Epigenetics Heritable changes in gene expression that do not involve changes in gene sequence

1
Epigenetics - Heritable changes in gene
expression that do not involve changes in gene
sequence Mediated by changes in chromatin
structure, which influence gene expression DNA
methylation generally (but not always!)
associated with silenced chromatin Histone
acetylation generally associated with open or
active chromatin Histone deacetylation and
histone methylation associated with HP1 protein
binding and silenced chromatin
Image from Pennisi 2000 Science 2931065
2
Genomic imprinting Maternal and paternal
genomes are not equivalent in development Isopare
ntal embryos in mice gynogenotes (2 ? genomes)
?normal embryo underdeveloped
placenta androgenotes (2 ? genomes) ?
overdeveloped embryo placenta Uniparental
disomies for some (but not all) chromosomes
result in developmental abnormalities similar to
gyongenotes or androgenotes
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Genomic Imprinting - Parent-of-origin
differences in gene expression Maternally
inherited allele silent paternally inherited
allele expressed Paternally inherited allele
silent maternally inherited allele
expressed Note its the expression not the
transmission of alleles that is affected in
imprinting Leads to functional haploidy for
imprinted genes A limited number of genes
exhibit parent-of-origin effects often genes
involved in embryonic growth and
development Observed in plants (primarily
endosperm) and mammals
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• Genomic imprinting example
• Prader Willi Syndrome (PWS) Anglemans Syndrome
  (AS) locus
• (Human 15q11 q13)
• PWS patients
• Inherit 15q11q13 deletion from paternal parent
• Have only maternal copy
• Retardation, sluggish, overweight
• AS patients
• Inherit 15q11q13 deletion from maternal parent
• Have only paternal copy
• Retardation, hyperactive, seizures, thin
• What conclusion can be drawn about parental
  requirements for the expression of this locus?

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Imprinting mechanisms at the PWS/AS
region (Modified from Walter and Paulsen Seminars
in Cell Developmental Biology 14101)
Fig. 3. Regulation of imprinting in the PWS/AS
region. Indicated by different colouring, the PWS
and AS ICs possess different epigenetic
conformations on the parental alleles. Thereby,
the two ICs act in a allele-specific way. On the
maternal allele, the AS IC activates maternally
expressed genes (green arrows) but represses
function of the PWS IC, and thereby paternally
expressed transcripts (red line). Oppositely, on
the paternal allele, paternally expressed
transcripts are activated by the PWS IC (green
arrows). Note the paternally expressed antisense
UBE3A transcript that might silence the sense
expression of paternal UBE3A .
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Imprinting mechanisms at the PWS/AS
region (Modified from Walter and Paulsen Seminars
in Cell Developmental Biology 14101)
Results in only the paternal expression pattern
missing maternally expressed information AS IC
identified by micro deletions that condition AS
when inherited through the maternal parent
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Imprinting mechanisms at the PWS/AS
region (Modified from Walter and Paulsen Seminars
in Cell Developmental Biology 14101)
Deletion of PWS IC
Deletion of PWS IC
Results in only the maternal expression pattern
missing paternally expressed information PWS IC
identified by micro deletions that condition PWS
when inherited through the paternal parent
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Features of the PWS/AS locus shared with other
imprinted loci Complex! Cluster of genes,
including paternally and maternally imprinted
genes Cis-acting imprinting control (IC)
regions Antisense expression of silenced alleles
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Parent-of-origin imprints must be re-set each
generation (from Walter and Paulsen Seminars in
Cell Developmental Biology 14101-110) e.g. A
simple maternally expressed locus (no mutations)
Fig. 1. Imprinting switching in female and male
germ lines. The expressed maternal allele is
shown in red, whereas the silenced paternal
allele is shown in blue. The maternal and
paternal imprints are erased in premature germ
cells (gray bars) and are subsequently replaced
by new parent-specific imprints that result in
allele-specific expression in the offspring.
