EPIGENETIC CONTROL OF GENE EXPRESSION

Introduction to the concepts of epigenetic control

-Epigenetics: extra layer of information in addition to the genetic information which must be
present to enable differentiation and development
-each cell type only expresses a restricted subset of genes
Ex: neurons make dopamine. Not hemoglobin or myoglobin
-how are only a restricted subset of genes expressed in each cell type?
-activity of transcription factors specific for each cell lineage. Transcription factors that are
specific to certain genes
-epigenetic marks
-epigenetic modifications can be considered as the punctuation marks in the genome
-demarcate the start and end of genes, provide structure to the chromosome, alter how we read
each gene, which leads to genes being expressed (active) or not expressed (silent/inactive) or
more subtle changes

Mitotic Heritability of Epigenetic Marks
-Epigenetic control is important throughout development, goes wrong in disease (can lead to early
embryonic lethality, placental insufficiency, germ cell tumors, tumorigenesis)
-identical twins are genetically identical, but can sometimes be epigenetically different
-identical twins can have different appearances, or both have a disease gene but only one has a
disease phenotype. Why? Is it due to environment (epigenetics)?
-Epigenetics definitions:
-Conrad Waddington: study of epigenesist; how genotypes give rise to phenotypes in
development
-Robin Holliday: temporal and spatial control of gene activity during development of complex
organisms
-EPIGENETICS IS THE STUDY OF MITOTICALLY HERITABLE CHANGES IN GENE
EXPRESSION THAT OCCUR WITHOUT CHANGES IN DNA SEQUENCE
-Mitotic heritability of epigenetic state helps to maintain cell identity. Epigenetic heritability
means that each cell will give the same epigenetic marks to its daughter cells which will lead to
tissue homogeneity.
-Heritability is countered by periods when epigenetic marks are removed.
-Epigenetic reprogramming in germ cells and early development.
-Active remodeling of epigenetic marks during differentiation

Chromatin and the Nucleosome
-Chromosome structure and specific epigenetic marks
-DNA exists wrapped around histone proteins. DNA+histones=chromatin, which allows DNA to
fit in the nucleus
-Packaging of DNA into a compressed form causes problems of accessibility for transcription,
replication and repair.
-Nucleosome = DNA + histones = 146 bp of DNA wrapped around a histone octamer (2 H2A, 2
H2B, 2H3, 2H4 and kept together by histone H1)
-Histones are positively charged (lots of lysine and arginine). Positively charged histones bind to
negatively charged DNA. N-terminal histone tails protrude from the octamer.

Chromatin Compaction-Heterochromatin versus Euchromatin
-Nucleosome chromatin is further packaged into a 30nm fiber, because the H1 histones interact
and fold together. This fiber then attaches to scaffolding proteins, which condenses the DNA
even more. This is how the DNA is found in interphase, when the cell is not dividing. In
metaphase, the scaffolding proteins are condensed again, which makes the chromosomes as
condensed as they can be in preparation for cell division.
-Euchromatin: open chromatin, less stained
-heterochromatin: closed chromatin, densely stained
-facultative: can differ by cell type or time (ex: tissue specific genes, inactive X chromosome)
-constitutive: same in all cell types. Performs a structural role (ex: centromeres, telomeres,
portions of Y and X chromosomes)
-heterochromatin functions: gene silencing (facultative), structural integrity of the genome
(constitutive)
-different epigenetic marks are associated with euchromatin and heterochromatin
-active histone tail modifications, and inactive histone tail modifications/DNA methylation

DNA methylation at CpG Islands
-Specific epigenetic modifications:
-DNA methylation: addition of methyl group on the DNA at cytosine. Added on to the 5
Carbon. Occurs almost exclusively on C followed by G (called CpG dinucleotide), because it is
pretty symmetric. DNA methyltransferases (DNMT3a/b) lay down the methylation marks during
division. When DNA is replicated only the parent strand has the methyl group. Then DNMT1
comes in, finds the hemi-methylated DNA (when one strand is methylated and one is not) and
methylates the daughter strand.
-CpGs clustered into CpG islands, often at promoters of genes (the start of the gene, where
transcription factors bind). CpG islands tend to be protected from methylation, but when CpG
islands are methylated, they almost always silence the gene. Some CpG islands are methylated
and therefore silenced, generally in X inactivation.
-How does DNA methylation lead to silencing? Methylated CpGs are associated with chromatin
condensing, (meCP1/2), methylated CpG is bound by methylated CpG binding proteins, and they
have a transcriptional repression domain, and because methylated CpG will stop a transcription
factor from binding (this is less common).

