Silencing/ DNA methylation/Imprinting
1.
Silencing mechanisms
Sir2/ HP1/HP1 and DNA methylation
budding yeast, fission yeast, mammals/plants
2.
Insulators (boundary elements/ enhancer blocker
Position effect variegation
3.
DNA methylation
de novo, maintenance , CpG islands
functions
methods of study
4.
DNA demethylation
plants
mammals
4.
Imprinting
Silencing
creates
large domains of chromatin that are
compacted and
less accessible to DNA-binding proteins
Silencers
Silencing proteins
Sir2
HP1
Polycomb group (PcG) proteins
DNA Methylation
noncoding RNAs
Boundary elements
S.c. S.p. A.th.D.m. Mamma
Hypoacetyl.
H3/H4
H3K9 me
HP1
DNA methyl.
-
+
+
+
+
+
-
+
+
+
+*
+
+
+
+
-
+
-
+
Polycomb
-
-
+
+
+
Sir2
+
+
+
+
+
* present but binds H3K27me notH3K9me
Heterochromatin
Condensed, deeply staining
Regular nucleosome spacing;
DNA mostly associated with histone core
Gene poor
Late replicating
Localized at nuclear periphery
Chromatin in silenced regions
Tight nucleosome arrays (short linkers)
Presence of certain histone modifications
i.e. methylated H3K9
Presence of DNA methylation
Bordered by boundary elements
Presence of non-histone proteins
HP1, Sirtuins, PcG, telosome
HP1 localization
Fanti and Pimpinelli, COGD, 2008
Purpose of heterochromatin
protect repetitive DNA from recombination
keep centromer/telomer intact (chromosome
integrity)
protect genome from transposons and other
‘selfish DNA’
prevent (not block) transcription
Silencing: lessons from yeasts
Saccharomyces cerevisiae or Saccharomyces pombe
Telomers
Centromers
Silent mating type loci
Mating type loci
Saccharomyces cerevisiae or Saccharomyces pombe
Haploid cells have one active mating type locus
(MAT, mat1)
Others are silenced in heterochromatin
When needed a mating type switch can occur
Yeast mating type loci
S. cervisiae
S. pombe
S.c. S.p. A.th.D.m. Mamma
Hypoacetyl.
H3/H4
H3K9 me
HP1
DNA methyl.
-
+
+
+
+
+
-
+
+
+
+*
+
+
+
+
-
+
-
+
Polycomb
-
-
+
+
+
Sir2
+
+
+
+
+
* present but binds H3K27me notH3K9me
silenced state initiated
deacetylation of H3/H4
3 binds H4K16 (unacetylated)
ultimerization of Sir proteins; spreading
ontinuous presence of Sir proteins
Molecular Cell, Vol. 8, 489–498,
September, 2001
Common Themes in
Mechanisms of Gene Silencing
Danesh Moazed
SIR
silencing
Function Sir2
NAD-dependent HDAC!
Molecular Cell, Vol. 8, 489–498, September, 2001
Common Themes in Mechanisms of Gene Silencing
Danesh Moazed
Other roles of sirtuins
Linked to caloric restriction/lifespan extension
Resistance to Neurodegenerative disease
Cancer (Tumor suppressor)
Yeast mating type loci
S. cervisiae
S. pombe
S.c. S.p. A.th.D.m. Mamma
Hypoacetyl.
H3/H4
H3K9me
HP1
DNA methyl.
-
+
+
+
+
+
-
+
+
+
+*
+
+
+
+
-
+
-
+
Polycomb
-
-
+$
+
+
Sir2
+
+
+
+
+
* present but does not bind H3K9me
$ PRC2 but not PRC1
Silencing S. pombe
Clr1
DNA binding
Clr3
HDAC
Clr4
HMT
Clr6
HDAC
Swi6
HP1
Rik1
DNA binding
(Hda1)
(Suv3-9)
(Rpd3)
(WD40 repeats)
Silencing S. pombe
HP1
Grewal and Elgin, COGD, 2002.