10
Parent-of-origin imprints must be re-set each
generation (adapted from Walter and Paulsen
Seminars in Cell Developmental Biology
14101-110) Failure to reset imprints
Fig. 1. Imprinting switching in female and male
germ lines. The expressed maternal allele is
shown in red, whereas the silenced paternal
allele is shown in blue. The maternal and
paternal imprints are erased in premature germ
cells (gray bars) and are subsequently replaced
by new parent-specific imprints that result in
allele-specific expression in the offspring.
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Epigenetics, development and nuclear
cloning (Reik et al. 2001. Science 293 1089
Rideout et al. 2001. Science 2931093)
Development of multicellular organisms Almost
all somatic cells have identical genetic
information Cell and tissue type is determined
by expressing a subset of that information and
silencing the rest Transformation of a
single-cell zygote to multicellular individual
occurs via a cascade of gene expression highly
regulated through space and time Chromatin of a
somatic cell is extensively remodeled compared to
that of a zygote
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Epigenetics, development and nuclear
cloning (Reik et al. 2001. Science 293 1089
Rideout et al. 2001. Science 2931093)
Chromatin remodeling in development Setting the
stage ... Extensive DNA de-methylation
immediately after fertilization DNA
re-methylation begins about the time of embryo
implantation Parent-of-origin imprints, set in
the gametes, are erased only in primordial germ
cells Parent-of-origin imprints are retained in
somatic tissues to direct some aspects of
development As the developmental program unfolds
... Embryonic stem cells chromatin retains
capability to express wide range of developmental
options Mature somatic cell chromatin has been
re-modeled to restrict developmental / gene
expression options
13
Epigenetics, development and nuclear
cloning (Reik et al. 2001. Science 293
1089 Rideout et al. 2001. Science 293)
primordial germ cells ?
mitosis spermatogonia / oogonia ?
mitosis primary spermatocyte / oocyte
? meiosis sperm / egg ?
fertilization zygote ? mitosis early
embryo ? mitosis ? ? germ
cells stem cells ?
mitosis adult somatic cells
? Parent-of-origin imprints erased
? Parent-of-origin imprints set
? Extensive de-methylation (Parent-of-origin
imprints retained)
? DNA re-methylation begins
? Chromatin plasticity
? Chromatin restricted
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Epigenetics, development and nuclear
cloning (Reik et al. 2001. Science 293 1089
Rideout et al. 2001. Science 2931093)
Nuclear cloning Enucleated mature oocyte
introduce a somatic or embryonic stem cell
nucleus Activate development with electric
current Implant embryo into surrogate
mother Relatively low success rate many births
with developmental abnormalities Donor chromatin
must be remodeled in the oocyte Developmental
abnormalities look like parent-of-origin
imprinting errors suggesting the imprints, which
should remain in place, are improperly altered /
remodeled in the mature oocyte
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Paramutation - Change in expression of one
allele brought about by association with another
allele
A paramutable allele after association with a
paramutagenic allele becomes a parmutant
allele Paramutant allele is often stable through
one or more subsequent generations Paramutant
allele becomes itself paramutagenic Neutral
allele is neither paramutable nor
paramutagenic Most alleles are neutral!
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Paramutation at the B locus of maize (Chandler
and Stam, Nature Rev Genet 5536)
The b1 locus encodes a basic helixloophelix
transcription factor that activates the
anthocyanin biosynthetic pathway and results in
the presence of purple pigment throughout the
plant92. Two b1 alleles are involved in
paramutation, B' and B-I these two variants have
a 1020-fold difference in transcription, but do
not differ in DNA methylation in the
promoter-proximal or coding regions80. The
paramutagenic B' state arises spontaneously from
the unstable, highly expressed paramutable B-I
state at frequencies of 0.11032. Unlike B-I, B'
is extremely stable.