DNA methylation at intergenic regions and repetitive elements
-Intergenic regions and repetitive elements are often methylated.
-DNA methylation is mutagenic. CpGs are under-represented in the genome because methylated
C is prone to deamination to T.
-What is the function of DNA methylation at intergenic regions? Maybe to maintain genomic
integrity. Methylation can help with genomic mutations (deletions, insertions) and can silence
cryptic transcription stat sites or cryptic splice sites
-What is the function of DNA methylation at repetitive elements? Probably to maintain genomic
integrity. Silences repeats to prevent transposition, mutates the repeats (methylated C to T) to
prevent transposition, silences repeats, which helps avoid transcriptional interference from strong
promoters, methylates repeats which can help improper recombination
-Genome defense model: if DNA methylation is mutagenic, the evolutionary benefit must be to
protect the genome from transposable elements
-DNA methylation and cancer. In cancer, there is a genome wide lack of methylation at repetitive
elements and sometimes too much methylation at CpG islands.
-DNA methylation is mitotically heritable and originally thought to be irremovable, except by
failure to maintain methylation by DNMT1
-DNA demethylation shown to occur in early development, in primordial germ cell development
and at later specific stages of differentiation.
-Around year 2000, shown this could happen without DNA replication so could also be an active
process.
-Passive DNA demethylation: dilution of DNA methylation with every cell division, when
DNMT1 is not expressed or not in the nucleus.
-Active DNA demehtylation: not simple removal of methyl group (C-C bond is very hard to
remove). Enzymatic removal via intermediates, using multiple different systems. TET and AID
proteins main players.
DNA METHYLATION:
-occurs at CpG dinucleotides in mammals
-associated with gene silencing when found at promoters
-helps to maintain genomic stability
-laid down by DNA methyltransferases
-mitotically heritable, due to features of DNMT1
-can be removed passively, or actively which involves TET proteins
-essential for viability, DNMT knockouts die in utero


GET WEEK 2 INFORMATION

History and Background of X chromosome inactivation
-Dosage compensation in mammals, worms, flies
-1949: Barr and Bertram found structure at nuclear periphery in female, but not male interphase
nucleiBarr body=heterochromatin. In 1959 Ohno found that the Barr body is the inactivated X
chromosome. In 1961 Lyon hypothesized that this X chromosome was inactivated randomly for
dosage compensation.
-normal diploid female has XX and 44 autosomes
-normal diploid male has XY and 44 autosomes.
-Trisomy has XXX, Klinefelter has XXY, tetraploid has XXXX
In females you have 1 inactive X, in males you have none. Trisomy people have 2 inactive Xs,
klinefelters have 1 inactive X, and in tetraploid female cells there are 2 inactive Xs. So Lyon
hypothesized that for every diploid set of autosomes, only 1 X can stay active. This doesnt
change due to sex or whether there is a Y chromosome.
-random X inactivation occurs at gastrulation in the embryo, then this epigenetic state is
mitotically inherited by daughter cells
-Gartler found that cancer is clonal because all the tumor cells had the same X inactivated,
showing that they all came ultimately from the same cell

Timing of random and imprinted X chromosome inactivation
Random X inactivation
-occurs in embryo proper around gastrulation (epiblast)
-inactive X can be either maternal or paternal copy, but once established is maintained in daughter
cells
Imprinted X inactivation
-Paternal X chromosome is selectively silenced (between 2-4 cell stage)
-Occurs in pre-implantation embryos and extra-embryonic tissues (placenta)
-Occurs in all cells of marsupials, argued to be the more evolutionarily more primitive mechanism
-trophectoderm (makes the placenta) maintains the imprinted X inactivation (paternal X is
silenced), but the blastocyst (makes the adult) becomes the gastrula and has random X
inactivation. This means the blastocyst has to remove the imprinted inactivation and randomly
inactivate one. So as a blastocyst there are 2 active Xs, and as primordial germ cells X
inactivation is again cleared.

Stages of X inactivation-counting and control of Xist expression
1. Counting of X chromosomes, find the X: autosome ratio to determine if
inactivation is required. Very little is known, there are controversial theories.
Consensus is that there are both X-linked and autosomal factors that determine the
X:A ratio, and the balance is what allows the cell to find the ration. Deletion of X-
linked regions (non-coding RNAs) near to Xist leads to aberrant numbers of inactive
X chromosomes, so they must be X-linked factors. Autosomal factors are still
unknown for the counting process.
2. Choice of which X chromosome to inactivate
3. Initiation of inactivation on chosen X chromosome, Xist expression from XIC (X
inactivation center)
4. Spreading of inactivation in cis along the chromosome
5. Establishment of inactivation, turning the Xist signal into silence
6. Maintenance of the inactive X, stably through the life of the cell and its progeny,
clonal cell populations
X inactivation is accomplished via a progressive accumulation of epigenetic marks on the inactive
X chromosome
-Counting and choice are about getting Xist expressed and the last stages turn the expression of
Xist into transcriptional silence
-Xist is the critical determinant for X inactivation
-17 kb, spliced and polyadenylated, constrained to the nucleus and non-coding (no protein
product)
-expressed from just one of the 2 X chromosomes, then it coats that chromosome
-expression is the first detectable event in X inactivation
-determines the chromosome that will become the inactive X
-Xist RNA coats the inactive X in cis
X Inactivation Center (XIC)
-XIC is necessary and sufficient for X inactivation
-all elements appear to be non-coding RNAs that regulate Xist
-Tsix, DXPas34, Xite and Tsx are involved in repressing Xist
-Ftx and Jpx are involved in activating Xist
-Rnf12 is a few hundred kb upstream of the XIC and it is subject to X inactivation, and encodes a
protein Rnf12 or Rlim (same protein, different names). There is a threshold level required of
Rnf12 that is required for X inactivation to proceed. Once X inactivation starts, the other X is not
inactivate, as insufficient Rnf12 to overcome the threshold. But, this doesnt explain everything
because if you completely delete Rnf12, inactivation still occurs (poorly).
-Rnf12 is an activator of Xist
-Rnf12: activates Xist, perhaps directly and inactivation ensues
-male cells have insufficient Rnf12 to activate Xist
-female cells where X inactivation has occurred, one copy of Rnf23 is silenced, so wont
accidentally silence second X chromosome
-Rnf12 must be involved in counting the number of X chromosomes per cell via its own
expression

