Epigenetics and Imprinting

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Epigenetics and Imprinting

• Dr Una Fairbrother

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Imprinting

• Describes the differential expression of genetic
  material at chromosomal/allelic level, depending
  on that material being maternal or paternal in
  origin.

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What is the imprint at a molecular level?

• An imprint is an heritable tag
• Usually associated with differential DNA
  methylation
• The met CpG usually on the silent chromosome
• In addition, histone aceylation and methylation
  has been identified

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Imprinting involves 3 distinct biological stages

• (a) Establishment of the imprint in gametes,
  according to the sex of the individual
• (b) Its maintenance during embryogenesis/adult
  somatic tissues
• (c) Erasure in the early germline

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Schematic of Imprinting
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Why imprinting?

• The advantages of biparental inheritance seem
  clear from the recessive nature of many disease
  mutations.
• Imprinting could help to maintain sexual
  reproduction.

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Imprinting in Development

• In mammals, imprinted genes have a vital role in
  development of the embryo and neonate.
• The division is not clear cut but
• placental development appears to rely more
  heavily on paternal expression
• embryonic growth appears to rely more heavily on
  maternal expression.

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Imprinted Genes Present a complex Picture Of
Expression

• In embryonic development, monoallelic expression
  is not always coincident with the onset of gene
  expression
• Can vary with development and differentiation.

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Imprinting, primary inactivation?

• Some evidence suggests that the imprint itself
  does not have to be a primary inactivation event
• The imprinting mechanism may involve additional
  trans-acting molecules.

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Imprinting and Disease

• Approx 80 imprinted genes in the human genome
  poss up to 600!
• Non-mendelian inheritance pattern with parent of
  origin effects
• Best characterised syndromes
• Angelman/Prader-Willi syndromes on 15q (UBE3A-
  AS)
• Beckwith-Wiedemann syndrome (BWS) 11p (IGF2)

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Species Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Human Chr 01 01 06 06 06 07 07 07 07 07 07 07 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 Gene ARHI, NOEY2 TP73 PLAGL1 ZAC LOT1 HYMAI IGF2R, M6PR (disputed) GRB10, MEG1 PEG10 DLX5 MEST, PEG1, MESTIT1, PEG1-AS COPG2 (disputed PEG1-AS, MESTIT1 CPA4 WT1 (disputed) H19 IGF2 IGF2AS, PEG8 INS, insulin TRPM5, LTRPC5, MTR1 KCNQ1, KvLQT1 KCNQ1OT1, LIT1, KvLQT1-AS, KvDMR1 KCNQ1DN, BWRT CDKN1C, p57KIP2 SLC22A1L, IMPT1, BWR1A, ORCTL2, ITM, TSSC3, IPL, BWR1C, TSSC5, HET ,ZNF215 2G3-8 SDHD
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Human Human Human Human Human Human Human Human Human 14 14 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 18 19 20 20 MEG3, GTL2 DLK1, PEG9 Paternally expressed non-coding transcripts on 15p (Prader-Willi syndrome region) MKRN3, ZNF127 NDN MAGEL2, NDNL1 SNRPN, SNURF PAR-SN HBII-13 PAR5, D15S226E HBII-85, PWCR1 IPW PAR1, D15S227E HBII-52 UBE3A, E6-AP, UBE3A-AS ATP10A, ATP10C Elongin A3 PEG3 NNAT, Neuronatin GNAS1, Gs alpha, NESP55, XLalpha s, GNAS-AS
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Features Of Imprinted Genes

• No direct sequence homology has been found among
  imprinted genes.
• Sequences carrying mat and pat gametic imprints
  resemble CpG islands containing GC rich regions
  of between 200 and 1500bp with a balanced CGGC
  ratio.
• Imprinted sequences contain or are closely
  associated with a region of direct repeats
  ranging in size between 25 and 120bp. They are
  found in all imprinted genes analysed so far and
  are evolutionarily conserved.

