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CHAIRE ÉPIGÉNÉTIQUE ET MÉMOIRE CELLULAIRE
Année 2017-2018 :
“Le chromosome X -
paradigme de la génétique et l’épigénetique”
12 février, 2018
Cours III
Dynamique de l’hétérochromatine facultative
Dynamics of facultative heterochromatin
E. Heard, February 12th, 2018
SUMMARY of last week
! Very diverse strategies used to achieve dosage equality between the sexes! Observed in
many XX and XY (or XO) animals (flies, worms, placental mammals) but not others
! Dosage does matter- failure to establish sex chromosome dosage compensate is lethal
during mouse development: perturbs mbryonic and extraembryonic development
! Dosage compensation is not necessarily chromosome-wide in all organisms
! Some genes are more dosage sensitive than others –this may vary between individuals
and in different tissues
! X-chromosome up-regulation of the single X relative to autosomes (Ohno’s Hypothesis) is
not a universal principle –applies to just some dosage sensitive genes in humans for ex.
! Sex determination and dosage compensation are triggered by the same pathway in
Drosophila and C. elegans -but not in mammals (XX dosage for XCI, Sry for sex)
! Targeting / modulating dosage compensation factors to the sex chromosomes involves
DNA elements (C. elegans , Drosophila) and non-coding RNAs (Drosophila, mammals)
! And diverse chromatin and chromosomal complexes
Facultative heterochromatin in dosage compensation
E. Heard, February 12th, 2018
Sex Chromosome Dosage Compensation : Triggering Differential Gene Expression & Chromatin E. Heard, February 12th, 2018 S. Ercan, J Genomics 2015; 3:1-19. doi:10.7150/jgen.10404
Flie
Sex Chromosome Dosage Compensation : Ma
Wo
Triggering Differential Gene Expression & Chromatin
Xist
RNA
E. Heard, February 12th, 2018 S. Ercan, J Genomics 2015; 3:1-19. doi:10.7150/jgen.10404
Sex Chromosome Dosage Compensation :
Triggering Differential Gene Expression & Chromatin
Maintaining differential activity states requires
chromatin changes in all three systems
E. Heard, February 12th, 2018 S. Ercan, J Genomics 2015; 3:1-19. doi:10.7150/jgen.10404
Differential States of Chromatin
l
m
n
“The Heterochromatin of Moss” f
w
“…heterochromatin refers to a part of a t
chromosome that remains heteropyknotic after e
o
telophase and thus behaves opposite to A
euchromatin” Heitz (1928) t
y
fl
Constitutive heterochromatin tends to be n
non-coding and repetitive (Pontecorvo, 1944) o
m
Facultative heterochromatin n
p
Emile Heitz (“facultas” = opportunity) can be either active n
or inactive (eg Barr body, 1949; Lyon, 1961) i
(1892-1965) t
u
c
Euchromatin Heterochromatin m
e
Facultative Heterochromatin: a
http://medcell.med.yale.edu/histology/
Potential for gene expression at some point in development a
and can be either condensed or decondensed depending on T
t
cell type d
It is reversible, and only appears depending on the stage of “
p
development or the cell type examined.
E. Heard, February 12th, 2018
The Inactive X chromosome l
A classic examples of facultative heterochromatin
m
n
f
From M. Lyon, 1974 w
The Barr Body
t
e
XY o
A
somes
t
ll;
inac7ve
y
osomes
are
fl
n
o
m
XX n
p
n
i
t
u
Bertram et Barr, 1949 c
m
e
a
a
‘‘Changes in quantity, quality or structural organization of heterochromatic elements T
may well alter the kind and/or degree of particular exchanges that occur, and in this t
d
way control the chromosome organization and the kind and the relative effectiveness “
p
of genic action” (McClintock, 1950). (see COURS 2017)
E. Heard, February 12th, 2018
The Inactive X chromosome
A classic examples of facultative heterochromatin
Identical (or almost) DNA sequences
Differential gene activity states
Heritable through cell divisions
Xa TF
at some
be either
epending Xi
Epiallele : epigenetically marked regulatory sequence
ars
elopment
Barr body
(Bertram and Barr, 1949)
TEM
E. Heard, February 12th, 2018 Rego et al, 2008
The Inactive X chromosome
A classic examples of facultative heterochromatin
Identical (or almost) DNA sequences
Differential gene activity states
Heritable through cell divisions
Xi
e
er Xa TF
ng
Epiallele : epigenetically marked regulatory sequence
nt
Barr body
(Bertram and Barr, 1949)
TEM
Heritable through cell divisions
Thanks to heterochromatic state…
E. Heard, February 12th, 2018 Rego et al, 2008
Reminder: What are the Roles of Chromatin?
