silencing/ dna methylation/imprinting

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 - + +* + +


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 - + +* + +


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


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


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*


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


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





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



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!

silencing/ dna methylation/imprinting

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