DNA methylation and epigenetics

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DNAmethylation and epigenetics
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Epigenetics

• Mitoticaly and/or meioticaly heritable changes in
  gene function, which are not based on changes in
  primary DNA sequence
• MechanismDNA methylation
• histon modifications (acetylation,
  methylation,..)
• Regulation of gene expression in development,
  parental imprinting
• Transgene and transpozone silencing,
• Paramutations,
• Conformational changes in chromatin (keeping
  heterochromatin)

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Mechanism of DNA mathylation

• in Eukaryots metylation of cytosin on C5(also
  adenin in bacteria)

H
H
H
H
O
N
N
H
N
N
N
T
C
C
SAM
N
H2O - NH3
O
N
O
N
O
thymine
cytosine
5-methyl cytosine
hot spot of mutagenesis
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DNA methylation target sequences
- both symmetric (palindromatic) sekvence and
asymmetric sequences 5 -------CG-------CNG---
-------C---C------G--C---- 3 3
-------GC-------GNC----------G---G------C--G----
5 Symmetric methylation pattern easily kept
after replication hemimethylated state
signal for maintainance methylation -----CG--
---------- CNG-----------C------- ---------CG
---- -----G C-------------G
NC-----------G ------- ---------G
C---- -----CG -------------CNG
-----------C-------- -----GC-------------GNC-
----------G-------- Asymmetric after
replication de novo methylation (by another
signal)
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DNA methylation in Eukaryots

• Non-methylated DNA Saccharomyces cerevisiae,
• Drosophila, Caenorhabditis, .
• mammals 3-8 cytosine residues, CpG sequences
• plants 25-30 cytosine residues ( genome size)
• Mainly in repetitive heterochromatinic regions
• (non-methylated islands of gene rich DNA in large
  genomes)

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Genom Arabidopsis - statistics
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Analysis of DNA methylation in Arabidopsis
Strong methylation in heterochromatin near
centromeras MetC even in coding sequences of
expressed genes! Metylation in promotors of
developmentally regulated genes
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Analysis of methylation in Arabidopsis Chromosom
e epigenetic landscape
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DNA methylation maintainance and de novo
Maintainance symmetric hemimethylated state
- chromatin state (histon modifications)
de novo both symmetric and asymmetric
(changes in methylation) maintainance
methylation of asymmetric sequences -
chromatin state (histon modifications) -
siRNA (RNA directed DNA metylation)
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Plant cytosine 5-methyltransferases in
Arabidopsis min. 10 genes in 4 families
MET (metyltransferase) CMT (chromomethyltransferas
e) DRM (domain rearranged methyltransferase) At5g2
5480 - related to mammalian Dnmt2 ?
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MET family - MET1 (ddm2, met1), MET2a,
MET2b, (MET3) - related to mammalian Dnmt1 -
maintainance methylation of symmetric CG -
asociated with replication - involvement of
nucleosom remodeling complex
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• CMT family
• CMT3,(CMT1), CMT2
• - unique for plants
• related to mammalian Dnmt1 chromodomain
• (interacting with chromatin) - chromomethyltransfe
  rase
• activity associated with methylation of histon
  H3K9
• - maintainance methylation of CNG and certain
  asymmetric seq.

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• DRM family
• - DRM2 (DRM1)
• - related to mammalian Dnmt3, but with rearranged
  domains,
• domain rearranged methyltransferase
• - de novo methylation of all sequences (CG, CNG,
  asymmetric)
• maintainance methylation of asymetric and CNG
• regulation of gene expression, silencing
• Targed sequences determined by presence of
  homologous
• siRNA

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RNA directed DNA methylation
NRPD nuclear RNA polymerases RDR RNA
dependent RNA polymerase DCL dicer-like protein
AGO4 ARGONAUTE DRM de novo metyltransferase DR
D chromatin remodelling protein
siRNA (24 nt) probably interact with nascent
ssRNA during transcription!!!
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Cytosine demethylation
1) Through replication without maintainance
methylation 2) DNA demethylases (cytosin and
methanol), DNA glykosylases (cleaving out
metylcytosine, replaced by cytosine) DME1
DEMETER parental imprinting (endosperm)
ROS1 REPRESSOR OF SILENCING somatic cells If
and where is the methylation state kept by
simultaneous action of methyltransferases and
demethylases is under discussion.
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Methylation mutants
Total decrease in 5metC (decrease in DNA
methylation) selection based on sensitivity of
repetitive centromeric sequences to
restriction by enzymes sensitive to DNA
mathylation ddm2 ddm1 symmetric
CG symmetric CG a CNG Mutation in gene for
SWI2/SNF2-like
protein MET1 - nukleosom remodelling
complex
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Phenotype of mutation in MET1 and DDM1

• - similar
• developmental alterations (flowering, shapes of
  flowers, leaves, fertility, .)
• u ddm1 phenotype observed after several
  generations of selfpolination (gradual loss of
  methylation pattern)
• demethylation and activation of silenced genes
  and transposons
• local hypermethylation and silencing

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Methylation mutants
Decreased methylation in asymmetric
sequences drm1, drm2 double mutant loss of
de novo methylation of asymmetric and some
CNG cmt3 loss of maintainance methylation
of CNG and some asymetric Strong phenotype
connected with developmental alterations observed
only in triple mutant in all three genes (?
partial redundancy)
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Methylation mutants