• The physiological template of the genome
• Packaging of the the genome in interphase and during mitosis
he
• Barrier to aberrant gene expression and reprogramming
• Facilitator of gene regulation and genome function
• Integration of environmental signals
e
• Carrier of cellular memory…cell cycle, mitosis and meiosis
nuclei)
he
is
localized
The f
but n
suffic
order
totali
that m
exten
E. Heard, February 12th, 2018Stable Differential Gene Expression States
Transitions between euchromatin and heterochromatin
TF
z
AAAA
RNA
Pol
II
Germ
lin
Epigenetic Barrier
Accessible
informa7on
Restricted
informa7on
Chromatin states and chromatin compaction change
during development and processes such as X-chromosome dosage
compensation.
How many types of epigenetic change, or chromatin states?
E. Heard, February 12th, 2018
What enables heritability and reversibility?Transitions between euchromatin and heterochromatin
Germ
lin
Epigenetic Barrier
Accessible
informa7on
COURS
Restricted
2014
informa7on
Chromatin states and chromatin compaction change
during development and processes such as X inactivation.
How many types of epigenetic change, or chromatin states?
What enables heritability and reversibility?
E. Heard, February 12th, 2018Facultative Heterochromatin E. Heard, February 12th, 2018 Trojer and Reinberg, 2007
Chromatin-based States and Partners
Histone Variants and Histone Modifications are:
Mediators of chromatin accessibility
Platforms for binding proteins
Histone modifying enzymes
can add or remove these
modifications
TETs
Dnmts
De novo: Dnmt3a,3b, 3L
Maintenance: Dnmt1
RNAs are important players: In DNA
some cases, histone associated
methylation ‘readers’ andwith
‘writers’ are in
repressed
complexes, possibly enabling self-perpetuation
lncRNAs or small interfering RNAs state of some genes, repeats:
and/or spreading in cis
(in heterochromatin) Self-templating, stable - but can be removed
eg HP1/H3K9me3; PRC2/H3K27me3 etc
(actively eg Tet-induced conversion to 5hme;
passively during DNA replication)
E. Heard, February 2nd, 2015Detecting Chromatin Marks using Antibodies
In the 1990’s, highly specific antibodies raised - discriminating between
e
chemically modified histones at specific amino acids, => histone
modifications could be detected by immunofluorescence (IF) and
teris7c
which
chromatin immunoprecitipation (ChIP).
ated
by
Bryan Turner, Dave Allis, Thomas Jenuwein
orm
etramers.
es
in
an
H3
and
Bryan Turner
The
H2A/
due
to
clude
]
The
H4
2B
l
four
e
only
in
Xi
Xi
DNA Anti-H4K12Ac
Chaumeil et al, 2002
Jeppesen and Turner, Cell, 1993
Unique tools to explore the differential states of chromatin and
E. Heard, February 2nd, 2015
generating Epigenomic mapsFacultative Heterochromatin E. Heard, February 12th, 2018 Trojer and Reinberg, 2007
Facultative Heterochromatin E. Heard, February 12th, 2018 Trojer and Reinberg, 2007
Molecular Characterization of Facultative Heterochromatin
ATAC
–Seq
(Chroma0n
accessibility-‐
RNA-‐Seq
E. Heard, February 12th, 2018 Adapted
from
Caloni
et
al,
2015
New insights into the molecular 3D architecture of chromosomes using
Chromosome Conformation Capture (« C ») techniques
“Hi-C”
Job Dekker Whole Genome
(UMass)
Combined with revolutionary microscopy techniques we can
now observe the dynamic 3D organisation and functions of
Lieberman-‐Aiden
et
al.
2009
the genome :
Compartments
“Active” or “inactive”
Distinctive epigenomic features
Vary between tissues
Cell-type specific
Lieberman-Aiden et al. 2009
TADs (100kb-1Mb scale)
Invariant (almost) between tissues
And ask questions about how Nora et al, 2012
facultative heterochromatin is Conserved (man/mouse) &
Dixon et al, 2012
organised over developmental time Also in other species?
E. Heard, February 12th, 2018 Sexton et al, 2012Molecular Characterization of Facultative Heterochromatin
Chromosome
conforma0on
capture.
Method
enabling
the
determina7on
of
interac7on
frequencies
between
pairs
of
genomic
loci.
