 mantiene la metilacin luego
de la replicacin
Jia et al. (2007) Nature

Despite its global depletion, CpG sites

are highly present in gene-rich regions
Deamination

NH 2
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O

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methyl-Cytosine

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purified and tested for resistance to the methylation-sensitive

restriction endonuclease HpaII. As shown in Fig. 3a, DNA of
mutant germ cells was less methylated than DNA from wild-type
germ cells or mutant somatic cells from testis. The DNA blot
hybridization of Fig. 3b shows that the promoter regions of LINE1 retrotransposons were largely demethylated, as were LTR
sequences of retrotransposons of the intracisternal A particle
(IAP) class (Fig. 3c). These findings are consistent with the overall
demethylation shown in Fig. 3a, because retrotransposons and their
remnants contain the large majority of the 5-methylcytosine present

La metilacin del DNA regula la

estabilidad de la cromatina y la expresin
gnica

Meiotic organization

Figure 3 Abnorma
from 17 d.p.p. tes
stained for GCNA1
endonuclease Hpa
contained DNA tha
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right show partial d
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covered positions 8
probe spanned the
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from plasmid pMR
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Bourchis et al. (2004) Nature

Alternative splicing
Figure 2 Meiotic catastrophe in spermatocytes derived from Dnmt3L-deficient
prospermatogonia. a, Normal meiotic synapsis in wild-type spermatocytes. Note the
formation of complete linear synaptonemal complexes except for the XY chromosome
pair, in which synapsis is restricted to the pseudoautosomal regions. b, Branching and
anastomosing synaptonemal complexes in Dnmt3L-deficient spermatocytes. Nearly all
the chromosomal regions are unpaired or engaged in non-homologous synapsis; a single
The new england journal of medicine
apparently normal synaptonemal complex is indicated by the white arrowhead.
Thec,new
of medicine
england
d, Formation
of highly journal
aberrant complexes
of synaptonemal proteins in the form of
interlocked rings (c) and complex three-dimensional structures (d) in Dnmt3L mutant
spermatocytes. Similar staining patterns were seen after
labelling with antibodies against
)*+'
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DNMT
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Pancreatic beta cell identity is maintained

by DNA methylation of Arx promoter
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Figure 1. Loss of DNA methylation in beta cells results in an increase in the number of glucagon
expressing cells

(A) Representative pancreatic sections from 3 months old Dnmt1fl/fl (control) and RIPDhawan (Glu;
et al. (2011)
Devand
cell.
Cre:Dnmt1fl/fl (RC:Dnmt1fl/fl) littermates were immunostained for glucagon
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CCGG sequences where the central CG dinucleotide is unmethylated.
The first Illumina adapter (AE) is ligated to the compatible cohesive
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Luminometric methylation assay

(LUMA)