Silencing S. pombe
Silent mating type loci (S. pombe)
DNA: centromer-like region (cenH)
boundary elements ( IR-L, IR-R)
Order of events:
1. Deacetylation (Class I (Hda1 Clr3), Class II (Clr6))
key residue H3K9
2. Methylation H3K9 Clr4
3. Binding HP1 Swi6, Chp2
4. Establishment of Heterochromatin
Silencing S. pombe
Silent mating type loci (S. pombe)
DNA: centromer-like region (cenH)
boundary elements ( IR-L, IR-R)
Order of events:
1. Deacetylation H3K9
2. Methylation H3K9
3. Binding HP1
4. Establishment of Heterochromatin/spreading
Trigger:
repetitive DNA, small noncoding RNAs
recruiting factors, silencers
A key silencing protein: HP1
(S. pombe Swi6, Chp2)
Identified biochemically as nonhistone chromosomal protein
Binds to centromers, telomers, silenced regions
In mammals HP1 comes in different isoforms
(HP, HP1, HP1)
Not all are repressive
HP1 structure
H1
Chromodomain: binds H3K9me
Hinge region: binds RNA
Chromoshadow domain:
dimerization, interaction with
other proteins such as Suv39
HP1 structure and binding
Vermaak and Malik, Ann. Rev. Genet., 2009
HP1 chromodomain binds H3K9me2/3
Jacobs and Khorasanizadeh, Science 2001
HP1 Binds to:
H3K9 me2/3 (chromodomain)
su(var)3-9 KMT (chromoshadow domain; spreading)
mammals: Suv39h1, SETDB1,G9a/GLP
itself (chromoshadow domain; spreading)
DMT3a, DMT1 (DNA methyltransferases)
HDACs
CAF1: replication linked recruitment
lamin B (nuclear periphery)
RNA (hinge region; recruitments, stabilize binding)
Su(var)3-9
Is HMT: methylates H3K9 (di/tri methylation)
has set domain and chromodomain (both
required for Su(var)3-9 binding to chromatin)
Binds HP1
HP1 heterochromatin: recruits other factors
DNA methylation
Grewal and Jia Nature Reviews Genetics 2007
Reversal of Heterochromatin formation
H3K9me demethylase
H3S10ph
Both cause HP1 removal
Methyl/phospho switch
Hirota et al. Nature 2005
Genes within heterochromatin
Uniquely regulated
Require heterochromatin for proper expression
Require HP1 for proper expression
Heterochromatin spreads: prevented by boundary
Gaszner and Felsenfeld Nature Reviews Genetics 2006
Defects in boundary
Silencing of adjascent genes:
transgene silencing
translocation
PEV (position effect variegation)
PEV in Drosophila
PEV in Drosophila
Drosophila white gene (responsible for red eye color)
is located in heterochromatin in the PEV flies
PEV strain
PEV strain
HP1 mutant
http://www.biology.wustl.edu/faculty/elgin/hetchrom.html
PEV
Gaszner and Felsenfeld Nature Reviews Genetics 2006
PEV in yeast
Lunyak COCB, 2008
PEV
Variegation due to imprecise establishment
or inheritance of the silenced state of euchromatic
gene
Dissecting Heterochromatin Biology
via genetic identification of
modifiers of PEV
Great system because is part on/part off
so can easily identify:
enhancers of PEV (more silenced)
suppressors of PEV (less silenced)
Types of PEV modifiers identified
Boundary: a type of insulator that prevents
the spreading of heterochromatin
Valenzuela and Kamakaka , Annu. Rev. Genet. 2006
Delete boundary: H3K9me3 and HP1 (Swi5) spread!
Noma et al. Science, 2001
How is boundary made?
1. Region of high transcriptional activity, low
nucleosome density, high acetylation
Counteract heterochromatin
2. Region with RNA secondary structure
Can be active/ inactive
Second type of insulator: enhancer blocker
Valenzuela and Kamakaka , Annu. Rev. Genet. 2006
Blocks transcription if BETWEEN
enhancer and promoter
Gaszner and Felsenfeld Nature Reviews Genetics 2006
DNA Methylation
and Imprinting
DNA Methylation
• Found in:
– Prokaryotes: E. coli
– Eukaryotes: Some Fungi
Plants
Vertebrates
not found in: S. cerevisiae, C.elegans or
Drosophila
DNA Methylation
http://www.med.ufl.edu/biochem/keithr/fig1pt1.html
Eukaryotic DNA methylation
• Mostly methylated cytosines at CpG*
• Plants are also methylated at CHG or CHH
60-90% of CpGs are methylated at cytosine
* CpA and CpT in ESCs
DNA Methylation function
• Generally a repressive mark
• Reduced DNA-binding of many proteins
• Condensed chromatin structure
• Binding site for methyl binding proteins
General roles
imprinting
X inactivation
differentiation
regulation of gene expression
aging and cancer
genome stabilty defense against transposons, viruses
counteracts recombination of repetitive DNA
chromosome segregation
Experimental Techniques- Bisulfite
Mutagenesis and Sequencing
Slide from Marisa Bartolomei
Experimental Techniques- Bisulfite
Mutagenesis and Sequencing
-Each row represents a different strand of DNA
-Each circle represents a CpG
-Filled in circles-methylated CpG
-Open circles- unmethylated CpG
Engel, 2004, Nat. Genet.