The figure illustrates paramutation at b1 B-I
(dark purple plant) is always changed to B'
(light purple plant) in B'/B-I heterozygotes. The
newly paramutated allele, B', is
indistinguishable from the B' parent it always
paramutates B-I in subsequent crosses (as shown
in the photos). Fine-structure recombination
mapping delimited a 6-kb region, which is located
100 kb upstream of the b1 transcription start
site, that was required for paramutation and B-I
enhancer activity24. In this region, B-I and B'
have seven tandem repeats of an 853-bp sequence
(black arrows) that is otherwise unique in the
genome neutral alleles only have one copy of
this sequence. The repeats are required for both
paramutation and high levels of transcription.
DNA sequences are identical in B-I and B',
indicating that epigenetic mechanisms mediate the
stable transcriptional silencing that is
associated with b1 paramutation. The tandem
repeats are differentially methylated and have
greater DNaseI hypersensitivity in B-I relative
to B'. The sequences required for paramutation
are indicated in the figure by red rectangles.
The B' and B-I states are represented by
different numbers and sizes of ovals to symbolize
the distinct epigenetic states within the tandem
repeats.
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Using genetics to identify cis sequences
important for paramutation (Stam et al. 2002
Genetics 162-917-930)
Used outside markers to identify recombinants in
the region upstream of the b locus that changed
the parmutagenic B allele into a neutral allele
(B-Peru) Found a region 93-106 kb upstream of b
locus transcription initiation site regulates
paramutation in cis Cis element has seven tandem
repeats of an 853 bp repeat in B and B-I (i.e.
no difference between paramutable and
paramutagenic allele) Neutral alleles have only
one copy of the repeat Hypermethylation of
repeats and open chromatin structure associated
with active expression (transcription) of
B-I Hypomethylation of repeats and closed
chromatin structure associated with repressed
transcription of B
18
Models for paramutation in maize (Chandler and
Stam, Nature Rev. Genet. 5532)
For simplicity, only allele interactions are
illustrated, but these models could also explain
interactions between non-allelic homologous
sequences. a Pairing model for trans-induction
of chromatin changes. Pairing between repeats is
hypothesized to reduce gene expression by
changing the subnuclear localization of the
repeats, establishing a distinct chromatin
structure, or both. b RNA-mediated
trans-induction of chromatin. Two mechanisms are
illustrated. 1) Long non-coding RNAs from one or
both strands are postulated to induce altered
chromatin in cis and in trans. 2) A second
possibility is a role for small interfering RNA
(siRNA). The dsRNA that is formed by
transcription from the two strands of the
repeated DNA is a target for Dicer, which
produces siRNA (dashed lines in figure). The
siRNA is then postulated to mediate chromatin
changes, which in turn alters the expression of
the adjacent gene. Possible mechanisms include,
but are not limited to, RNA-directed DNA
methylation40, 41, 97, 98 and RNA-directed
histone modification8, 49, 99. RdRP activity,
which synthesizes complementary strand RNA using
siRNA primers, results in increased amounts of
siRNAs from throughout the repeats
19
Using genetics to dissect paramutation (Dorweiler
et al. 2000 Plant Cell 122101-2118 Alleman et
al. Nature 442295)
B/B X B-I/B-I gt all F1 progeny B/B
(efficient paramutagenic conversion of B-I to
B) Self pollinate F1 progeny, expecting all
B/B (extreme stability of B alleles once
created) Look for exceptions to stability F2
families with ¼ intensely colored seedlings (due
to loss of unlinked function needed to maintain
paramutation, B changing back to
B-I) Identified one mutant mop1 (modifier of
paramutation) Disrupts paramutation at other loci
(r and pl ) as well as b Recently cloned by
positional approaches encodes an RNA dependent
RNA Polymerase (RdRP) favors RNAi models
20
RNA-based gene silencing (RNAi)
Best elucidated in C. elegans (Fire et al. Nature
391 806) Transcriptional gene silencing Post-tran
scriptional gene silencing Interference with