Control of Xist expression by pluripotency factors
Control of Xist expression:
-Long, noncoding RNAs encoded within the XIC
-Rnf12, X-linked gene which encodes a protein activator of Xist
-pluripotency factors: help maintain the pluripotent state of embryonic stem cells and other
pluripotent cells. Ex: primordial germ cells (have 2 active X chromosomes)
-Pluripotency factors silence Xist expression by:
1. Repressing Xist expression directly, by binding Xist intron 1
2 Activating Tsix via binding DXPas 34 and Xite, Tsix represses Xist
3. Repressing Rnf12 as Rnf12 activates Xist
Upon differentiation, pluripotency factors are reduced, relieving the silencing of Xist

Stages of X Inactivation-choice of which X to inactivate
Choice: which X will express Xist?
-X chromosomes transiently pair via the XIC and surrounding regions followed by Xist transcript
stabilization on a single X chromosome
-transient pairing may allow exchange of information to allow choice (exchange of bound factors)
-pairing is critical for counting and choice
-ectopic pairing via autosomal multicopy transgenes of pairing region
-ectopic pairing out-competes X:X leading to failure of Xist upregulationfailure to inactivate
Skewed choice and skewed inactivation
-studies in mice show genetic differences in a region near to the XIC (called XCE) result in
skewed X inactivation (preference to inactivate one X chromosome over the other
-skewed X inactivation has consequences for many diseases (X-linked disorders) for example,
Rett syndrome: an X-linked neurodevelopmental disorder caused by mutation in a methyl binding
domain protein (MeCP2) which is X-linked. This is lethal in males before birth, but heterozygous
females survive. Phenotype of female Rett syndrome patients is variable, dependent not only on
the specific mutation, but also on skewing of X inactivation. If the mutated chromosome is
silenced more/less often, it will cause the cell to be diseased more/less often respectively.

Stages of X inactivation-initiation and spreading of silencing
-Initiation requires Xist expression and coating of the chromosome
-Xist expression remains throughout the life of a cell and continues to coat the inactive X
chromosome
-X inactivation is initially Xist dependent and reversible, but over developmental time becomes
fixed and more reversible, therefore: Xist is required to initiate, with only a minor role in
maintenance of X inactivation
-limited number of cell types capable of initiating X inactivation, even with Xist expression.
There are other factors involved in enabling heritable, transcriptional silencing
Theories of how Xist coats the whole inactive X chromosome:
-Progressive recruitment into transcriptionally silent compartment: early event after Xist
expression is exclusion of RNA Pol II and silencing of repeat rich portions of the X chromosome.
Formation of silent nuclear compartment occurs before gene silencing. As inactivation spreads, it
coincides with genes moving into this silent compartment
Genes that escape X inactivation:
-differing number of escape genes between species
-escapees cluster in pseudo-autosomal regions, shared with Y chromosome that allow XY pairing
-escapees reside outside the silent nuclear compartment
-double dose of non-pseudo-autosomal escapee genes in females vs. males. Could this be a
function in evolution of differences between the sexes?

Stages of X inactivation-establishment of silencing
Earliest events after Xist expression and creation of the transcriptionally silent compartment are
alterations of the epigenetic marks on the X chromosome. You lose active histone marks and
accumulate repressive histone marks.
-Xist recruits PRC2 via A-repeats, catalyzes H3k27me and recruits PRC1 which catalyzes
H2AK119Ub
Repeat A (RepA) is required for the silencing function of Xist. It binds the histone
methyltransferases

Stages of X inactivation-maintenance of silencing (Dnmt1 and Smchd1)
-imprinted and random X inactivation have variations in requirements for maintenance. Bothe
require progressive layering of redundant mitotically heritable epigenetic marks, to lock in
epigenetic silencing.
-factors involved in maintenance identified by reactivation of a previously silent X chromosome
when the factor is removed
Dnmt1, DNA methylation and maintenance of XCI
-Dnmt1 is the maintenance methyltransferases
-DNA methylation is one of the final steps of random XCI, locks in the silent state
-Used Dnmt1 knockout compared to Dnmt wildtype embryos. Used X-linked lacZ transgene,
makes blue pigment when exposed to specific substrate. Sectioned embryo, looked for proportion
of blue cells in embryo (random XCI) and the placenta (for imprinted XCI)
-Dnmt1 is required to maintain random but not imprinted X inactivation. Imprinted X
inactivation is more labile than random, perhaps since DNA methylation does effectively lock in
the silent state
-Smchd1 is bound to the inactive X chromosome at E13.5, well into the maintenance phase of X
inactivation.
-Smchd1 null females die at around E11, approximately 4-5 days after the initiation of X
inactivation. Both imprinted and random X inactivation are abnormal, accompanied by placental
defect. Xist expression and PRC2 recruitment is normal. Little DNA methylation of the inactive
X and failure of transcriptional silencing on inactive X
- X green fluorescent protein (GFP) transgene expression in Smchd1 mutant female embryos.
Forced inactivation of the paternal X by Xist knockout, so GFP is not expressed
-Smchd1 is required for maintenance of both imprinted and random X inactivation
-X reactivation upon reduction of Smchd1, Dnmt1, MacroH2A and other factors in differentiated
cells:
-Mouse embryonic fibroblasts (MEFs) from E13.5 embryos) the maintenance phase of XCI)
-XGFP on inactive X chromosome
-microRNAs against Smchd1, Dnmt1 or MacroH2A results in GFP expression i.e. X reactivation
only when inhibit DNA methylation further with drug 5-azacytidine which inhibits Dnmt1
-several layers of epigenetic marks ensuring heritable epigenetic silencing

