Silencing/ DNA methylation/Imprinting1. Silencing mechanisms
Sir2/ HP1/HP1 and DNA methylationbudding yeast, fission yeast, mammals/plants
2. Insulators (boundary elements/ enhancer blockerPosition effect variegation
3. DNA methylationde novo, maintenance , CpG islandsfunctionsmethods of study
4. DNA demethylationplantsmammals
4. Imprinting
Silencingcreates
large domains of chromatin that are
compacted and
less accessible to DNA-binding proteins
SilencersSilencing proteins
Sir2HP1Polycomb group (PcG) proteins
DNA Methylation
noncoding RNAs
Boundary elements
S.c. S.p. A.th.D.m. MammalsHypoacetyl. + + + + +H3/H4H3K9 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 proteinsHP1, Sirtuins, PcG, telosome
Fanti and Pimpinelli, COGD, 2008
HP1 localization
Purpose of heterochromatin
protect repetitive DNA from recombination
keep centromer/telomer intact (chromosomeintegrity)
protect genome from transposons and other ‘selfish DNA’
prevent (not block) transcription
Saccharomyces cerevisiae or Saccharomyces pombe
Telomers
Centromers
Silent mating type loci
Silencing: lessons from yeasts
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
Mating type loci
S. cervisiae S. pombe
Yeast mating type loci
S.c. S.p. A.th.D.m. MammalsHypoacetyl. + + + + +H3/H4H3K9 me - + + + +HP1 - + +* + +
DNA methyl. - - + - +
Polycomb - - + + +
Sir2 + + + + +
* present but binds H3K27me notH3K9me
silenced state initiateddeacetylation of H3/H4
Molecular Cell, Vol. 8, 489–498, September, 2001Common Themes inMechanisms of Gene SilencingDanesh Moazed
Sir3 binds H4K16 (unacetylated)Multimerization of Sir proteins; spreading
Continuous presence of Sir proteins
silencing
Molecular Cell, Vol. 8, 489–498, September, 2001Common Themes in Mechanisms of Gene SilencingDanesh Moazed
SIR
Function Sir2NAD-dependent HDAC!
Other roles of sirtuins
Linked to caloric restriction/lifespan extension
Resistance to Neurodegenerative disease
Cancer (Tumor suppressor)
S. cervisiae S. pombe
Yeast mating type loci
S.c. S.p. A.th.D.m. MammalsHypoacetyl. + + + + +H3/H4H3K9me - + + + +HP1 - + +* + +
DNA methyl. - - + - +
Polycomb - - +$ + +
Sir2 + + + + +
* present but does not bind H3K9me$ PRC2 but not PRC1
Clr1 DNA binding
Clr3 HDAC (Hda1)
Clr4 HMT (Suv3-9)
Clr6 HDAC (Rpd3)
Swi6 HP1
Rik1 DNA binding (WD40 repeats)
Silencing S. pombe
Grewal and Elgin, COGD, 2002.
HP1
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 H3K92. Methylation H3K9 Clr43. Binding HP1 Swi6, Chp24. 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 H3K92. Methylation H3K93. Binding HP14. Establishment of Heterochromatin/spreading
Trigger: repetitive DNA, small noncoding RNAsrecruiting factors, silencers
Silencing S. pombe
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
Chromodomain: binds H3K9me
Hinge region: binds RNA
Chromoshadow domain: dimerization, interaction with other proteins such as Suv39
H1
HP1 structure
Vermaak and Malik, Ann. Rev. Genet., 2009
HP1 structure and binding
Jacobs and Khorasanizadeh, Science 2001
HP1 chromodomain binds H3K9me2/3
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
Grewal and Jia Nature Reviews Genetics 2007
DNA methylation
HP1 heterochromatin: recruits other factors
Reversal of Heterochromatin formation
H3K9me demethylase
H3S10ph
Both cause HP1 removal
Hirota et al. Nature 2005
Methyl/phospho switch
Genes within heterochromatin
Uniquely regulated
Require heterochromatin for proper expression
Require HP1 for proper expression
Gaszner and Felsenfeld Nature Reviews Genetics 2006
Heterochromatin spreads: prevented by boundary
Defects in boundarySilencing of adjascent genes:
transgene silencing
translocationPEV (position effect variegation)
PEV in Drosophila
PEV strain PEV strain HP1 mutant
Drosophila white gene (responsible for red eye color)is located in heterochromatin in the PEV flies
http://www.biology.wustl.edu/faculty/elgin/hetchrom.html
PEV in Drosophila
PEV
Gaszner and Felsenfeld Nature Reviews Genetics 2006
Lunyak COCB, 2008
PEV in yeast
PEVVariegation due to imprecise establishment or inheritance of the silenced state of euchromatic geneDissecting 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
Valenzuela and Kamakaka , Annu. Rev. Genet. 2006
Boundary: a type of insulator that prevents the spreading of heterochromatin
Noma et al. Science, 2001
Delete boundary: H3K9me3 and HP1 (Swi5) spread!