Experimental Techniques- Methylation
sensitive Restriction Enzyme
Other techniques
Miho, Nature Reviews, 2008
De Novo vs. Maintenance DNA
methylation
Maintenance Methylation
Chen, Current topics in developmental biology, 2004
Maintenance Methyltransferases
methylate hemi-methylated DNA after replication
copy existing pattern onto new strand
Dnmt1 mammals
MET1 Arabidopsis
CTM Arabidopsis
generally ubiquitously expressed
Dnmt1 is essential (mutants are embryonic
lethal)Li, Cell, 1992
Bestero
Bestor, Hum. Molec. Genet., 2000
Slide from Doris Wagner
DNA Methyltransferases
DRM2 Arabidopsis
De novo methylation
DRM
Arabidopsis
DNA methylation throughout
development
Imprints
paternal
maternal
Modified from Reik, Theriogenology, 2003
DNA Demethylation
Active vs. Passive
Active demethylation
Enzyme Demethylates DNA independent of
Replication
Passive Demethylation
DNA methylation is not maintained through
replication.
Methylation marks get “diluted” out
Active and Passive DNA demethylation in
preimplantation mouse embryo
1 cell
Paternal= Active
Maternal=Passive
Passive
2 cell- 4 cell
Mayer, Nature, 2000
Lesson from Plants
Law and Jacobsen, Nature Review Genetics 2010
Demethylation in Mammals
Carey et al. Drug discovery today, 2011
DNA Methylation and transcription
1. Promoter regions
CpG islands (CGIs): non-methylated
CpG poor promoters: can be methylated, repressive
CGIs that are methylated do exist: long term silencing
imprinted loci; also seen in cancer
CpG Islands (CGI)
Definition: presence of normal (genome average) level of CpGs in promoters
G +C
observed/
expected CpG*
CGI
65%
close to 1
bulk DNA
40%
<1
*Num of CpG/(Num of C × Num of G) × Total number of nucleotides in the sequence
Kept unmethylated by:
transcription factor binding
actively demethylated
DNMT cannot methylate well (NDR, H2A.Z, H3K4me2/3)
DNA Methylation and transcription
2. Gene bodies
A. DNA methylation is required for Efficient transcription
• Prevents access to cryptic transcription start sites found in gene bodies
B. Is important for Splicing
• DNA methylation and nucleosomes predominantly found in exons
also H3K9me3
• DNMT3 substrate is nucleosomal DNA
• DNAme and H3K9me3 recruit one another
DNA Methylation and transcription
3. Enhancers
variable methylation level found
= diffferentially methylated regions
• prevent TF binding (E2F, NF-KB, AP2)
• Recruit Methyl binding proteins
• TF binding may alter DNAme (CTCF)
cause and effect relationship not very clear
Methyl-CpG-Binding proteins
Bogdanovic, Chromosoma, 2009
Methyl-CpG-binding proteins
MeCP2: first identified, has MBD (methyl binding domain),
binds mSin3a (HDAC)
MBD1: interacts with SETDB1 (H3K9 HMT)
MBD2 : in MeCP1 complex:
1: heterochromatin, binds HP1, Suv39, p150 (CAF1)
2: also in NuRD (HDAC)
MBD3: NuRD (HDAC) complex component
MBD4: DNA glycosylase; Cme-T repair
KAISO: no MBD, binds methylated DNA via Zn-finger
Important for amphibian development
DNA Methylation patterns and
epigenetic memory
Bird, GenesDev, 2002
DNA Hydroxymethylation
In mouse cerebellum, hematopoesis, and ESCs
10% of all meC in mammals
Promoters and gene bodies
pluripotency
role in bivalent domains in ESCs
differentiation of hematopoeitic cells
TET enzymes; bind to CGIs
one pathway for demethylation for example of the paternal genome
(hmC by TET1) followed by passive loss or deamination/mismatch repair or
other pathways
DNA Methylation
The key EPIGENETIC mark for meiotic or transgenerational inheritance!
Of critical importance in carcinogenesis
Genomic Imprinting
Both parental genomes are necessary!