Karimi M. et al, (2011) Experimental Cell Res

Downlo

2005 Natur

Humain (CEPH) pedigree 1332 (fig. S3) reWe modified the Affymetrix 500,000 (500K) clone after MSRE treatsolved the parental origins of 1948 heterozyDifferential DNA methylation is important
for the epigenetic
of gene
single-nucleotide
polymorphismregulation
(SNP) mapping
ment.expression.
This is due to methInput
IP
Figure
2of Chromosomal
ofgous
DNA
array X(12)
to allow allele-specific
analysis
ylation at
differences profile
at an
SNPs in the mother GM10849 and/or her
Allele-specific1 methylation of the inactive
chromosome
has been
demonstrated
arrays,
Reversible methylation of cytosine is a major epigenetic modification microarray. Using whole-genome as well as promoter-specific
DNA methylation (13). We digested
genomic MSRE
site
label with
Cy5 1. In label
withcytosine
Cy3 methylationpromoter
methylation
using
apresent
tiledwithin
whole daughter
human genome
GM13130. Sequence analysis sugislands,
butof the
overall
of
methylation
on
the
active
X
(Xa)
and
R =profile
0.88
we present CpG
a methylation
unique
sequencespattern
of the human
in multicellular
organisms
mammals,
DNA with a cocktail of five methyl-sensitive the amplicon (fig. S1).
genome in
and transformed cells.
occurs almost exclusively at CpG dinucleotides, which are under-inactive
BAC
array.
(a)
Methylation
levelsgested
are determined
that 1269 (65.1%) of them were suitX primary
(Xi) chromosomes
is unknown.
We performed
allele-specific
analysis
of
more
Mismatch
(mm)than
probes
restriction
enzymes (MSREs)
(14).
Together,
0.5
represented in the genome with the exception of CpG islands.
single
frequent cutters recognize
~40%
of all
able
forDNA
our methylation assay, in that they
informative loci along the humanthese
X chromosome.
The Xa displays
morecontain
thanainput
two mismatch
by cohybridizing
and bound
(IP)
RESULTS
These are small CpG-rich regions that, in many cases, are associated1000
at a site distinct from the
CG dinucleotides
the genome (15),
Unbiased
detection
of methylated DNA
by immunoprecipitation
with promoter regions. Cytosine methylation results in transcriptionaltimes
had
at least one methyl-sensitive restriction
as much
allele-specific
methylation
as Xi. Thisinmethylation
is allowing
concentrated
at
gene
labeled
with
different
fluorescent
dyes.
polymorphic
site
and
serve
an
efficient
analysis
of
regions
with
both
high
DNA
repression either by interfering with transcription factor binding Current strategies to identify chromosomal sites of
AR
T I methylaC LBefore
E S X inactivation, all of these Xa gene
0
site on
their
amplicons (table S2).
bodies, affecting
multiple neighboring
CpGs.
controls. (B) PresentaMethylated assequences
were labeled
with
Cy3respective
and
and
low
GC content.
After this pretreatment,
restriction
or by inducing a repressive chromatin structure2. DNA methylation tion rely primarily on the use of methylation-sensitive
tions of Xa (black) and Xi
These
informative
SNPs
were distributed
siteshigh-molecular-weight
are biallelicallyDNA
methylated.
a bipartite
methylation-demethylation
enzymes. These require
and of
are200Thus,
is required to complete embryonic development3 and has beenbodymethylated
fragments
to 1100
base pairs
containhave a higher
fluorescence
(yellow)
monoallelic meth-in the green channel.
limited byresults
the sequence
of the chosen
enzyme.
directly implicated in genomic imprinting4 and X-chromosomeprogram
ingFortheexample,
polymorphic
were polymerase
along
the chromosome
(fig. S4) with an averin context
Xa-specific
hypomethylation
at gene sites
promoters
and
hypermethylation
ylation
fractions
in
four
The
ratio
of
bound
over
input
is
calculated
for
only 3.9% 0.5
of all CpGs in human nonrepetitive DNA
reside
at
sites
inactivation2.
chain reaction (PCR) amplified, and the reage distance of 120.1 kb between succeeding
gene
bodies.used
These
relationship
between global and
methylation
clones and
two individuthe frequently
HpaIIresults
enzyme7. suggest
Alterations in DNA methylation are associated with many humanat for
Conversion a
of
unmethylated
sulting
amplicons were then labeled
hy- on
each spot