X-chromosome Inactivation Summary
Replication timing:
-temporal segregation of replication for euchromatin or heterochromatin
-likely relates to ability to transfer epigenetic marks onto newly formed chromatin
-specific histone modifier enzyme complexes or histone deacetylases only associate with the
replication fork in late S (synthesis) phase
-heterochromatin and Xi is replicated late in S phase
-euchromatin is replicated early in S phase
-XCI is initiated by Xist, a long non-coding RNA expression, which coats and silences in cis
-The XCI pair, which influences the choice of which X will upregulate Xist
-Xist creates a transcriptionally repressive nuclear compartment via nuclear reorganization,
involved in spreading and silencing
-Xist recruits various epigenetic modifiers, histone variants and chromatin proteins which result in
a progressive accumulation of repressive epigenetic marks on the inactive X to lock in the
inactive state

Dosage Compensation in flies and worms, compared with mammals
-Mammals: inactivation of one X in females, upregulation of active X in males and females.
Upregulation (controversial) involves a long noncoding RNA (called XACT), histone
acetyltransferase (homolog of same fly HAT)
-Worms: inactivation of both X chromosomes in hermaphrodites by half (one regular sized X, vs.
2 half-sized Xs). Involves SMC proteins, condensins or condensing-like normally involved in
chromosome condensation in mitosis. Smchd1 involved in mammalian dosage compensation is an
SMC protein
-Flies: upregulation of single X chromosome in males (one huge X, vs. 2 smaller Xs). Involves
long, noncoding RNAs, roX1 and roX2. Involves histone acetyltransferases and RNA/DNA
helicase. Involves creation of an active nuclear compartment, with an enrichment of transcription
factories
-Dose of X-linked genes between males and females, but also relative to autosomes. Why
downregulate/inactivate and upregulate single active X in mammals?

Position effect variegation and screening for epigenetic modifiers
-position effect variegation (PEV): position of a gene relative to heterochromatin results alters
expression of the gene. Variegated expression: mosaic expression of a gene i.e. sometimes the
gene is on and sometimes off within the same tissue
Heterochromatin spreading:
-common feature of heterochromatin in all organisms
-spreading of heterochromatin is not limited to pericentromeric heterochromatin, occurs for
telomeres, repetitive elements etc.
-heterochromatin spreading is limited by boundary elements

-screening for genes involved in epigenetic control in the fly
Using mammalian variegation to find mammalian genes involved in epigenetic control:
-transgene variegation in mice is an epigenetic effect
-GFP transgene, directed to express in red blood cells
-known (fly homologs) and novel proteins identified, novel proteins are unique to higher
organisms and involved in higher organism specific processes (ex: XCI)

Epigenetic Reprogramming of the maternal and paternal genomes
-Epigenetic Reprogramming
-clearing of epigenetic marks in germ cell development and early embryonic development
-disruption of epigenetic reprogramming in IVF, somatic cell nuclear transfer
-reprogramming of somatic cells to induced pluripotent stem cells
-Genomic Imprinting
-parent of origin specific gene expression
-reprogramming of imprinted genes (by bisulfite sequencing)
-imprinting disorders
Epigenetic marks need to be cleared between generations to restore totipotency
-Two phases of epigenetic reprogramming
1. Pre-implantation period , early development: within 6 hours after fertilization the
paternal genome is rapidly and actively demethylated. The maternal genome is gradually
and passively demethylated. Both genomes have most demethylation at the blastocyst,
quickly are remethylated after the blastocyst
2. Primordial germ cell development. Both the paternal and maternal genomes are
demethylated at mostly the same rate, but are remethylated as primordial germ cells at
different rates because eggs take slightly longer to mature

Epigenetic reprogramming of imprinted genes and repetitive elements
Epigenetic reprogramming
-clearing and resetting of epigenetic marks in PGC (primordial germ cell development) and early
embryonic development
-paternal and maternal genomes treated differently
-other specific regions of the genome undergo variant epigenetic reprogramming
-repetitive elements ex: IAPs
-imprinted genes
Repetitive elements are spread throughout the genome, generally you want them to be silent so
as not to cause trouble. They tend to maintain a high level of methylation throughout the periods
of epigenetic reprogramming.
Imprinted genes undergo reprograming in PGC, but not in early development. They are cleared
a bit after the rest of the genome, but are restored at essentially the same rate.
-Genomic imprinting: monoallelic gene expression (i.e. only one of the 2 alleles is
expressed), allele specific expression, based on parent-of-origin of the allele (ex: paternal
allele is always expressed and maternal is always silenced). Known to be critical for
embryo viability, shown by creating embryos with 2 paternal or 2 maternal genomes which
die before implantation. You need to have epigenetic differences. Expression of imprinted
genes is controlled by imprint control regions (ICRs). The imprint itself is associated with
DNA methylation at the ICR. The way each ICR brings about imprinted gene expression
differs by locus (long noncoding RNAs, enhancer/insulator blocking). ICR methylation
established in primordial germ cells to achieve parent-of-origin specific marks, maintained
in the embryo.

