DNA Methylation_ Imprinting and X-Inactivation

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					DNA Methylation, Imprinting and

             Nov. 2 2010
           Lara Abramowitz
              DNA Methylation
• Found in:
  – Prokaryotes: E. coli
  – Eukaryotes: Some Fungi
           not found in: S. cerevisiae, C.elegans or
    drosophila (though drosophila does have
DNA Methylation

    Eukaryotic DNA methylation
• Mostly methylated cytosines at CpG
• Plants are also methylated at CNG

60-90% of CpGs are methylated at cytosine
      DNA Methylation function
• Generally a repressive mark

• Reduced DNA-binding of many proteins

• Condensed chromatin structure

• Binding site for methyl binding proteins
       General roles
parental genome regulation/imprinting

X inactivation


regulation of gene expression

aging and cancer

defense against transposons, viruses
counteracts recombination of repetitive DNA

important in 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.
Other techniques

                   Miho, Nature Reviews, 2008
De Novo vs. Maintenance DNA

                 Maintenance Methylation

       Chen, Current topics in developmental biology, 2004
DNA Methyltransferases
           Maintenance Methyltransferases
     methylate mainly hemimethylated DNA after replication
     copies existing pattern onto new strand

     Dnmt1 mammals
     MET1 Arabidopsis
     CTM Arabidopsis
     generally ubiquitously expressed
     Dnmt1 is essential (mutants are embryonic
     lethal)Li, Cell, 1992


Bestor, Hum. Molec. Genet., 2000

Slide from Doris Wagner
De Novo Methyltransferases
 can methylate naked (unmethylated) DNA
 Dnmt2 very little activity
 Dnmt3a imprinting
 Dnmt3b differentiation, cancer
         CpG islands, X-chromosome
 Dnmt3L imprinting (lacks catalytic sites)

 DRM Arabidopsis

 expression regulated in development
DNA Methylation throughout
   mouse development

                        Germline specific

              Hajkova, Epigenetic reprogramming in mouse germ cells, 2002
        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

                       2 cell- 4 cell

                        Mayer, Nature, 2000
Active DNA Demethylation
                                 Lesson from Plants


Modifies from Gehring, Trends in Genetics, 2009
Demethylation in Mammals

    Contradictory Data
    In vitro experiments
    Irrelevant in vivo experimental systems
How does DNA Methylation
interfere with transcription?
       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 complex component

MBD4: DNA glycosylase; Cme-T repair

KAISO: no MBD, binds methylated DNA via Zn-finger
Important for amphibian development
Methyl-CpG-Binding proteins

                     Bogdanovic, Chromosoma, 2009
DNA Methylation patterns and
    epigenetic memory

                 Bird, GenesDev, 2002
                     CpG Islands

Kept unmethylated by:             Often found in promoter regions
transcription factor binding
 actively demethylated
 DNMT methylated islands poorly
         DNA Methylation and Cancer

                                 Jones, Nature, 1999
LOH= loss of heterozygosity
DNA Methylation and Cancer

                       Jones, Nature, 1999
Genomic Imprinting
         Genomic Imprinting
The unequal expression of the maternal and
  paternal alleles of a gene.
Both parental genomes are necessary!
~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
Genomic imprinting

Parent of origin has to be marked…
                The Mark…
• Established in the Germline

• Stable

• Erasable
         DNA Methylation!!!!
• Differential methylation during
• Reversible
• Stably inherited
• Repression of transcription
Differential Methylation
DNA methylation throughout

                   Modified from Reik, Theriogenology, 2003
         Hypothetical example-
Establishment and maintenance of DNA
          methylation imprint
       sperm                                                egg
~100%                                                         ~0% methylation
methylation at this         Fertilization                     at this specific
specific imprinted                                            imprinted locus

         ~50% methylation
         at this specific                   Somatic cells of offspring
         imprinted locus

 methylation at this                Germline of male offspring
 specific imprinted
  Differential DNA methylation
established during gametogenesis