thefrom
array
and used as a measure of
diseases and are a hallmark of cancer5. A decrease in the total amountexpression
cytosine withpotentiality.
bisulfite followed by sequencing provides
an unbiased
SNPs. Moreover, 351 SNPs mapped to 135 difbridized
to the array. Thus, an unmethylated als. The parental origin of
methylation.
(b)
Comparison
of
enrichments
from
Xa
for
each
clone
is
indialternative, but it is laborious and cannot be easily
of cytosine methylation is observed in many human neoplastic tissues, and sensitive 1
MSRE site present on a given amplicon will
ferent
genes (for
an average of 2.6 SNPs per
but the genomic context of this hypomethylation has not been applied to screening a large set of sequences or samples8. To circumcated, and the numbers
genetically
unrelated
primary
fibroblasts.
Shown
lead
to
allele-specifically
reduced
intensity
cor6
gene), representing ~9% of the known X-linked
is robust
NAlimitations,
methylation
is essential
many
identified . At the same time, aberrant promoter hypermethylation vent these
we developed
methylatedfor
DNA
immuno-after the MSRE treatment. The
of assay
occurrences
of monoresponding
to the resident SNP (Fig.
This allelic
(MeDIP), which
permits highly
efficient
enrichment
has been observed in sporadic cancer and is thought to contribute to precipitation
is 1A).
aof
pairwise
comparison
of
levels
methylation
are methylation
genes (table
S2).
the
DNA never
developmental
processes,
including
mainallowed
us toreplicate
use either analyses
genotype calling
or same
1.5
DNA. In this assay, an antibody specific
for methycarcinogenesis by inactivating tumor-suppressor genes5. In light of the of methylated
marked
blueand
(paternal)
(log
ratio)
in male
female fibroblasts
for the active and inactive X copies
We resolved
reveal 1allele-specific
status
changtaining1.5
Xi0.5
statemethylated
(13).
2 methylation
copy-number
algorithms
to identify transitions
is the
usedsilenced
to 1
immunocapture
genomic
relevance of DNA methylation for normal development and disease, lated cytosines
0 Xi0.5
or
red
(maternal).
Results
online material (SOM)
Microarray
from
a heterozygous
state toone
a hemizygous
statethe from
fragments.
The resulting
in the
immunoprecipitated
we know little about its genomic distribution. This partly reflects thespecific
all autosomal
BACs.
similarity
isof indicated
in clones
from
single cells
of GM10849
ing from
allele
to
other
methylation
is enrichment
seen
at CpG
islands
(49),
the [supporting
Nsp The
I or the
Sty I halves
theoriginated
500K arrayby
are presented.
(C) Frequency
of switching validation
from
included DNA
Female
(WI38)
DNAfibroblasts
detection (Fig.
1a); thus,
limitations of existing techniques for analyzing DNA methylation at fraction is determined by standard
heterozygous
to hemizygous(R
callafter
MSRE treatment for each chromosome. Data are presented for
all PCR products befo
but
the
global
distribution
of
Xi-specific
metha
high
correlation
coefficient
0.88).
cific
Human Genetic Research and Department of
usingforexisting
specific sequences. Here we used an immunocapturing approach to MeDIP can be combined with large-scale analysisCenter
informative 500K SNPs for GM13130 clone 7 and GM13130 clone 16. Potential monoallelic methylation at
Medicine, Massachusetts General Hospital,(c)
Harvard
Medtreatment (table S1), and
along the chromosome is unknown.
DNA microarrays.
enrich methylated DNA and combine it with detection by DNAylation
Methylation
profile
of human
16 resulting from MSRE site polymorphisms
parentally
imprinted
regions, aschromosome
well as technical artifacts
or
ical School, 185 Cambridge Street, Boston, MA 02114,
ses (fig. S2).
0.4
Fig. 1. HypermethylaAlthough silenced chromatin regions areUSA.
usually
allelicline)
bias ofand
the genotyping
algorithm,
were filtered out by requiring at least one clone switch from AB
E-mail: hellman@chgr.mgh.harvard.edu
(A.H.); (red
in female
male (blue
line)
Genotyping individua
to A0, and one
cloneChess
switch from AB to B0 per each SNP.
chess@chgr.mgh.harvard.edu
(A.C.)
tion of the
active X chroAsaf Hellman
Andrew
hypermethylated,
studies
have
sugFriedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058
Basel, Switzerland. British cytogenetic
Columbia Cancer Research
Center,
Vancouver,
British
tions of Centre dEtude
fibroblasts
after and
local
averaging.
Gray and black
1
2
1
1
3
2
Columbia, Canada. Department
of Pathology, Dresden
University of Technology,
Dresden, Germany. Correspondence
should be addressed
to D.S. (dirk@fmi.ch).
mosome. (A) An example
Michael Weber , Jonathan J Davies , David Wittig , Edward
Michael Haase , Wan
L Lam
&
gestedJaOakeley
global ,hypomethylation
of the
Xi (10);
Humain (CEPH) pedigre
boxes reflect R and G banding, respectively.
1 2005; doi:10.1038/ng1598
Published
online
10 July
beler
Dirk Schu
solved
the parental origi
Differential DNA methylation
theFEBRUARY
epigenetic regulation
of gene expression.
www.sciencemag.org
SCIENCEis important
VOL 315for 23
2007
1141
also, studies of mammalian hypoxanthine-guanine of hybridization intensities
0
A
methylation
map
for
the
complete
human
gous SNPs in the mother
(brighter yellow indi-Allele-specific methylation of the inactive X chromosome has been demonstrated at
phosphoribosyltransferase
(HPRT)
genes
have
daughter GM13130. Seq
promoter
CpG islands,
but the overall pattern
of methylation
on the active X (Xa) and
Cytosine methylation is required for mammalian development and is often perturbed in human cancer. To determine how this 8 5 3cates higher signal)
genome
is shown
in Supplementary
Figure
1
NATURE GENETICS VOLUME 37 [ NUMBER 8 [ AUGUST 2005
for
shown
that atcells,
least
site
within the approach
tranepigenetic modification is distributed in the genomes of primary
and transformed
we one
used an
immunocapturing
gested that 1269 (65.1%
inactive X (Xi) chromosomes is unknown. We performed allele-specific analysis of more than
the
six
probe
sets
queonline.
(d)
Methylation
profile
of
a
30-Mb
region
followed by DNA microarray analysis to generate methylation profiles
of
all
human
chromosomes
at
80-kb
resolution
and
for
able for our methylation
1000
informative
loci
along
the
human
X
chromosome.
The
Xa
displays
more
than
two
REPORTS
scribed region of the gene is methylated only
a large set 0.4
of CpG islands. In primary cells we identified broad genomic regions of differential methylation with higher levels
rying a particular
SNP
had at least one methy
times
as
much
allele-specific
methylation
as
Xi.
This
methylation
is
concentrated
at
gene
positioned
at
Xq.
The
X
chromosome
is
globally
on
Xa
in
opposition
to
the
Xi-specific
methin gene-rich neighborhoods. Female and male cells had indistinguishable profiles for autosomes but differences
on the using
X
ificity
and
B cell line expression
levels
showed
The
CpGaffecting
frequencymultiple
in gene-body
amplicons
and GM13130
reverse
transcription (RT) When
site on their respective a
bodies,
neighboring
CpGs.(red
Before
X inactivation,
all of these
Xa gene
(rs16999756).
hypomethylated
in
female
fibroblasts
line)
chromosome. The inactive X chromosome (Xi) was hypermethylated
at only
subset
gene-rich
regions
and,
unexpectedly,
ylation
of athe
5ofCpG
island
(6,
9,
11).
This
expressed
andmethylation-demethylation
silenced genes underdisplaying
Xa methylation
is biallelically
similar to methylated.
the that both
PCR to examine allele-specific
mRNA expresThese informative SN
bodymethylated
sites are
Thus,
a bipartite
examining
the compared
perfectoverall hypomethylated relative to its active counterpart. The chromosomal methylation profile of transformed
cells
was
similar
with
male
fibroblasts
except
go atXa-specific
methylation
(table S9). Furaverage
S8), hypomethylation
well (blue
below line),
sion to
(table
S3). Replication
timing analyses fur- genome-wide
unexplained divergence led us
conduct
a match
along the chromosome (f
program
results
in (table
Xa-specific
gene
promoters
and hypermethylation
to that of primary cells. Nevertheless, we detected large genomic segments with hypomethylation in the transformed cell
(pm)
probes,
one
thermore,
genes
subject
to
Xa
methylation
do
the
frequency
seen
in
CpG
islands.
Neverthether
confirmed
the
direction
of
X
inactivation.
Chr 16
Mb Furthermore, analysis of 6,000 CpGcomprehensive