Location of Imprinted genes in the genome and bisulfite sequencing
~150 imprinted genes in humans
-imprinted genes exist in clusters; each cluster has its own ICR
-unlike X inactivation, imprinted gene expression for a particular cluster is not always found in
all tissues, despite presence of ICR methylation
-imprinted gene expression common in the placenta and the brain
How is DNA methylation measured?
-bisulfite sequencing: bisulfite chemical conversion of DNA, to differentiate C from
me
C.
amplification of DNA by PCR. Clonal sequencing of products. Add bisulfite to DNA and
5-methylcytosine stays the same, but cytosine changes to uracil. Then amplify it by PCR,
and see how much of the DNA is methylated (stayed as 5-methylcytosine) and how much
became uracil (was originally unmethylated cytosine)
-sequencing: isolated each DNA fragment by inserting into plasmid backnone (clones), then
sequence each clone. This allows for identification of polymorphisms that allow
determination of which allele is being examined (paternal or maternal). Each allele is
treated differently, advantage for parental imprinting or XCI, for example

Kcnq1 and H19/lgf2 ICR mechanisms of action and Beckwith Weidemann syndrome
How do imprint control regions (ICRs) bring about imprinted gene expression?
-ICR DNA methylation does not necessarily correlate with silencing of gene expression
-methylation of an ICR will tell you if its maternally or paternally imprinted, but not
whether its active or not
-Mechanism of action of the ICR varies with each cluster:
-Kcnq1 cluster, human chromosome 11-long noncoding RNA
-H19/lgf2 cluster, human chromosome 11-enhancer blocking
-Beckwith WIedemann syndrome: Kcnq1 and H19/lgf2 clusters
-Snrpn cluster, human chromosome 7-long noncoding RNA
-Angelman and Prader-Willi syndromes
Kcnq1 locus: controlled by long noncoding RNA in cis
-ICR forms part of the promoter for long noncoding RNA Kcnq1ot1
-Methylation of ICR on maternal allele silences Kcnq1ot1
-Kcnq1ot1 long noncoding RNA recruits G9a and PRC2 that perform H3k9me and
H3K27me, silencing in cis
-compare with Xist
-Cdkn1c is maternally expressed, cell cycle regulator, involved in growth restriction
H19/lgf2 cluster: enhancer blocking
-CTCF is an insulator protein, insulates lgf2 from downstream enhancers
-DNA methylation at ICR blocks binding of CTCF binding
-Without CTCF, DNA methylation spreads to H19 promoter to silence and enhancers can
access Igf2 to activate
Beckwith Wiedemann syndrome
-imprinted disorder, results from various abnormalities inlinked Kcnq1 and H19/Igf2
clusters 11q15.5. Ex: loss of Cdkn1c (tumor suppressor, growth suppressing)
-caused by maternal allele behaving like paternal allele
-mutation/deletion to cause loss of imprinting
-uiparental disomy, two copies of one parental chromosome, here the paternal UPD
-Epigenetic disruption for loss of imprinting (rare)
-Fetal and post-natal overgrowth (loss of Cdkn1c, overdose Igf2 and/or other genes)
-macroglossia: large tongue
-Predisposition to embryonic/childhood tumors, but not adult tumors
-many imprinted genes are involved in growth. Loss of imprinting is a common feature of
cancer
-Maternal transmission from carrier mother to affected offspring (50% of cases since one
mutant allele)
-Parent-of-origin specific inheritance, not sex-specific
ICRs have different modes of action, which control parent-of-origin expression at the imprinted
cluster
Loss of DNA methylation at ICRs is common in cancer
Aberrant expression of imprinted genes results in specific diseases