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Imprinting Clusters

• Imprinted genes are clustered
• Best understood on chromosome 15 and 11
• Complex coordinated regulation based on an
  imprinting centre (IC) or an imprinting control
  element (ICE)
• Coodinates in cis expression of neighbouring
  genes in one domain

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Action of Imprinting Centres

• Allele discriminating action of imprinting
  centres based on epigenetic modifications of
  chromatin
• Typically via methylation of cytosines (DNA)and
  histone acetlylation and methylation
• Acetylation of histones associated with
  transcriptionally active chromatin
• Methylation of histones and DNA associated with
  inactivation of transcription

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Imprinting and Transcription

• Methylation and acetylation lead to allele
  specific accessibility to transcriptional
  machinery
• And to DNA binding proteins acting as enhancers
  or insulators
• Setting and stability of imprinted gene
  expression controlled by ICs with multiple levels
  of DNA and chromatin modifications

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More complexity IGF2

• IGF2 (chromosome 11) has been shown to be
  imprinted very early on, in 8 cell human embryos
• recruits different promoters to confer mono or
  biallelic expression at different developmental
  stages.

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.And KVLQT1

• KVLQT1 (chromosome 11) has been reported as
  having a number of alternatively spliced
  transcripts
• shows both mono and biallelic expression patterns
  in different tissues
• Important in identification of imprinted genes
  associated with imprinted disorders

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Conservation

• Obviously evolutionary conservation is a good way
  to identify fundamentally important genes and
  sequence motifs
• A horse mare-donkey cross gives rise to a mule
  but a donkey-stallion gives rise to a hinny
• The callipyge gene phenotype beautiful buttocks
  is only expressed when paternally inherited Hum
  Mol Genet 1998 7No10 review

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Imprinting in animal models

• Once thought to be restricted to mammals, genomic
  imprinting has been documented in angiosperm
  plants 1970, zebrafish 1995, insects, and C.
  elegans 2004
• There may be genomic imprinting in drosophila,
  but transgenes have shown that imprinting switch
  regions act as silencers in flies
• In marsupials methylation on the X chr
  preferentially inactivates paternal X
• Mouse studies are one of the commonest in the
  literature

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Of Mice and Men

• Mouse models have been helpful in identifying the
  genes involved in Prader-Willi and Angelman
  syndrome
• The PWS mouse model has a partial maternal
  duplication of the region of mouse chromosome 7
  homologous to human 15q11-13
• The AS mouse model has a paternal duplication for
  the same region

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Imprinting in Mice Not Identical

• There are differences in imprinting between mice
  and humans
• Imprinting of H19, Igf2, p57kip2 and Snrpn is the
  same
• IGF2R appears to be monoallelic in mice and
  biallelic in humans, though this may be
  polymorphic

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Imprinting on Human Chromosome 15

• IC regulates chromatin structure, DNA methylation
  and gene expression in a 2Mb region in 15q11-13
• Mutations in IC cause chromosome to be stuck as a
  single gender and not assume the sex imprint of
  its host.
• gt7 imprinted transcripts in AS region, paternally
  expressed
• UBE3A is only coding transcript maternally
  expressed

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Angelman Syndrome (AS)

• incidence of 1/20,000 live births
• severe mental retardation
• Lack of speech (commonly only a three to six word
  vocabulary)
• Ataxic gait
• Hand flapping
• Happy disposition including inappropriate
  laughter
• Diagnostic EEG
• Others

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Chromosome 15 deletion in As and PWS

• Initially identified by the same cytogenetic
  lesion i.e. the deletion of 15q11-13

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Prader-Willi Syndrome (PWS)

• Clinically distinct syndrome
• frequency of about 1 in 25,000 and
• probably the most common syndromal cause of human
  obesity Clinical features including
• Mild mental retardation
• Obesity
• Short stature

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AS Oppositely Imprinted to PWS
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How Does AS Arise?