age distance of 120.1 kb
gene bodies.region
These results
suggest
a relationship
theatgene-rich
at the
telomeric
end.between global methylation and
analysis
of the
and Xiwas
allele can observe bothinalleles
residing in gene-poor10
areas.
islands showed that
only a small
set ofXa
promoters
not
share
a
common
biological
function
or
less,
nearby
CpGs
within
different
amplicons
We
selected
four
clones,
one
paternal
active
SNPs. Moreover, 351 SN
expression potentiality.
methylated differentially, suggesting that aberrant methylation of
CpG island
promoters in malignancy
might
be less
frequent
specific
methylation
patterns of
the
human
X active
(e) distantly
Xi
is hypomethylated
in gene-poor
regions
and
expression
pattern
(17).
We
cannot
rule
out
a
present
in
the
same
gene
(Fig.
2
and
(Xap)
and
one
maternal
(Xam)
from
each
in
genomic
DNA,
but
ferent genes (for an aver
than previously hypothesized.
chromosome. 0.3
specific
need
for
shutting
down
spurious
trantable
S4),
and
neighboring
CpGs
within
indiindividual, for further analysis.
By
definition,
gene), representing ~9% o
hypermethylated
in
gene-rich
regions
relative
to
after
the
MSRE
treatment.
The
assay
is
robust
NA
methylation
is
essential
for
many
only one allele in each
scription
(e.g.,analyses
from transposable
elements)
(fig. S7) tend
to have
the same
clones (500K)
must originate from four indepen- vidual amplicons
replicate
of the same
DNA at
never genes (table S2).
developmental
processes,
including
mainWe modified the Affymetrixthese
500,000
0.4
clone
after
MSRE
treatthe
active
X.
Plotted
is
the
average
methylation
regions
(i.e., genes),
but the absence
of
Xa-specific taining
methylation
pattern. Xi state (13). Xi-active
dent
X inactivation events.
as well
as promoter-specific
arrays,
Reversible methylation of cytosine is a major epigenetic modification microarray. Using whole-genome
We resolved the active
reveal
allele-specific
methylation
status changthe silenced
single-nucleotide0.2
polymorphism
(SNP)
mapping
ment. which
This isheterodue to
methlevel
for
allmethylation
X-linked
depending
sequences
of the
in multicellular organisms1. In mammals, cytosine methylation we present a methylation profile of uniqueAs
bias allele
toward
Silencing
tissue-specific
not
a prelude
to human
determining
ing on
from one
to repetitive
the otherelements
[supporting in clones originated from si
specific
isBAC
seen genes
atclones
CpG does
islands
(49),a methylation
(12)
to allow
allele-specific
analysis
of
genome
in primary
and transformed
cells.
occurs almost exclusively at CpG dinucleotides, which are under-array
ylation
differences
at
an
explain
Xa-specific
gene-body
methylation;
zygous SNPs switch to a hemizygous state aftergene
but the
global
distribution
of Xi-specific
meth-embedded in genic amplicons (table S9) is a
count
for male
(blue)
and
female
0.1 (13). We digested
represented in the genome with the exception of CpG islands.DNA methylation
indeed,
mining
public
for tissue
spec- probable hint against this hypothesis. The list
digestion with
the MSRE MSRE
cocktail,site
we present
first fil- within
genomic
ylation
along
thedatabases
chromosome
is unknown.
0
(red) fibroblasts.
These are small CpG-rich
regions that, in many cases, are associated RESULTS
tered
out
SNPs
that
would
show
loss
of
heteroAlthough silenced chromatin regions are usually Fig. 1. Hypermethylawithdetection
a cocktail
of fiveDNA
methyl-sensitive
the amplicon (fig. S1).
Unbiased
of methylated
by immunoprecipitation
with promoter regions. Cytosine methylation results in transcriptionalDNA
zygosity
as a result of polymorphism in their
hypermethylated, cytogenetic studies have sug- tion of the active X chroCurrent strategies
to 0identify
chromosomal
sites Together,
of DNA methyla-Mismatch (mm) probes
repression either by interfering with transcription factor bindingrestriction
Fig.
2. Genes are preferenzymes
(MSREs)
(14).
linked MSRE sites (fig. S1). We then analyzed
gested
hypomethylation of the Xi (10); mosome. (A) An example
or by inducing a repressive chromatin structure2. DNA methylation tion rely primarily on the use of methylation-sensitive restriction