                               Li, Nature, 2002
Techniques- Allele specific Expression
             F1 Hybrid!
             C57BL/6       M. castaneous


     F1 Hybrid
Experimental assays- allele specific
        Rnase Protection

                      Bartolomei, Nature, 1991
Examples of imprinted loci
                         H19/Igf2 locus-
                         Insulator Model

                                                            Wan, Advances in Genetics, 2008

ICR= Imprinting control region
                                                     Igf2= Fetal Mitogen
DMR/DMD= Differentially methylated region (domain)
                                                     H19- ncRNA of unknown function
       Without the ICR

                                              Thorvaldsen, GenesDev, 1998

       ICR is required for imprinting
Igf2r/Air locus- long ncRNA mediated

                                            Ideraabdullah, Mut. Res., 2008

Igf2r= female scavenger receptor for Igf2
Slide from Marisa Bartolomei
     ncRNA mediated vs. Insulator

• 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
• Imprints can activate or silence gene expression
• 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
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

 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
Example Pedigree of an imprinted

X- inactivation
                Sex Determination- Mammals

       Equal expression?- Dosage

• Drosophila- upregulates male X expression

• Nematode- downregulate female X expression

• Mammals- inactivate 1 female X
     Barr and Bertram, 1949

Female        Male            Female

             In Cats!
                  Inactive X
• Most of the X chromosome is silenced

• Cytologically distinguishable

• Highly condensed X= Barr Body
When does X-inactivation occur?-Mouse

                        Early in Development (preimplantation
                        Imprinted Xp Inactive
                        Remains this way in trophectoderm

                         Random X-inactivation in embryonic

                       Iderraabudllah, Mut. Res. 2008
Random X-inactivation!
Components of X chromosome inactivation (XCI)

     1. Counting X : autosome ratio
     limited amount of blocking factor binds to one X chromsome
     keeps it active (not yet identified!); X-pairing, stochastic?
     2. Choice (which X in random inactivation)
     XCE strength (X controlling element)
     blocking factor binding site ?
     also strength of Tsix promoter cis element: Tsix levels
     3. Initiation/propagation of inactivation
     temporal series of events
     4. Maintenance of silent state
     late events are required
• Always only 1 active X

       XY            XX      XXX
• XIC- X-inactivation center

• Discovered by a series of translocations and

• Sequence that coordinates differentiation and
  counting signals, inactivation spreads in cis
  from XIC
X Inactivation Center


Xic            Tsix


 Mouse X Chromosome
                        Slide from Marisa Bartolomei

                                           Heard, Current Opinion in Cell Biology, 2004

Only expressed from inactive chromosome

Long ncRNA that coats inactive X

Necessary to initiate X inactivation

Sufficient to Silence chromosomes in cis
How does Xist silence?

                 Chow, Current opinions in cell biology, 2009
                         Tsix (mouse)

                                                  Heard, Current Opinion in Cell Biology, 2004

• Antisense to Xist transcribed from active X

• Negatively regulates Xist during initiation

• Mutations that disrupt TsiX expression result in that mutant chromosome
  always being inactive
          Other components of XIC

                                             Heard, Current Opinion in Cell Biology, 2004

Xce- Genetically mapped element that influences X chromosome choice

Xite- noncoding RNA, enhancer of TsiX
• Heterochromatin formation
      -DNA methylation
      -Novel histones (enrichment of macroH2A
  on inactive X)
      -Change in histone modifications
      -Late replicating DNA

           Heard, Current Opinion in Cell Biology, 2004
Heard, E., Current Opin. In Cell Biol 2054 15:482-489
            X-linked disorders
• Hemophilia- deficiency to control blood clotting.
  Sex-linked recessive therefore mostly found in
• Rett Syndrome- mutation in Mecp2.
  Neurodevelopmental disorder. X-linked
  dominant. Found in females (males die).
• Fragile X syndrome- Mental impairment. X-linked
  dominant. Effects both males and females
  (though in females depends on x-inactivation

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