Genomic Imprinting
The unequal expression of the maternal and
paternal alleles of a gene.
~100 imprinted genes clustered throughout the genome
Why Imprinting?
Genetic conflict hypothesis
Paternal allele: aggressive in obtaining maternal
resources for particular offspring
Maternal allele: equal allocation of resources to all offspring
Many paternally expressed genes encode growth factors,
Many maternally expressed genes inhibit growth factors
trophoblast hypothesis
Avoid depletion of maternal resources
(after spontaneous oocyte activation)
Make process fertilization dependent
DNA Methylation
•
•
•
•
Differential methylation during
gametogenesis
Reversible
Stably inherited
Repression of transcription
ICR: Imprinting Control Region
DMR: Differentially methylated region
Differential Methylation
ICR/DMR
Hypothetical exampleEstablishment and maintenance of DNA
methylation imprint
sperm
~100%
methylation at this
specific imprinted
locus
~50% methylation
at this specific
imprinted locus
~100%
methylation at this
specific imprinted
locus
Fertilization
egg
~0% methylation
at this specific
imprinted locus
Somatic cells of offspring
Germline of male offspring
DNA methylation throughout
development
Imprints
paternal
maternal
Modified from Reik, Theriogenology, 2003
Differential DNA methylation
established during gametogenesis
Li, Nature, 2002
Techniques- Allele specific Expression
F1 Hybrid!
M. castaneous
C57BL/6
X
F1 Hybrid
Examples of imprinted loci
H19/Igf2 locusInsulator Model
Wan, Advances in Genetics, 2008
ICR= Imprinting control region
DMR/DMD= Differentially methylated region (domain)
If CTCF is bound = enhancer blocker
Igf2= Fetal Mitogen
H19- ncRNA of unknown function
Igf2r/Air locus- long ncRNA mediated
imprinting
Ideraabdullah, Mut. Res., 2008
Igf2r= female scavenger receptor for Igf2
Slide from Marisa Bartolomei
ncRNA mediated vs. Insulator
mediated
• ncRNA mediated imprinting seems to be a
more widespread mechanism of imprinting
• Insulator mediated imprinting is more ancient
(found similar mechanism in marsupials)
Features of Imprinted loci
• The imprinting mechanism acts in cis
• Imprinted genes are clustered and are
controlled by a single imprinting control
region (ICR)
• The ICR acquires an imprint in one gamete
(often DNA methylation)
• Imprinted gene clusters contain at least 1 long
ncRNA
Imprinted loci conserved in humans
and associated with disease
Ideraabdullah, Mut. Res. 2008
Example diseases:
Angelman Syndrome, Prader-Willi Syndrome, Beckwith-Wiedemann
Syndrome, Silver-Russell Syndrome
Causes:
Genetic mutation in expressed gene (i.e. deletion)
Genetic mutations at the ICR that result in epigenetic defect
Epimutations, with no genetic change
Uniparental Disomy
DNA methylation throughout
development
The requirement is to erase
essentially all methylation in the PGC
is thought to be linked to the low number
of transgenerational inheritance events in
mammals
Pressure less strong on plants, imprinting
mainly occurs in extra-embryonic tissue
Imprints
paternal
maternal
Silencing/ DNA methylation/Imprinting
1.
Silencing mechanisms
Sir2/ HP1/HP1 and DNA methylation
budding yeast, fission yeast, mammals/plants
2.
Insulators (boundary elements/ enhancer blocker
Position effect variegation
3.
DNA methylation
de novo, maintenance , CpG islands
functions
methods of study
4.
DNA demethylation
plants
mammals
4.
Imprinting
Glossary I
Sir2, Sir1T
NAD-dependent HDAC
Swi6, HP1
H3k9me binding, heterochromatin
Clr4, Su(var)3-9
KMT, generates H3K9me
PEV
position effect variegation
due to juxtaposition of euchromatic
gene and heterochromatin
Boundary
insulators that prevents
heterochromatin spreading
Enhancer blocker
insulator that prevents
enhancer from acting on the
wrong promoter
Glossary II
DNMT1 (CMT, MET1)
maintenance DNA
methyltransferases
DMNT3 (DRM2)
DNA demethylation
de-novo DNA methyltransferases
plants: glycosylases plus base
excision repair
mammals: deamination plus
mismatch repair
hmcC TET pathway
ICR/DMD
Imprinting control region/
differentially methylated domain
CHH and CA or CT
always de novo methylation!