E. Heard, February 12th, 2018Chromosome changes in Worm Dosage Compensation
011
t
P
Selec7v
pe
demeth
genome
H4K20m
• DC proteins lead to higher levels of histone H4 lysine elevate
20 monomethyl (H4K20me1) on hermaphrodite Xs reduced
• H4K20me1 functions in repressing X-chromosome • H4K20me2 JmjC demethylase subfamily revealed by compac
gene expression (by studying mutants SET1=HMTase) DPY-21 structure and activity chromo
• Therefore, histone modification is an important aspect • In somatic cells, DPY-21 enriches H4K20me1 on X weaken
These
fi
of the mechanism of dosage compensation. chromosomes to repress gene expression
of
• H4K20me1 thought to impact chromatin structure • H4K20me1 enrichment controls the higher-order
demons
chroma
regulation, structure of X chromosomes order
ch
• => Dosage compensation may lower gene • In germ cells, DPY-21 enriches H4K20me1 on autosomes structur
expression on hermaphrodite X chromosomes by to promote chromosome compaction. of
gene
compacting them? (Brejc et al, Cell 2017)
(Vielle et al, Plos Gen. 2011)
E. Heard, February 12th, 2018Chromosome changes in Worm Dosage Compensation
Condensin-driven remodeling of X-chromosome topology during
011
dosage compensation
X chromosome Autosome
t
P
Selec7v
pe
demeth
genome
H4K20m
elevate
reduced
compac
• Dosage-compensated X chromosomes consist of self-interacting domains (~ 1 Mb) chromo
weaken
resembling mammalian Topologically Associating Domains (TADs). These
fi
of
• TADs on X have stronger boundaries and more regular spacing than those on autosomes. demons
• Many TAD boundaries on X coincide with the highest-affinity rex sites, and these chroma
order
ch
boundaries become diminished or lost in mutants lacking DCC binding, causing the
structur
structure of X to resemble that of autosomes. of
gene
• Loss of H4K20me1 (in dpy-21 mutants) leads to reduced TAD boundaries, and greater long
range interactions (H4K20me1 reinforces the strength of these boundaries?)
E. Heard, February 12th, 2018Chromosome changes in Worm Dosage Compensation
Condensin-driven remodeling of X-chromosome topology during dosage compensation and
011
during mitosis: First insights into the molecular architecture of facultative heterochromatin
X chromosome Autosome
t
P
Selec7v
pe
demeth
genome
H4K20m
elevate
reduced
compac
chromo
! Condensin and H4K20me1 are also found associated with mitotic chromosomes in weaken
many organisms These
fi
of
! H4K20me1 enrichment is also associated with the inactive X in mammals
demons
chroma
! Are these condensin-driven domains (TADs?) on the C. elegans X chromosomes a order
ch
structur
cause or simply a consequence of DCC-induced down regulation? of
gene
! Is the impact on gene down-regulation of H4K20me1 via chromosome structural
changes
th
or rather through local effects on the chromatin?
E. Heard, February 12 , 2018Facultative Heterochromatin of the inactive X
Differential treatment of identical DNA sequences in the
same nucleoplasm
Ac7ve
X
chromosome
Inac7ve
X
chromosome
Xist
RNA
X
Xi
RNA
Pol
II
Pc?
H3K27
H2A
H3K9
Ac
Ac
Ac
me3
Ub
me2
Barr
body
H3K4
Ac
H3K4
H4K20me1
5-‐methyl
cytosine
me3
me2
OMX)
DAPI
H4K20me1
Xist
RNA
O.
Masui
Xi
Xa
E4.5
DAPI
X
Chromosome
Paint
Teller
et
al,
Nucleus,
2013
E4.0
E. Heard, February 12th, 2018At least Two Flavors of Heterochromatin on the Xi
in Normal Human Cells
H3K27me3
enriched
Xi
domains
H3K9me3
enriched
Xi
domains
ChIP-‐seq
on
Normal
cells
(HMEC)
Chadwick
et
al,
2004
Different human cell types
Have different distributions of
H3K9 or H3K27me3 enriched
heterochromatin
E. Heard, February 12th, 2018Chromatin Signature of Facultative Heterochromatin
on the human inactive X
H3K27me3
enriched
Xi
domains
H3K9me3
enriched
Xi
domains
Chadwick
et
al,
2004
E. Heard, February 12th, 2018Facultative heterochromatin on the Xi
is disrupted in human cancer cells
The Sex Chromatin in Human
Malignant Tissues
K. L. Moore and M. L. Barr, 1957
ChIP-‐seq
on
normal
cells
(HMEC)
Chadwick
et
al,
2004
ChIP-‐seq
:
H3K27me3
Normal
cells
Luminal
HER2
+
Basal-like
Global defects in H3K27me3 organization for the Xi in breast
cancer cell lines (consistent with “loss” of Barr body)
E. Heard, February 12th, 2018 Chaligné
et
al,
Genome
Res.
2015
Facultative heterochromatin on the Xi
is disrupted in human cancer cells
Healthy breast tissue Luminal A / IDC Grade II Luminal A / IDC Grade II
T1 T2
XIST RNA RNAPII H3K27me3 XIST RNA RNAPII H3K27me3 XIST RNA RNAPII H3K27me3
. .