able
targets aforglobal
Xa-specific
contain
a
single
mismatch
these
frequent
cutters
recognize
~40%
of
all
the
methylation
status
of
the
remaining
913
in3
also,
studies
mammalian hypoxanthine-guanine of hybridization intensities
is required to
complete embryonic development and has been enzymes. These require high-molecular-weight DNA and are
methylation.
(Top)ofEnrich0.4
0.1in context
SNPs
(table
S4).at a site distinct from the
(HPRT) genes have (brighter yellow indidinucleotides
the genome
(15),
allowing
by the sequence
of the formative
chosen
enzyme.
For
example,
directly implicated in genomic imprinting4 and X-chromosomeCGlimited
mentphosphoribosyltransferase
of Xa-methylated
A pattern
appeared
when
we related
the
shown
that
at
least
one
site
within the tran- cates higher signal) for
only
3.9%
of
all
CpGs
in
human
nonrepetitive
DNA
reside
at
sites
inactivation2.
polymorphic
site
and
serve
SNPs
at
gene
regions.
an efficient analysis of regions with
both
high
to Xa of
and
Xi: We always observed a sig- The scribed
Alterations in DNA methylation are associated with many human for the frequently used HpaII enzyme7.SNPs
Conversion
unmethylated
region
ofXa-the gene is methylated only the six probe sets quedistributions
of
as
controls.
(B)
Presenta0.2
rying a particular SNP
low with
GCbisulfite
content.
After
this
pretreatment,
nificant
excess an
of unbiased
monoallelically methylated methylated
diseases and are a hallmark of cancer5. A decrease in the total amountandcytosine
followed
by sequencing
provides
on Xa in
opposition
SNPs
(black) to the Xi-specific meth- (rs16999756). When
2loci onand
4 cannot
of8 Xaratio
(black)
Xiall informative
and sensitive
it isbase
laborious
be 6
easilytionsXa:Xi
of cytosine methylation is observed
in many
Chr
Xq human neoplastic tissues,fragments
the
Xa. The
average
was and
of alternative,
200 to0 but
1100
pairs
containof the SNPs
5 CpG island (6, 9, 11). This
and ylation
8
examining the perfect10 Mb
Gene
count
or samples
but the genomic context of this hypomethylation has
not been applied to screening a large set of sequences
. To circum(yellow) methylation
monoallelic meth2.4 (Fig.
1B).
The
allele-specific
unexplained
led us to conduct a match (pm) probes, one
(gray)
are shown.divergence
SNPs
ing the polymorphic sites were
polymerase
identified6. At the same time, aberrant promoter hypermethylation vent these limitations, we developed we
methylated
immunoobservedDNA
is unique
to the
X chromosome,
asin were
comprehensive
analysis
of the Xa and Xi allele can observe both alleles
ranked according
to
ylation
fractions
four
reaction
(PCR)
theenrichment
reprecipitation
(MeDIP),
whichamplified,
permitsshown
highlyand
has been observed in sporadic cancer and is thought to contribute tochain
byefficient
control
analyses
of autosomes,
whichindividudistance
frommethylation
the near- patterns of the human X in genomic DNA, but
specific
clones
from two
of methylated
DNA. Inwere
this assay,
an antibody
specific
for
carcinogenesis by inactivating tumor-suppressor genes5. In light of thesulting
amplicons
then
labeled
andlower
hy-methyshow
a vastly
level of switching and no est chromosome.
gene (table S6).
only one allele in each
relevance of DNA methylation for normal development and disease, lated cytosines is used to immunocapture methylated genomicals. The parental origin of
probably
reflecting
de novo
methylation
of promoters
undergoing
genomic
regions (data not shown). We then applied
local
averaging
to
parent-of-origin
effects (Fig.
1C and fig S5).
(Bottom)
complete
WeThe
modified
the Affymetrix
500,000 (500K)
theresulting
array.
Thus, an
clone after MSRE treatfragments.toThe
enrichment
in unmethylated
the immunoprecipitated
we know little about its genomic distribution. This partly reflects thebridized
Xa
for
each
clone
is
indilist
of
the
SNPs
showing
Because
this
global
Xa
hypermethylation
single-nucleotide polymorphism (SNP) mapping ment. This is due to methfraction
is determined
standard
DNA amplicon
detection
1a); thus,
limitations
of existingchromosomal
techniques for analyzingmaps
DNA methylation
atMSRE
site
present
a given