protein translation General mechanisms widely
conserved (but not in S. cerevisiae) Many paths
of entry Likely evolved as a defense mechanism
against viruses and transposons
Silencing of a green fluorescent protein (GFP)
reporter in C. elegans occurs when animals feed
on bacteria expressing GFP dsRNA (a) but not in
animals that are defective for RNAi (b). Note
that silencing occurs throughout the body of the
animal, with the exception of a few cells in the
tail that express some residual GFP. The signal
is lost in intestinal cells near the tail
(arrowhead) as well as near the head (arrow). The
lack of GFP-positive embryos in a (bracketed
region) demonstrates the systemic spread and
inheritance of silencing. (Mello Conte, Nature
431338)
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Mechanisms of RNAi (Mello Conte, Nature 431338)
In some cases dsRNA functions as the initial
stimulus (or trigger), for example when foreign
dsRNA is introduced experimentally. In other
cases dsRNA acts as an intermediate, for example
when 'aberrant' mRNAs are copied by cellular
RdRP. Transcription can produce dsRNA by
readthrough from adjacent transcripts, as may
occur for repetitive gene families or high-copy
arrays (blue dashed arrows). Alternatively,
transcription may be triggered experimentally or
developmentally, for example in the expression of
short hairpin (shRNA) genes and endogenous
hairpin (miRNA) genes. The small RNA products of
the Dicer-mediated dsRNA processing reaction
guide distinct protein complexes to their
targets. These silencing complexes include the
RNA-induced silencing complex (RISC), which is
implicated in mRNA destruction and translational
repression, and the RNA-induced transcriptional
silencing complex (RITS), which is implicated in
chromatin silencing. Sequence mismatches between
a miRNA and its target mRNA lead to translational
repression (black solid arrow), whereas near
perfect complementarity results in mRNA
destruction (black dashed arrow). Feedback cycles
permit an amplification and longterm maintenance
of silencing. CH3, modified DNA or chromatin
7mG, 7-methylguanine AAAA, poly-adenosine tail
TGA, translation termination codon.
22
Mechanisms of RNAi
Double stranded RNAs via _ Encoded inverted
repeats, shRNAs, miRNAs _ Cellular or
viral-encoded RNA dependent RNA polymerase
(RdRP) _ Antisense transcripts Double-stranded
RNAs cleaved to short interfering RNAs (siRNA) or
mature miRNAs by RNaseIII type enzymes
(Dicer) siRNA/miRNA guides RNA induced
silencing complex (RISC) to cleave target RNAs
(post-transcriptional silencing) miRNA guides
RISC to inhibit translation of target RNA
(translational silencing) siRNA/miRNA guides
chromatin methylation/modification
(transcriptional silencing)
23
Mechanisms of RNA-based gene silencing
Transcriptional silencing siRNA can mediate
methylation and chromatin changes in the nucleus
to achieve transcriptional silencing Viroids
(non-coding RNAs with secondary structure)
can mediate methylation of viroid- homologous
transgenes (Wassenegger et al. Cell
76567) Argonaute, Dicer and RdRP mutants
disrupt methylation and silencing of centromere
sequences, and consequently disrupt centromere
function in S. pombe (Volpe et al. 2003.
Chromosome Res ii137-146).
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Gene silencing as a defense mechanism

• Post-transcriptional gene silencing, cytosolic
  defense against RNA viruses (Vance and Vaucheret,
  2001. Science 2922277)
• In plants an RNA-based silencing signal can move
  systemically
• Many examples of transgene transcripts that
  protect against plant virus infection
• Two different plant viral suppressors of
  post-transcriptional gene silencing have been
  discovered
• Transcriptional gene silencing, nuclear defense
  against transposable elements
• (Hirochika et al. 2000. Plant Cell 12357)
• Introduced tobacco Tto1 retrotransposon into
  arabidopsis
• _Transposed and increased in copy number
• _ Eventually became methylated and transposition
• ceased
• Introduced the ddm1 mutation into the transposon
  silenced line
• _ the ddm1 mutation disrupts methylation
• _ Tto1 became de-methylated
• _ Active transposition resumed