. .
In total, 17 tumors (50%)
show escape of ≥1 of the
three X-linked genes studied
(11/24 luminal, 2/3 HER2
and 4/8 TN)
Global defects in chromatin organization for the Xi in breast cancers are
accompanied by aberrant reactivation of X-linked genes
E. Heard, February 12th, 2018 Chaligné
et
al,
Genome
Res.
2015
Chromatin Signatures of Facultative Heterochromatin
on the inactive X in different mammals
Shafagh
A.
Waters,
Paul
D.
Waters
E. Heard, February 12th, 2018Chromatin Signatures of Facultative Heterochromatin
on the inactive X in different mammals
Stochastic monoallelic
gene expression on the X
chromosomes:
Xist
Not chromosome-wide?
gene
E. Heard, February 12th, 2018 Courtesy
of
Julie
Chaumeil
(Ins9tut
Cochin)
Chromatin Signatures of Facultative Heterochromatin
on the inactive X in different mammals
Immunofluorescence with Antibodies against different Histone Modifications
• In the 3 groups, the Xi shows an enrichment in H3K27me3 and a lack of
RNA PolII and active marks (H3K9Ac, H4Ac, H3R17me)
E. Heard, February 12th, 2018 Chaumeil
et
al,
2011
Chromatin Signatures of Facultative Heterochromatin
on the inactive X in different mammals
• In the 3 groups, the Xi shows an enrichment in H3K27me3 and H3K9me2.
• In elephant and mouse, the Xi shows also an accumulation of H4K20me1.
• The marsupial Xi shows no enrichment in H4K20me1 but an accumulation
of H3K9me3 and H4K20me3 (common features of constitutive
heterochromatin at centromeres).
• The marsupial Xi also shows no DNA methylation at promoters
• Reported to have « leaky » XCI (ie frequent escape from XCI)?
• BOTH SIMILARITIES AND DIFFERENCES BETWEEN SPECIES
E. Heard, February 12th, 2018 Chaumeil
et
al,
2011
Expression and Chromatin status of the inactive X
in Marsupials
Opposum: Monodelphis domestica
E. Heard, February 12th, 2018Expression and Chromatin status of the inactive X
in Marsupials
Opposum: Monodelphis domestica
E. Heard, February 12th, 2018 Wang
et
al,
2014
Expression and Chromatin status of the inactive X
in Marsupials
Opposum: Monodelphis domestica
# Parent-of-origin allele-specific expression, DNA methylation, and histone modifications
in fetal brain and extra-embryonic membranes in the opossum (Monodelphis domestica).
# Most X-linked genes (152 of 176 genes with trackable SNP variants) had paternally
imprinted silencing, with nearly 100% of transcripts derived from the maternal allele
# 24 loci (14%) escaped inactivation, showing varying levels of biallelic expression.
# No association between X-linked gene expression and promoter DNA methylation,
E. Heard, February 12th, 2018 Wang
et
al,
2014
Summary of Chromatin Marks
Marsupial XCI shares some basic features with afrotherian and eutherian XCI:
Late replication,
Lack of active chromatin marks (eg histone hypoactylation )
And shows some differences:
Lack of DNA methylation at promoters H3K9m
Lack of H4K20me1
H3K27
H4K20
H3K9m
H3K27
H4K20
E. Heard, February 12th, 2018 From
Waters
and
Waters,
2015
Summary of Xi status in Somatic Cells of
Mice, Humans and Marsupials
Diversity in heterochromatin marks: facultative heterochromatin is a
means to an end – the need to dosage compensate
H3K9m
Xist RNA Rsx RNA H3K27
H4K20
RNA DNA DNA
Pol II methylation
Pc? methylation
Pc? Pc?
Ac H3K4me2, H2AK119Ub H3K27me3 H3K27me3
H3K9me3 H2AK119Ub? H3K9m
H3K9me2 H3K9me3
me3
H3K27
H4K20
H3K36me3 H4Ac H3Ac MacroH2A H4 H4K20me3 MacroH2A H4K20me3
H4
K20me1 K20me1
Active X chromatin Inactive X chromatin Inactive X chromatin
Barr body Barr body
Late replicating Late replicating
MOUSE MARSUPIAL
HUMAN
E. Heard, February 12th, 2018Long Non-Coding RNAs trigger X inactivation
in Mice, Humans and Marsupials?
Brown et al, (1991) A gene from the region of the human X
inactivation centre is expressed exclusively from the inactive X
chromosome. Nature 349, 38-44
Brown et al (1992) The human XIST gene: analysis of a 17kn
inactive X-specific RNA that contains conserved repeats and is
highly localised within the nucleus. Cell, 71, 527-542.