• Reactivation of expression of silenced gene
• - both ddm mutants decreased methylation
• - hog1 decreased methylation
• mom1, sil1, sil2 no change in methylation!!
• Silencing of gene expression is not neccessarily
  connected with DNA methylation (remember also
  species without methylation)

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Analysis of methylation

• Direct
• spektrophotometric, HPLC
• - metC
• anti metC antibodies (in situ on chromosoms )
• - localization of metC rich regions of
  chromosoms
• Restriction endonucleases
• - detection of metC on certain position
• hydrogensulphite sequencing
• - determination of C and metC in certain PCR
  amplicon
• Indirect e.g. reactivation of silenced genes

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Restriction endonucleases
A -----------C CGG-----------------------
------------CmetCGG--------------------------C
CGG---------------------- B -----------C
CGG-----------------------------------C
CGG--------------------------C
CGG---------------------- -
restriction PCR primer
electrophoresis, Southern
PCR,
electrophoresis
A B
A B
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Hydrogensulphite sequencing
(NaHSO3) 1. denaturation ---------C---------C--
------CC---C------C------------C-- 2.
Modification of Cyt to Ura (5metC remains
unchanged) ---------C---------U--------UU---C-----
-U------------U-- 3. PCR, sequencing original
sequence ---------C---------C--------CC---C------C
------------C-- ---------G---------G--------GG---G
------G------------G-- modified
sequence ---------C---------U--------UU---C------U
------------U-- ---------G---------A--------AA---G
------A------------A--
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Demethylation of DNA

• - 5-azacytidine inhibition of MET1
• - dihydroxypropyladenine (DHPA) reduced levels
  of SAM
• (antisense) inactivation of MET1, DDM1, CMT3,

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Mutagenesis connected with methylation
C T
replication reparation
T
A
New gene evolution duplication, methylation,
mutation, new function, reactivation,
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Steric effects of DNA methylation
Modified DNA double helix
modulation of histon
modifications Altered interaction of associated
proteins - regulators of transcription -
proteins responsible for structure and
modifications of chromatine
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MBD proteins (metyl-CpG-binding
domain) Compression of chromatin Interaction
with histon modifying proteins (HDAC)
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DNA methylation and chromatin state
interconnections

• correlation between hypermethylation, late
  replication and histon hypoacethylation
• methylation is heritable signal for histon
  deacethylation and chromatin condensation
• similar phenotype of mutants with blocked DNA
  methylation (met1) and histon deacethylation
  (AtHD1)
• - methylation of H3K9 (KYP) signal for
  methylation of CNG
• - methylation of H3K4 typical for euchromatin
  (active DNA)

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Posttranslational modifications of histons
histon methyl transferases KYP H3K9
met histon demethylases histon
acetyltransferases histon deacetylases histon
kinases a phosphfatases, Complexity of histon
code exceeds complexity of genetic code
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(No Transcript)
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Role of DNA methylation

• heritable modulation of transcription
  (predominantly methylation of promoters)
• - defence against invasive DNA (transposons)
• - regulation of gene expression (ontogenesis,
  diferenciation, cell memory)
• - parental imprinting
• modulation of chromatin structure
• - timing of DNA replication
• - regulation of homologous recombination
  (meiosa)
• tool of evolution
• - polyploid plant genomes (large changes in
  methylation)
• - new genes (mutagenesis)

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• Pharental imprinting
• different epigenetic modification (methylation)
  of alels inherited from mother and father gamete
  (different expression in zygote hemizygous
  state)
• in Angiosperms and mammals (parallel evolution)
• role in keeping balaced embryo nutrition
  (endosperm growth) in the mother organism
• demethylation
• or methylation
• of certain genes
• (aleles from certain
• pharent)
• in endospern
• (? not in embryo
• as in mammals)

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Transgene silencing
Pozition effect (location on chromosom, character
of neighboring sequences) - heterochromatin -
MARs a SARs (mitosis/scaffold associated/attached
regions) Homology Dependent Gene Silencing
- siRNAs
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Homology Dependent Gene Silencing

• TranscriptionalGeneSilencing no transcription
  (promotor methylation)
• PostTGS mRNA not
  translated to protein
• PTGS can exchange to TGS!
• Induction of silencing
• dsRNA (sense antisense RNA, hairpin, RdRP)

()
()
(-)
(RdRP RNA dependent RNA pol.)
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siRNA formation
Signal dsRNA Cleavage of dsRNA DICER
(DICER-like) Efector siRNA in complex
with ARGONAUTE
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Gene Silencing - dsRNA formation
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Homology Dependent Gene Silencing
PTGS degradation of mRNA (interference with
translation) TGS RNA dependent DNA methylation
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Gene Silencing systemic transport of siRNAs
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Epigenetic regulation of gene expression in plant
development

• - miRNA (21 nt)
• encoded as pre miRNA (cleaved by DCL1
  DICER-LIKE)
• in Arabidopsis more than 100 well characterized
• (in total probably thousands ???)
• targets regulatory proteins
• e.g. transcription factors, components of
  ubiquitination
• pathway,

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miRNAs in plant development
Inhibited expression of certain miRNA
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miRNAs in plant development
Expression of miRNA-resistant variants of target
genes (the same protein encoded by different
nucleotide sequence)