will
X to(Fig.
inactivation.
Yet
most
of
the
X
chromosome
is
gene-poor
and,
create
complete
of DNA
methylation
inbyon
male
and
Xa-specific
methylations
is
contrary
the
reports
of
Xi-specific
methcated,
and
the
numbers
array (12) to allow allele-specific analysis of ylation differences at an
specific sequences. Here we used an immunocapturing approach to MeDIP can be combined with large-scale analysis using existing
lead to microarrays.
allele-specifically
intensity
cor- we
in all
fouris
clones
from(13). We digested at
ylation
ataccording
CpG islands,
rule out
DNA
methylation
genomic
enrich fibroblasts
methylated DNA (Fig.
and combine
it with Supplementary
detection by DNA DNA Fig.
to
ourto experiments,
hypomethylated
Xi.
Previous
female
2c and
1 online). reduced
These
MSRE site present within
ofsought
occurrences
of monois presented,
possibility
that the clones we analyzed GM13130
responding to the resident SNPthe(Fig.
1A). This
DNA with
a cocktail of five methyl-sensitive the amplicon (fig. S1).
methylation
the methylated
allele (MSREs) (14).
reports
hadallelic
already
hinted
atarerestriction
reduced
methylation
of Xi
in metaphase
profiles illustrate the high similarity of DNA
between
have unusual
Xi methylation
patterns. Two
in- with
Mismatch (mm) probes
enzymes
Together,
allowedmethylation
us to use either
genotype
calling
or
marked
blue
(paternal)
indicated
for
each
clone
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland. British Columbia Cancer Research
Center,
Vancouver,
British
formative SNPs reside on amplicons that
a single mismatch
these
frequent
cutterswith
recognize
~40%
of all contain
spreads,
an
antibody
against
maleColumbia,
andCanada.
female
autosomes.
Department
of Pathology, Dresden University of Technology,copy-number
Dresden, Germany. Correspondence
shouldto
be addressed
to D.S.
(dirk@fmi.ch). as measured by immunostaining
algorithms
identify
transitions
(maternal
in
pink
and
red (maternal).
overlap with CpG islands: or
rs17139585
at phos- Results
CG dinucleotides in the genome (15), allowing at a site distinct from the
13
paternal
in
blue).
Published online 10 July 2005; doi:10.1038/ng1598
from a heterozygous state to a phoglycerate
hemizygous
state
polymorphic
and serve of switching from
kinase 1 and from
rs864570
calcium/
5-methylcytosine
or Iby
immunodetection
of unmethylated
HhaII
an
analysis
of regions
with
theatNsp
or the
Styefficient
I halves
of the
500K
arrayboth
arehigh
presented.
(C) site
Frequency
as controls. (B) Presentacalmodulin-dependent serine
protein
kinase.toAshemizygous
and low
GC
content.
After
this pretreatment,
14
heterozygous
call
after
MSRE
treatment
for
each
chromosome.
The inactive X chromosome is globally Center
hypomethylated
restriction
sites
. In addition,fragments
there isof at
least one reported example of Data are presented for all
these twoofSNPs
are monoallelically
for Human Genetic Researchexpected,
and Department
200 to 1100 base pairs contain- tions of Xa (black) and Xi
NATURE GENETICS VOLUME 37 [ NUMBER 8 [ AUGUST 2005
8 5 informative
3
500K
SNPs for
GM13130
clone 7sites
andwere
GM13130
clone 16.
Potential
methylation at
15. monoallelic
(yellow)
monoallelic
methmethylated
on
their
Xi
alleles,
indicating
that
our
ing
the
polymorphic
polymerase on
Medicine,
Massachusetts
General
Hospital,
Harvard
MedPromoter hypermethylation at the inactivated X chromosome (Xi) is a low-copy parentally
repeat sequence
that isasunmethylated
exclusively
Xifractions
in site
four polymorphisms or
imprinted regions,
well(PCR)
as technical
MSRE
abnormal in their Xi methylachain reaction
amplified,artifacts
and the resulting
re- ylationfrom
ical School, 18512Cambridge Street, clones
Boston,were
MAnot
02114,
from
two individua characteristic of mammalian dosage compensation
Our(A.H.);
extends
findings
by providing
evidence
forclones
global
. It is unclear
tion. Analysis
ofanalysis
allele-specific
methylation
of genotyping
allelic
bias of these
the
werethen
filtered
outandbyhyrequiring
at least
one clone switch from AB
sulting algorithm,
amplicons
were
labeled
USA. E-mail: hellman@chgr.mgh.harvard.edu
than previously hypothesized.