Xist evolved in eutherians, from a protein-coding gene, Lnx
E. Heard, February 12th, 2018Long Non-Coding RNAs trigger X inactivation
in Mice, Humans and Marsupials?
E. Heard, February 12th, 2018Long Non-Coding Rsx RNA triggers X inactivation
in Marsupials?
E. Heard, February 12th, 2018 Wang
et
al,
Genome
Res.
2014
• X inactivation in mammals (Lyon, 1961): How similar are the mechanisms?
Sex chromosome Dosage Compensation in Mammals
Random XCI
Imprinted
XCI
Imprinted &
Random XCI
Random XCI (Xist)
(XIST)
Stochadtic
Monoallelic
Silencing?
5X / 5Y
Partial dosage
Compensation?
(no Rsx, no Xist)
Imprinted XCI
From Hore and Graves, 2007
(Rsx)
E. Heard, February 12th, 2018Sex chromosome Dosage Compensation in Mammals
An imprinted form of X inactivation may have evolved more than once
Rodents
Imprinted Xist
1st paternal XCI
then Random XCI
See discussion
Okamoto et al,
Nature 2005
E. Heard, February 12th, 2018How does XIST work?
(X-Inactive-Specific-Transcript)
A Multi-Tasking Molecule
Scaffold for repressor recruitment?
RNA-DNA binding? Chromatin changes ?
Nuclear compartmentalisation ?
E. Heard, February 12th, 2018
E. Heard, February 12th, 2018Long non-coding RNAs:
from spurious transcription to functional entities
Claes
Wahlestedt,
2013
Adapted
from:
Wang,
KC
and
Chang
HY,
Molecular
Mechanisms
of
Long
Noncoding
RNAs.
Mol
Cell.
2011
Sep
E. Heard, February 12th, 2018 16;43(6):904-‐14.
How does XIST work?
(X-Inactive-Specific-Transcript)
Gene
silencing
+++
+++
+++
+++
Chromosome
coa7ng
A
F
B
C
D
E
hnRNP
U
binding
•
Poor
sequence
conserva7on
between
mammals
-‐
except
for
repeats
A-‐F
•
17
000
-‐
19
000
nt,
spliced,
untranslated,
nuclear
transcript
•
RNA
expressed
from
and
“coats”
the
inac7ve
X
chromosome
in
cis
(not
trans)
•
Xist
is
essen0al
for
X
inac7va7on
in
cis
(KOs,
transgenes
in
mouse
embryos,
ES
cells)
•
Xist
can
only
induce
silencing
during
an
early
developmental
7me
window
•
Xist
binds
broadly
across
the
X
chromosome,
exploi7ng
3D
structure
for
ini7al
binding
•
Es7mated
~2000
molecules
of
Xist
RNA
per
nucleus
•
Conserved
“A”
repeats
ensure
silencing
func7on
•
Mul7ple
Xist
domains
required
for
coa7ng
including
C
repeats
•
Xist
RNA
reported
to
recruit
chroma7n
factors
eg
Polycomb
group
proteins,
macroH2A…
12th, 2018
E. Heard, February
When does Xist trigger chromosome-wide silencing?
Xist RNA as a trigger for XCI
Tet
Inducible Xist cDNA :
Xist
Cellular Memory?
Epigenetic Marks?
Xist-dependent Xist-independent
INITIATION IRREVERSIBLE INACTIVATION
Days of in
vitro
differentiation 0 1 2 3 4 5 6 7 8 9
(ES cells) Xist RNA
coating begins in XX cells
Transcriptional silencing begins
(Northerns, allele-specific RT PCR, RNA FISH)
Wutz et al Cell 2001, Nat. Genet. 2002
E. Heard, February 12th, 2018What are the Functional Domains of Xist RNA?
Tet
Inducible Xist cDNA :
(Wutz and Jaenisch, 2001) Xist
Y X
Functional nullisomy for
the X chromosome
Y X
Cell death
TRE-Xist
Y X
Gene activity
Survival
E. Heard, February 12th, 2018What are the Functional Domains of Xist RNA?
Wutz et al, Nature Genetics 2002
silencing association in cis
Xist A-repeats are required for gene silencing
but NOT for chromosome coating, mH2A and
PcG protein recruitment…
Associated proteins ?
Mechanism of action ?