Snrpn ICR mechanism, Prader Willi and Angelman syndromes
How do imprint control regions (ICRs) bring about imprinted gene expression?
-mechanism of action of the ICR varies with each cluster
Snrpn cluster, controlled by lon noncoding RNA
-Prader-Willi syndrome/Angelman syndrome cluster
-Imprinted expression of Ube3a only present in the brain
-split imprinting center
-PWS-IC methylation determines Snurf/Snrpn lon noncoding RNA expression, produces
snoRNAs and Ube3a-antisense
-Snurf/Snrpn only extends to Ube3a-as in the brain
-Different mechanism for long noncoding RNA, appears true at other imprinted clusters
with long noncoding RNAs
-AS-IC allows establishment of DNA methylation at PWS-IC in oocytes
-AS-IC is not differentially methylated between parental alleles
Angelman syndrome: brain growth retardation, microcephaly, severe mental retardation, puppet-
like, jerky arm movement, seizures
-caused by failure to express Ube3a. commonly caused by deletion or inappropriate silencing of
the maternal copy of 15q11-13 (similar to BWS, maternal transmission)
-deletion that removes AS-IC, leaves PWS-IC means PWS-IC fails to be methylated in oocytes,
somatic tissue have unmethylated PWS-IC on both alleles, no UBE3A expression in the brain
-Caused by failure to express UBE3A
-can result from uniparentaly disomy, specific mutation in UBE3A, failure to methylate
PWS-IC (epigenetic defect)
Prader-Willi syndrome: low muscle tone, failure to thrive as infants, small, hypogonadism,
mental retardation, obsessive compulsive behavior, usually over-eatingobesity, paternally
transmitted, now treated with growth hormone for children, then caloric restriction
-commonly caused by deletion or inappropriate silencing of the paternal copy of 15q11-13
-leads to no expression of paternally expressed genes within this cluster (snoRNAs have a
role in determining brain phenotype)
-deletion of PWS-IC leads to maternal-like allele (snurf/snrpn start site removed)
-Each cluster has at least one imprint control region (ICR), which displays differential
methylation established in PGCs
-mechanism of action of ICR differs
-DNA methylation to silence a long noncoding RNA that binds and recruits epigenetic
modifiers in cis
-DNA methylation to silence a long noncoding RNA whose antisense transcription, or the
act of transcription is required to silence
-DNA methylation as a means of blocking insulator factor binding
Imprinted disorders are associated with misexpression of genes within specific imprinted
clusters, parent of origin dependent inheritance, genetic (deletion, mutation, uniparental disomy)
or epigenetic basis of disruption to imprinted gene cluster

Disrupted epigenetic reprogramming
-assisted reproductive technologies (ART): IVF, Intracytoplasmic sperm injection (ICSI)
-somatic cell nuclear transfer for cloning
-reprogramming of somatic cells to induced pluripotent stem cells
-Not yet entirely clear the effect of ICSI or IVF on epigenetic reprogramming. Many studies
show an increase in BWS and AS, particularly following ICSI
These cases of imprinting disorders result from epigenetic abnormalities
-angelman syndrome and Beckwith Wiedemann syndrome-both maternally transmitted
-imprinting disorders due to epigenetic anomalies are very rare (1/300,000) and increased 3-5
fold, the absolute risk is still very low even in ART children
-broader epigenetic abnormalities not necessarily disease-specific
What is the role fo the fertility defect in producing any anomaly? Maternal age, underlying
defect that causes fertility problems?
-related epigenetic abnormalities in non-humans (cattle, mice) following ART performed for
husbandry reasons or experimentally. Is it a procedural issue? Is it due to disruption during
sensitive periods of epigenetic reprogramming (early development and PGC/GC development)?
These stages are also very important in ART. The eggs might be removed before they are fully
mature, before they get epigenetic marks. Altered maternal effect proteins due to oocyte harvest
and erosion of DNA methylation imprints in early development?

Disrupted epigenetic reprogramming in somatic cell reprogramming and cloning
Epigenetic reprogramming in somatic cell nuclear transfer (cloning). Cloning is performed for
husbandry reasons. Very low efficiency (few %) production of live births. Large offspring
syndrome-placental and fetal overgrowthimprinting defect, due o disrupted epigenetic
reprogramming?
-imprinted genes contribute to growth, especially in the placenta
-somatic nucleus hasnt been through PGC epigenetic reprogramming, ICR not reset, and
germ cell genome packaging is not achieved
-ICR methylation eroded during early development reprogramming, lack of maternal effect
proteins binding ICRs?
-PGC clearing and resetting is totally skipped
-Oocyte cytoplasm attempts to allow early developmental reprogramming, but this occurs in
culture (cf IVF, ICSI)
Genome-wide transcriptional differences, even in surviving clones, indicative of broad=scale
epigenetic abnormalities even in clones deemed successful.
Increased efficiency of cloning with compounds that alter epigenetic state (i.e. inhibit the
epigenetic machinery)
Somatic Cell reprogramming
-production of an embryonic stem cell-like pluripotent cell from a somatic cell, induced
pluripotent stem cells (iPS cells)
-therapy: patient-derived source of stem cells, in vitro differentiation into different
organs/cells
-ethics: not embryo derived, couldnt form an embryo on their own (cant make placenta)
-epigenetic landscape, need to:
-remove lineage-specific epigenetic marks
-restore pluripotent epigenetic marks
-restore open pluripotent chromatin state
-retain imprints at ICR
-remove XCI (females)
-some cell lineages more readily reprogrammed, likely related to flexibility of epigenetic
marks
-Reprogramming achieved with a combination of transcription factors, decreases the barrier, still
very inefficient (few % cells)
-reprogramming efficiency can be improved by adding or removing particular epigenetic
enzymes

-Periods of epigenetic reprogramming (early development, primordial germ cell development)
are sensitive periods
-alterations or removal of the normal process result in epigenetic abnormalities, particularly
aberrant imprinting
-modifications to procedures are improving the efficiency of epigenetic reprogramming in all
these scenarios