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 BETWEENenhancer and promoter
Gaszner and Felsenfeld Nature Reviews Genetics 2006
DNA Methylation and Imprinting
DNA Methylation
• Found in:– Prokaryotes: E. coli– Eukaryotes: Some Fungi
Plants Vertebratesnot 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 rolesimprinting
X inactivation
differentiation
regulation of gene expression
aging and cancer
genome stabilty defense against transposons, virusescounteracts 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
Slide from Doris Wagner
Bestero
Bestor, Hum. Molec. Genet., 2000
methylate hemi-methylated DNA after replicationcopy existing pattern onto new strand
Dnmt1 mammalsMET1 Arabidopsis CTM Arabidopsisgenerally ubiquitously expressedDnmt1 is essential (mutants are embryonic lethal)Li, Cell, 1992
DNA Methyltransferases
DRM Arabidopsis
De novo methylation
DRM2 Arabidopsis
DNA methylation throughout development
Imprintspaternalmaternal
Modified from Reik, Theriogenology, 2003
DNA DemethylationActive 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
Paternal= Active
Maternal=Passive
Mayer, Nature, 2000
1 cell
2 cell- 4 cell
Passive
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 silencingimprinted loci; also seen in cancer
CpG Islands (CGI)
Kept unmethylated by: transcription factor bindingactively demethylatedDNMT cannot methylate well (NDR, H2A.Z, H3K4me2/3)
Definition: presence of normal (genome average) level of CpGs in promoters
*Num of CpG/(Num of C × Num of G) × Total number of nucleotides in the sequence
CGI bulk DNAG +C 65% 40%
observed/ close to 1 <1 expected CpG*
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 exonsalso 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 proteinsMeCP2: 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-fingerImportant for amphibian development
DNA Methylation patterns and epigenetic memory
Bird, GenesDev, 2002
DNA HydroxymethylationIn mouse cerebellum, hematopoesis, and ESCs
10% of all meC in mammals
Promoters and gene bodies
pluripotencyrole in bivalent domains in ESCsdifferentiation 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
Genetic conflict hypothesisPaternal allele: aggressive in obtaining maternal resources for particular offspringMaternal allele: equal allocation of resources to all offspring
Why Imprinting?
trophoblast hypothesisAvoid depletion of maternal resources (after spontaneous oocyte activation)Make process fertilization dependent
Many paternally expressed genes encode growth factors, Many maternally expressed genes inhibit growth factors
DNA Methylation
• Differential methylation during gametogenesis
• Reversible• Stably inherited• Repression of transcription
ICR: Imprinting Control RegionDMR: Differentially methylated region
Differential Methylation
ICR/DMR
Hypothetical example-Establishment and maintenance of DNA
methylation imprintsperm egg
Fertilization~100% methylation at this specific imprinted locus
~0% methylation at this specific imprinted locus
Somatic cells of offspring
Germline of male offspring
~50% methylation at this specific imprinted locus
~100% methylation at this specific imprinted locus
DNA methylation throughout development
Imprintspaternalmaternal
Modified from Reik, Theriogenology, 2003
Differential DNA methylation established during gametogenesis
Li, Nature, 2002
Techniques- Allele specific Expression F1 Hybrid!
X
M. castaneous C57BL/6
F1 Hybrid
Examples of imprinted loci
H19/Igf2 locus- Insulator Model
Wan, Advances in Genetics, 2008
ICR= Imprinting control regionDMR/DMD= Differentially methylated region (domain) Igf2= Fetal Mitogen
H19- ncRNA of unknown function If CTCF is bound = enhancer blocker
Igf2r/Air locus- long ncRNA mediated imprinting
Igf2r= female scavenger receptor for Igf2
Ideraabdullah, Mut. Res., 2008
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
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
Ideraabdullah, Mut. Res. 2008
DNA methylation throughout development
Imprintspaternalmaternal
The requirement is to eraseessentially all methylation in the PGCis thought to be linked to the low number of transgenerational inheritance events in mammals
Pressure less strong on plants, imprintingmainly occurs in extra-embryonic tissue
Silencing/ DNA methylation/Imprinting1. Silencing mechanisms
Sir2/ HP1/HP1 and DNA methylationbudding yeast, fission yeast, mammals/plants
2. Insulators (boundary elements/ enhancer blockerPosition effect variegation
3. DNA methylationde novo, maintenance , CpG islandsfunctionsmethods of study
4. DNA demethylationplantsmammals
4. Imprinting
Glossary ISir2, Sir1T NAD-dependent HDAC
Swi6, HP1 H3k9me binding, heterochromatin
Clr4, Su(var)3-9 KMT, generates H3K9me
PEV position effect variegationdue 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 IIDNMT1 (CMT, MET1) maintenance DNA
methyltransferases
DMNT3 (DRM2) de-novo DNA methyltransferasesDNA demethylation plants: glycosylases plus base
excision repairmammals: deamination plus mismatch repairhmcC TET pathway
ICR/DMD Imprinting control region/ differentially methylated domain
CHH and CA or CTalways de novo methylation!