E. Heard, February 12th, 2018What are the Functional Partners of Xist RNA?
Gene
silencing
hnRNP
U
binding
region
region
Xist
RNA:
A
B
C
D
E
AAAA
F
Regions
involved
in
Xist
RNA
coa7ng
:
+++
+++
+++
+++
E. Heard, February 12th, 2018What are the func0onal partners of Xist RNA? E. Heard, February 12th, 2018 Chu et al, Cell 2015
Xist RNA Functional Partners
Gene
silencing
region
Xist
RNA:
A
B
C
D
E
AAAA
F
Silent
nuclear
Gene
silencing
compartment
Fus
Chromosome
coa7ng
Chroma7n
modifica7on
E. Heard, February 12th, 2018 Chu
et
al,
Cell
2015
Xist RNA Functional Partners: a few examples
Gene
silencing
hnRNP
U
binding
region
region
Xist
RNA:
A
B
C
D
E
AAAA
F
Regions
involved
in
Xist
RNA
coa7ng
:
+++
+++
+++
+++
•
HnrnpU
(SAF-‐A)
is
required
for
Xist
localisa7on
(as
previously
shown)
•
Spen
(Drosophila
Split
ends
homolog
)
interacts
via
the
A-‐repeat
domain
of
Xist
and
is
required
for
gene
silencing
•
Wtap
–
RNA
methyla7on
machinery
•
Polycomb
PRC1
factors
Pcg5,
Rybp
–
but
no
PRC2
factors
•
HnrnpK,
par7cipates
in
Xist-‐mediated
gene
silencing
and
recruitment
of
non-‐canonical
polycomb
PRC1
complex
but
not
Xist
localiza7on
•
LBR
–
Lamin
B
receptor
–
nuclear
organisa0on?
E. Heard, February 12th, 2018 Chu
et
al,
Cell
2015
SPEN
recruit
his
HNRNPK
e
X
AC37
some31.
e
and
t
ncing
les
in
ncers
epigene7c
E. Heard, February 12th, 2018Identification of the Protein Partners of Xist RNA and the Factors that
are implicated in Xist-mediated Silencing
Holy Grail
Or Pandora’s box?
Xist
RNA
partner
isola0on:
Chu
et
al,
Cell
2015
McHugh
et
al,
Nature
2015
Chen
et
al
Science
2016
Minajigi
et
al,
Science
2015
Gene0c
screens
for
Xist
func0ons:
Moindrot
et
al,
Cell
Rep.
2015
Monfort
et
al,
Cell
Rep.
2015
E. Heard, February 12th, 2018The first molecular handles for Xist RNA function herald a
a new era of Xist RNA and X-inactivation research
Xist
RNA
partner
isola0on:
Chu
et
al,
Cell
2015
McHugh
et
al,
Nature
2015
Chen
et
al
Science
2016
Minajigi
et
al,
Science
2015
Gene0c
screens
for
Xist
func0ons:
Moindrot
et
al,
Cell
Rep.
2015
Monfort
et
al,
Cell
Rep.
2015
E. Heard, February 12th, 2018 Simao
Teixeira
da
Rocha
Does Xist RNA silence genes via nuclear reorganisation? E. Heard, February 12th, 2018
Does Xist RNA silence genes via RNA methylation?
Rbm15/Rbm15b
E. Heard, February 12th, 2018Does Xist RNA silence genes via RNA methylation?
YTHDC1
binds
XIST
m6A
residues
and
promotes
XIST-‐mediated
gene
silencing.
NB
Xiao
et
al.
(2015):
m6A
reader
protein
YTHDC1
impacts
mRNA
splicing
E. Heard, February 12th, 2018SUMMARY
Xist RNA and the initiation of X inactivation
• Xist non-coding RNA is a multi-tasking molecule essential for
initiation of XCI
• It induces gene silencing, spatial reorganisation of the X
chromosome and chromatin changes
• Mass-spec analysis of proteins bound to Xist RNA provide the
first molecular handle for exploring its functions
• The first regions Xist targets contain the first genes silenced
• Subsequent spreading due to « relay » elements, or chromatin
proteins, or spatial dynamics?
E. Heard, February 12th, 2018Where does Xist RNA bind on the X chromosome? E. Heard, February 12th, 2018
Where does Xist RNA bind on the X chromosome?
The
Xist
lncRNA
Exploits
Three-‐Dimensional
Genome
Architecture
to
Spread
Across
the
X
Chromosome
Jesse
M.
Engreitz
et
al.