Introduction of transgenerational epigenetic inheritance
Influence of the environment on epigenetic control
-sensitive periods of exposure, requirement for mitotic heritability
-diet, maternal care, chemical exposure
Transgenerational epigenetic inheritance through the gametes
-examples and potential mechanisms
Important questions:
1. What proportion of the genome is sensitive to the environment?
2. What proportion of people are sensitive to environmental disruption?
3. What proportion of changes are meiotically or mitotically heritable?
Environmental effects on epigenetic control:
-sex determination in turtles is not genetic, but temperature dependent
-vernalisation (cold) to allow flowering of some plants (bulbs)
-diet: Worker bees and queen bees are genetically identically, royal jelly causes the
difference between workers and queens, it must be an epigenetic difference
-Sensitive periods when altered environments have an effect on epigenetic control
-transgenerational epigenetic inheritance definition and considerations
Transgenerational epigenetic inheritance through the gametes
-inheritance of phenotypes or gene expression patterns from parent to offspring by passage
through the germ cells, not explained by genetic differences
-suggests epigenetic marks are transmitted from parent to offspring
-incomplete epigenetic reprogramming?
-messenger molecule transmitted in the gametes causing altered reestablishment of
epigenetic marks?
-distinction from effects not transmitted via gametes, brought about due to altered placenta,
mothering style, newborn nutrition via milk

The Dutch famine human epidemiological studies
Human epidemiological studies on diet-Dutch famine
-1944-1945. There was a German blockade of food supplies into western Netherlands, followed
by harsh winter meaning access was cut, average 580 calories per adult per day towards the end
of the famine. Ended with liberation in May 1945, Sweden gave food
Dutch famine Birth cohort study:
-exposure to famine during peri-conceptional period lead to increased mental and metabolic
disorders/altered glucose tolerance in the offspring leading to increased diabetes, obesity
and cardiovascular disease
-no such defects when children of the same parents were conceived in periods not subject to
famine, or when famine was later in gestation
-sensitive periods during embryogenesis/gametogenesis, when marks are established rather than
maintained
-increased disease propensity associated subtle changes in DNA methylation of a small number
of genes IGF2, GNAS and MEG and other metabolic genes i.e. some evidence of epigenetic
alterations and at some sensitive regions similar to IVF data
Original data suggested transgenerational effect of Dutch famine, but follow up, better controlled
data suggests there is no transgenerational aspect
-Barker hypothesis: used to explain the extensive and permanent effects of the in utero/neonatal
environment on adult health and disease
-dutch famine: under-nutrition and low birth weight correlates with obesity, type II diabetes,
CVD in adults
-data for hypertension
-effects exacerbated when food is plentiful in adults
-thrifty phenotype hypothesis: programmed to store energy if exposed to low food supply in
utero/early life
-proposed to have an epigenetic basis

Human epidemiological studies on diet
Overkalix study:
-correlation between increased grandparental food supply and a decrease in grandchilds
longevity
-sensitive period for food supply is slow growth period
-age 9-12 for grandfathers
-age 8-10 in grandmothers, plus fetal/infant life for grandmothers
-grandparental effects were sex specific
-paternal grandfathers food supply linked to grandsons mortality (CVD, diabetes)
-paternal grandmothers food supply linked to granddaughters mortality
Complicated, confounded by sex-specific parental effects and grandparental effects, not all seen
in all cohorts
-no current replication data (ongoing in Britain and elsewhere) or molecular data
Do grandparental effects due to some environmental change mean transgenerational epigenetic
inheritance through the gametes?
-lack of molecular data or controls (cross-breeding, control for genetic alterations or timing
of effects)
-consider exposure of germ cells
Grandmaternal effects:
-Effect must pass an additional generation to be potentially transgenerational epigenetic
inheritance through the gametes for grandmaternal effects
Grandpaternal effects:
-Only one subsequent generation altered in grandfather (germ cell development)
-grandpaternal effects on grandsons-Y chromosome? Genetic?
Human studies on paternal effects due to environment: prepubertal smoking
-onset of paternal smoking during prepubertal stage (slow growth period) is linked to
increased BMI in sons (not if smoking started later)
-germ cell epigenetic marks (Y chromosome?) potentially compromised by exposure
-complicated as smoking has mutagenic effects
Transgenerationl epigenetic inheritance through the gametes in humans is very difficult to study
because:
-underlying genetic differences may account for altered epigenetic state and re-establishment of
different epigenotypes
-formal proof of epigenetic inheritance via the gametes studies are not feasible
-often difficult to study the relevant cell type.
So, most studies are epidemiological in nature. If this is true, large impact on our interpretation
of inheritance of phenotypic traits