Gene BodySpecific Methylation on

the Active X Chromosome

Methylation level
(log2 ratio)

Methylation level
(average log2 ratio)

ed from www.sciencemag.org on July 12, 2010

(log2 ratio)

Methylation level

2005 Nature Publishing Group http://www.nature.com/naturegenetics

Chromosome-wide and promoter-specific analyses

identify sites of differential DNA methylation in normal
cand transformed human cells

Downloaded from www.sciencemag.org on July 12, 2010

Male fibroblasts (HFL-1)

Contradicciones acerca del estado de

metilacin del cromosoma X inactivo

Dnde encontramos metilacin del

DNA?

Metilacin del DNA del marco de lectura es

una caracterstica de los genes de clulas
mamferas
a

Figure 2 C
position rel
Key
En mamferos, la mayora de las regiones promotoras
no
estn
Top
0.8
and weakly
quartile
metiladas.
of gene-bod
0.6
Median
(a) and MS
El incremento
gradual de los niveles de metilacin del DNA dentro
de
Bottom
0.4
of BSPP-de
quartile
los cuerpos
de genes est asociado con los niveles transcripcionales de
position sh
0.2
weakly exp
dicho0gen.
regions of t
30,000 20,000 10,000
0
0.2
0.4
0.6
0.8
10,000 20,000 30,000
1
averages of
Inside gene (fraction from start)
Downstream (bp)
Upstream (bp)
position for
cell line sh
Key
1.0
0
expression
Highest
1
expression
0.8
five equally
2
level. The c
3
0.6
was normal
Lowest
4
expression
control cou
5
0.4
gene length
6
are seen in
0.2
7
counts vers
8
0.2
0.4
0.6
0.8
1.0 10,000 20,000 30,000
30,000 20,000 10,000 0.0
site. (d) Ex
Inside gene (fraction from start)
Downstream (bp)
Upstream (bp)
seen in the
counts vers
0
0
1.0
1.0
ends of gen
Low
expression
High
expression

Fraction methylated

0.8

er site

Estimate

er site

Estimate

served.

Average observations per site

Estimated average methylation

Ball et al. (2009) Nat. Biotech.

0.8

GM06990

HELP
HELP

00 -_
-3.87
-3.87 __
_
90
90

990
990

CpG island

Figure 1

Refseq genes

0 _

Low

5meC

Figure 1

00 __
Dnase
Dnase hypersensitive
hypersensitive _
_
4.00
4.00

200000000
200000000

High

_
-3.77
-3.77 __
_
90
90

CpG island

Refseq genes

Replication
Replication

GM06990

00 --

0 _

Dnase hypersensitive

Replication

Dnase hypersensitive

Replication

HELP

chr2:
150000000
chr2:
150000000
__
4.83
4.83

HELP
HELP

ast
ast

-3.87 _
_
90

0 -

0 _
Dnase hypersensitive
_
4.00

Replication

-3.77 _
_
90

-3.87 _
_
Fibroblast
90

Regiones de replicacin temprana

presentan
Downloaded
from genome.cshlp.org o
altos niveles de metilacin

Replication
Replication
00 __
Dnase
Dnase hypersensitive
hypersensitive
Refseq
Refseq genes
genes
CpG
CpG island
island

Fi g u r e 2. D N A hypermethylation correlates with earl

were drawn with two-dimensional histograms. Cytosine
are averaged in 100-kb sliding windows. The cumulativ
Suzuki
et.al
(2011) Genome
coded levels to generate
the
contours.
Early Res.
replicated
broblast and lym phoblastoid cell types.

Existe una relacin contexto-especfica

de la metilacin del DNA con la expresin
gnica

TLN1 presenta patrn similar de

metilacin en GM06990 y Fibroblastos

hipometilado
hipermetilado

BLNK slo se expresa en lymphoblastoids

hipometilado
hipermetilado

La metilacin en el cuerpo gnico aporta

mayoritariamente al porcentage de
metilacin genmico
With gene body

Without gene body

Suzuki et al. (2011) Genome Res.

Self-organization map correlates 5-aza-CdR

demethylation effect with highly expressed genes
b
DNA methylation
Gene expression

c
DNA methylation
5-aza-CdR demethylation

d
DNA methylation
Gene expression

Gene Expression
5-aza-CdR demethylation

Ramos et al. (2015) Epigenetics Chromatin

Novel intragenic pSer5 peaks after 5-azaCdR treatment tend to have lower
methylation levels and a shift in gene
expression

Ramos et al. (2015) Epigenetics Chromatin

Novel intragenic pSer5 peaks after 5-azaCdR treatment tend to have lower
methylation levels and a shift in gene
expression

Ramos et al. (2015) Epigenetics Chromatin

Novel intragenic pSer5 peaks after 5-azaCdR treatment tend to have lower
methylation levels and a shift in gene
expression

Ramos et al. (2015) Epigenetics Chromatin

Conclusiones
La mayora de los promotores en el genoma de
mamferos no se encuentra metilados.
Eucromatina presenta mayores niveles de metilacin,
asociados a la expresin gnica.
Es necesario estudiar de forma global los patrones de
metilacin en los genomas.

Gracias!
Preguntas: mramos@regenero.cl