Science
341,
(2013);
E. Heard, February 12th, 2018Xist RNA exploits 3D genome architecture to spread
across the X chromosome
Engreitz
et
al,
2013
RNA
An7sense
Purifica7on
(RAP):
Mapping
of
Xist
lncRNA
interac7ons
with
chroma7n
Lieberman-‐Aiden
et
al,
2009
Comprehensive
mapping
of
long-‐range
interac7ons
reveals
folding
principles
of
the
human
genome
From
Gendrel
and
Heard,
2014
E. Heard, February 12th, 2018Nuclear Organisation of the Inactive X
Enlightening the contribution of the dark matter to the X chromosome
inactivation process in mammals
E. Heard, February 12th, 2018
Author links open overlay panelMiguelCasanovaTharvesh
MoideenLiyakat AliClaireRougeulleXist RNA forms a silent nuclear compartment and triggers spatial
reorganisation of the Xi during XCI
CTCF-driven
Transcrip
sequest
Xist A-repe
(Spen, WTA
interbal
Outer
edge
Inside
Transcrip0on
machinery
(RNA
PolII)
Xist
RNA
RNA
Pol
FIISH
/
Gene
I
IF/RNA
DNA
FISH
FISH
DNA
Xist
RNA
Xist RNA
Xa
Genes undergoing inactivation are internalised into the
X-‐linked
gene
Xist RNA compartment, expressed genes (escapees) 1ary
transcript
remain external
Xist
RNA
RNA
Pol
II
E. Heard, February 12th, 2018 Chaumeil et al, Genes Dev. 2006; Chow et al, Cell 2010Investigating the molecular architecture of the active and inactive X
chromosomes using Hi-C
Lieberman-‐Aiden
et
al.
2009
chr1
chr4
chr2
chr5
chr3
chr6
…
chr6
HOX
cluster
Intrachromosomal
Topologically
associa0ng
Whole
genome
map
contact
maps
domains
Compartments
with
dis7nc7ve
TADs
(100kb-‐1Mb
scale)
palerns
of
epigenomic
features
Invariant
(almost)
between
7ssues
Variable
between
7ssues,
Nora
et
al,
2012
Cell-‐type
specific
Conserved
(man/mouse)
E. Heard, February 12th, 2018 Job
Dekker
Dixon
et
al,
2012
Allele-‐specific
RNA
seq
and
Hi-‐C
in
clonal
F1
129/Cast
ESCs
and
NPCs
F1
Hybrid
ES
cell
line
Neural
Progenitor
cells
(NPC)
Isolate
NPC
clones
(129xCast
=>
1
SNP
/~100bp)
Random
X
chromosome
inac0va0on
(XCI)
100%
cells
with
129
or
cast
Xi
Plate
low
Genera7o
Xa129XiCast
129
Cast
Differen7a7on
Subcloning
•
M
ChrX
Xi129XaCast
•
X
Xa129XaCast
(Gendrel
et
al,
Dev.
Cell
2014)
Dekker
Lab:
Ye
Zhan,
Bryan
Lajoie
Heard
Lab:
Mikael
A}a,
Luca
Giorge}
Allele-specific HiC analysis of the active and inactive X chromosomes
NPCs
• The Xi is divided into two megadomains separated by a 200kb boundary region
including the CTCF-rich, DXZ4 TAD
macrosatellite (Deng et al, 2015; Rao et al, 2014, Giorgetti et al, 2016)
insulation score
• Only escapee regions show long range interactions in trans (Splinter et al, 2011)
• Xi is devoid of compartments and TADs (Minajigi et al, 2015), except at a few regions…
th
E. Heard, February 12 , 2018 Bryan LajoieUnique Chromosome Organisation of the inactive X
Luca
Giorgec
(FMI,
Basel)
Giorgetti et al, 2016 Nora et al, Nature 2012
Implications of Xi organisation
for gene expression
MORE NEXT WEEK
(COURS IV et V)
E. Heard, February 12th, 2018Steps in X-chromosome inactivation
Ac0ve
gene
Xist
RNA
coa0ng
Maintenance
Reac0va0on
from
the
Xi
Ini0a0on
of
gene
silencing
PRC2,
PRC1
(Cbx7),
Lineage-‐specific
TFs?
PRC1-‐like
(Rybp)
Specialized
chroma9n
state?
LBR, SAF-A… Xist
RNA
macroH2A,
ATRX,
SMCHD1
CDYL,
BAHD…
SPEN
?
?
RBM15
TF
RNA
Pol
II
RNA
Pol
II
DNA
methyla7on
Pc?
? RNA
Pol
II
H3Ac
H4Ac
TF H4Ac
H3Ac
H2AK116Ub
H3K27me3
TF H3K27
me3
H3K4me3
H3K4me2
H3K36
H3K4me3
H3K4me2
H3K36
me3
Chu
et
al,
Cell
2015
me3
McHugh
et
al,
Nature
2015
MacroH2A
H4
K20me1
H3K9me2
? H3K4me
H2AK
H3K9me2
H3/H4Ac
116Ub
Minajigi
et
al,
Science
2015
Escape
from
XCI
(eg
Jarid1c,
Utx)
Some
genes
escape
XCI
XCI-‐resistant
TFs?
Insulator
elements?
cons0tu0vely
&
autonomously
Specialized
chroma9n
state?