Mouse and rat studies on paternal effects of chemical exposure
Vinclozolin: used as fungicide in fruit and vegetable production, wine making. Its an endocrine
disruptor, anti-androgenic effect (works against androgens)
If you expose a pregnant mother to vinclozolin during mid-gestation pregnancy, when PGCs of
embryos are developing, 90% of male offspring have low fertility rates, up to F3, F4
F3 females also display phenotypes, so its not just spermatogenesis that is sensitive to
vinclozolin. Similar results with methoxychlor (a pesticide that replaced DTT, an estrogenic
endocrine disruptor)
Effects are only passed down the male line-why?
-DNA methylation differences observe in the sperm, possibly responsible for
transgenerational effects
Vinclozolin appears to have genetic and epigenetic effects, since appears to promote at least one
copy number alteration (insertion/deletion). Effects of vinclozolin are dependent on strain
background of mice, i.e. underlying genetic factors required for sensitivity. This suggests thtat
there is some genetic basis for your subjectivity to environmental factors
Epigenetic alteration by rat maternal behavior:
-llifelong alterations in the nature of stress response
-cross-fostering experiments show not a genetic difference and not establilshed in the
gametes
-due to epigenetic changes at the glucocorticoid receptor on the hippocampus
Overall considerations:
-period of exposure-sensitive periods, germ cell exposure, exposure after birth
-mitotic heritability of any epigenetic difference brought about by environmental change is
required to see a sustained alteration
-what proportion of the genome is sensitive to environmental change (unknown, being
studied)
-what proportion of the population is genetically susceptible (unknown, being studied)
-could genetic alterations be responsible
-what proportion of changes may be transgenerationally heritable? (hard to study in humans)

Transgenerational epigenetic inheritance via the gametes
-Linaria vulgaris heritable epimutation. 2 flower types described in 1744. No genetic difference
found, in 1999 underlying epigenetic cause was found: heritable DNA methylation at Lcyc
-mammalian examples of transgenerational epigenetic inheritance via the gamets
-First observed for transgenes, more often for those derived from foreign DNA, bacterial
sequences
-compare with imprinted genes that are inherited in a particular epigenetic state, but reset
each generation (not transgenerational)
-via the gametes, so phenotype not established during embryogenesis or after birth
-when is the phenotype established?
-examine effect of uterine environment
-cross-fostering if phenotype is established late, to take into account mothering stype or
parental genetics
Why should we discriminate between genetic and epigenetic inheritance?
-consequences on our interpretation of genetic information and heredity
-consequences for analysis of mechanism

The Agouti viable yellow allele in mice
-mouse: metastable epialleles
-Agouti viable yellow (A
vy
) allele (also consider environmental influence
-Axin fused (Axinfu) allele
-mechanism of transgenerational epigenetic inheritance?
-incomplete clearing?
-messenger molecules, paramutation-like effects in mice
-humans?
-overkalix
-constitutional epimutation of Mlh1?
Agouti viable yellow allele is caused by an IAP ( intracisternal A particle, repetitive element that
jumps in upstream), that is sensitive to epigenetic state.
-agoutiswitch from dark to yellow pigment production (++)
-constitutive agouti yellow coat, obesity and type II diabetes
-variable expressivitydifferent phenotypes in the context of isogenicity
-mosaic expression results in variegated, mottled coat color, relates to expression state set
during gastrulation, cf calico cat
Variegation and variable expressivity of Avy produces a spectrum of phenotypes, due to
stochastic establishment of epigenetic statee
-epigenetic state established in early development is set for the life of that organism due to
mitotic heritability
-alleles of this kind are termed metastable epialleles
A
vy
allel inheritance patterns:
-Paternal transmission: clearing and resetting of epigenetic marks between generations. It
doesnt matter what color the father is, the offspring are statistically the same to have each color
-Maternal transmission: some memory of the phenotype and epigenotype of the mother. Why?
Intrauterine environment? Yellow mothers are obese and diabetic. When is the effect
established? Different behavior of the mother?
Separate experiments ruled out contributions due to differences in oogenesis in the yellow
female, before fertilization/zygote transfer. A
vy
allele is not altered by intrauterine
environment. So we know that the effect is due to the gametes. A
vy
allele displays
transgenerational epigenetic inheritance through the gametes

Environmental effects on the Agouti viable yellow allele
Altered maternal diet effects on Avy allele.
-effect seen when mother does not carry the allele
-early development (E0.5-8.5) sensitive period-when epigenetic reprogramming occurs,
importantly epigenetic marks are reestablished post-implantation
-preconception is also a sensitive period-alteration to oogenesis?
-mother carries the allele no change in her coat color
-period of establishment of epigenetic marks, rather than maintenance of epigenetic marks,
is sensitive to these dietary changes
-folate choline, betaine, vitamin B12, ethanol act as methyl donors or alter methionine/1 carbon
cycle, these factors increase S-Adenosyl methionine (SAM), the molecule from which the methyl
group is donated for methylation of DNA, dietary supplements appear to have subtle site specific
effects (like at Avy) and probably subtle global effects
-effects of folate supplementation in humans (?) prevent spina bifida
-Genistein (compound found in soy protein) alters DNA methylation at Avy in all germ layers, -
indicating early embryonic effects. It is a phyto-estrogen (plant derived estrogen), effects appear
independent of SAM, so not all dietary effects are just via alteration of methyl donor availability
-BPA (bisphenol A) found in many polycarbonate plastics. Effects of BPA on human health is
controversial, it is anti-androgenic (endocrine disruptor), effects can be counteracted with methyl
donors or genistein
Diet during (early) pregnancy can alter epigenetic makeup and adult health, due to mitotic
heritablilty of altered epigenetic state
-sensitive periods of exposure during establishment of epigenetic marks
-diet can alter aviailability of methyl donors, alter endocrine factors (endocrine disruptors)
or act via unknown mechanisms

The Axin fused allel in mice and metastable epialleles