• AS caused by loss of function of a gene/genes
  from the maternal chromosome 15q11-13
• (ICH study) 60 AS patients have large
  cytogenetic deletion
• 4 have uniparental disomy
• 4 have mutations in the imprinting centre (IC)
• 5 remaining, screened for mutations in the AS
  gene, UBE3A (E6AP) (50 familial, 10 sporadic)

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AS Oppositely Imprinted to PWS
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Recurrence risk

• typical large deletions are de novo and are
  expected to have less than 1 risk
• paternal UPD, (no parental translocation), less
  than 1
• transmission of a structurally or functionally
  unbalanced chromosome complement can lead to
  15q11-q13 deletions or to UPD and will result in
  case-specific recurrence risks.
• no large deletion or UPD, probably 50 due to
  maternal IC or UBE3A mutation see overhead
• Risks confounded by mosaicism

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UBE3A (E6AP) and AS

• UBE3A (ubiquitin protein ligase) complex
  expression patterns.
• 5 end of gene alternatively spliced in a
  variety of human tissue cell lines and in human
  foetal tissues including brain
• gt 7 isoforms have been identified monoallelic
  expression is tissue specific and isoform
  specific
• Human fibroblast and lymphoblast tissues and also
  adult mouse tissues show biallelic expression.

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Monoallelic expression of UBE3A

• Monoallelic expression of an isoform found in
  mouse brain tissue
• depression of expression is also evident in the
  hippocampus of a paternal UPD mouse
• Evidence for decrease of expression in human
  brain
• Clinical description of AS indicates a
  developmental brain disorder and so differential
  expression of the gene would be expected in brain
  tissue.

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UBE3A Has Even More Complex Splicing

• Normal UBE3A decreased in AS brain (MATERNAL)
• A new larger transcript decreased in PW brain
  (PATERNAL)
• Larger transcript is antisense and spans half
  UBE3A with additional down stream sequence

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• Also another sense strand was detected which lies
  in between the 2nd and 3rd polyadenylation signal
  of UBE3A
• Imprinted and transcribed in the same way as
  UBE3A
• All other tissues antisense transcript not
  expressed
• The expression of the antisense transcript in
  brain may force the UBE3A transcript to be
  monoallelic in brain
• Some as yet uncharacterised AS patients could
  have a mutation in either the down stream sense
  transcript or the antisense transcript, see
  overhead

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Mouse models

• Mouse models have been used to identify imprinted
  regions by engineering UPDs for mouse
  chromosomes, in part or in full
• Extensive mapping of imprinted mouse genes and
  their human homologues has been undertaken
  http//www.mgu.har.mrc.ac.uk/imprinting

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Similarities to Human Disease

• AS mouse Neuro behavoural differences, mild
  ataxia, abnormal limb clasping, hyperactivity,
  diminished brain weight (10 smaller)
• The PWS mouse increase in postnatal loss, small
  skeleton, grossly obese by 6 months

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Differences to Human disease

• AS mouse no detection of language difficulties
  (obviously), mouse also showed gross obesity -
  could be present in a subset of AS patients as
  late onset
• PWS mouse no detection of mild mental
  retardation.

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Usefulness of AS Mouse Model

• When gene was identified, AS mouse brains could
  be looked at and a monoallelically expressed
  transcript of UBE3A was found to be decreased as
  compared to the normal and the PWS mouse

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Outstanding questions

• How and when during germline development are old
  imprints removed and new ones introduced?
• Which demethylating activities and chromatin
  factors are involved?
• How does the spreading of epigenetic information
  in clusters work, - germline-specific,
  postzygotic phenomenon or both?
• How are imprints maintained when there is
  genome-wide active and passive demethylation in
  the early embryo?
• How many fundamentally different arrangements of
  imprinted genes and imprinting control elements
  are there in the genome?

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Cont

• How conserved is imprinting between mammalian
  species?
• How precisely do imprinted genes affect
  extraembryonic and embryonic development, and the
  nutritional exchange with the mother?
• Are there interactions of imprinted genes
  (particularly antagonistic ones) in known, or in
  novel, physiological pathways?
• In addition to growth and behaviour, are there
  other developmental processes and mechanisms in
  which imprinted genes have a decisive role, and
  how will these fit with evolutionary theories?