(eg
(eg
JUarid1c,
Li
aLnd
tx,
Jarid1c,
Carrel,
i
and
Carrel,
PPNAS
NAS
2008)
008)
Patrat
Corbel
Some
genes
escape
in
a
lineage
or
0ssue-‐specific
? RNA
Gendr
fashion
Pol
II
TF H3K27 Eg
Mecp2
escapes
XCI
in
neural
stem
cells
in
the
SVZ
(eg
Atrx,
Patrat
et
al,
PNAS
2009)
? me3
Escapees
a re
o len
involved
in
TGCs
-‐
Corbel
et
al,
Development,
in
2013
chroma0n-‐associated
Reviewed
In
NPCs
-‐
Gendrel
et
al,
Dev.
in:
fCunc0ons
ell,
2014
H3K9me2
Giorge}
eg
Jarid1c,
Gendrel
and
HUeard,
Atrx
=
c
tx
=e
H
hroma0n
t
aistone
l,
Nature,
Ann.
R
r
ev.
2016
demethylases
D ev.
Biol.
2014
emodeller
?
Insulator
elements
Rocha
E. Heard,and
Heard,
February 12 N, SMB,
th 2018 2017
Steps in X-chromosome inactivation
Ac0ve
gene
Xist
RNA
coa0ng
Maintenance
Reac0va0on
from
the
Xi
Ini0a0on
of
gene
silencing
PRC2,
PRC1
(Cbx7),
Lineage-‐specific
TFs?
PRC1-‐like
(Rybp)
Specialized
chroma9n
state?
LBR, SAF-A… Xist
RNA
macroH2A,
ATRX,
SMCHD1
CDYL,
BAHD…
SPEN
?
?
RBM15
TF
RNA
Pol
II
RNA
Pol
II
DNA
methyla7on
Pc?
? RNA
Pol
II
H3Ac
H4Ac
TF H4Ac
H3Ac
H2AK116Ub
H3K27me3
TF H3K27
me3
H3K4me3
H3K4me2
H3K36
H3K4me3
H3K4me2
H3K36
me3
Chu
et
al,
Cell
2015
me3
McHugh
et
al,
Nature
2015
MacroH2A
H4
K20me1
H3K9me2
? H3K4me
H2AK
H3K9me2
H3/H4Ac
116Ub
Minajigi
et
al,
Science
2015
Escape
from
XCI
Epigene0c
instability
and
(eg
Jarid1c,
Utx)
aberrant
escape
in
cancer
Some
genes
escape
XCI
XCI-‐resistant
TFs?
Insulator
elements?
cons0tu0vely
&
autonomously
Specialized
chroma9n
state?
(eg
(eg
JUarid1c,
Li
aLnd
tx,
Jarid1c,
Carrel,
i
and
Carrel,
PPNAS
NAS
2008)
008)
Some
genes
escape
in
a
lineage
or
0ssue-‐specific
? RNA
RNA
fashion
Pol
II
Pol
II
TF H3K27 TF H3K27
(eg
Atrx,
Patrat
et
al,
PNAS
2009)
? me3
me3
in
TGCs
-‐
Corbel
et
al,
Development,
2013
In
NPCs
-‐
Gendrel
et
al,
Dev.
Cell,
2014
Reviewed
in:
H3K9me2
H3K4me
H2AK
H3K9me2
Giorge}
et
al,
Nature,
2016
Gendrel
and
Heard,
Ann.
Rev.
Dev.
Biol.
2014
E. Heard,and
February
Op.
Gen.
Dev.
2015
12thC,urr.
2018
?
Insulator
elements
H3/H4Ac
116Ub
Chaligné
et
al,
Genome
Res.
2015
Galupa
Heard,
Genes that can escape from X inactivation
is
Human
X
Mouse
X
2005
• A few escapees have Y-linked homologs, most do not
• Escape may be accidental (epigenetic instability) or purposeful (requirement of a double
dosage in XX)
• Escape may underlie some sex chromosome dosage effects on several sex-biased
metabolic, immune and neurological phenotypes
E. Heard, February 12th, 2018Variable escape from X inactivation leads to
even greater female cellular mosiacism
In humans, up to 25% of X-linked
genes can escape from X inactivation
(ie are biallelic)!
10% of these escape constitutively
15% of these genes show variability between
individuals – and tissue specificity
Carrel and Willard (2005) Nature 434, 400-404
Consequences on physiology, behaviour, disease?
COURS IV and V
E. Heard, February 12th, 2018CHAIRE ÉPIGÉNÉTIQUE ET MÉMOIRE CELLULAIRE
Année 2017-2018 :
“Le chromosome X -
paradigme de la génétique et l’épigénetique”
19 février, 2018
Cours IV
Les troubles neurologiques liés au chromosome X
E. Heard, February 12th, 2018You can also read
