Transcript Document

Epigenetics
Immunology of Histocompatibility
May 2nd 2006
Jonathan Duke-Cohan (DFCI/HMS)
This presentation draws heavily upon some superb images
prepared by others and freely available on the Web. In every
instance, the copyright of the original artists is acknowledged,
and the artwork is being used here solely for teaching
purposes.
To all the anonymous authors of these graphics, thank you!
Epigenetics
Epigenesis:
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the unfolding development of an organism, and in particular the
development of a plant or animal from an egg or spore through a
sequence of steps in which cells differentiate and organs form;
in contrast to “preformation” or the “homunculus” theory
Epigenetics:
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C.H. Waddington in 1942 defined it as “the branch of biology which
studies the causal interactions between genes and their products
which bring the phenotype into being”.
Epigenetic inheritance:
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Epigenetic inheritance is the transmission of information from a cell or
multicellular organism to its descendants without that information being
encoded in the nucleotide sequence of the gene.
Epigenetic phenomena
Example - Cellular proliferation:
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The progeny of a fibroblast cell division are fibroblasts
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The progeny of a stem cell may or may not be a stem cell
How?
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Transcriptional factor presence determined by parent cell?
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Mechanisms of maintaining gene repression/activation through
generations?
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Environmental cues? (e.g. hormones, cytokines, pO2, osmolarity)
Phenotype or “trait” is the end result, but we’re really talking
about inherited control of gene expression
Epigenetic phenomena
Example 2 – Chromosomal dosage and compensation
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Women are XX, men are XY
How are levels of all essential X-encoded gene products similar
between men and women if women have twice the number of alleles?
Mary Lyon – 1961
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in cells with multiple X chromosomes, all
but one is inactivated during mammalian
embryogenesis – the “Lyon effect”
X-inactivation; which X? Usually random
… but always paternal in marsupials
and variable in calico cats representing
regional expression of
differing pigmentation
genes on alternate X
chromosomes
Epigenetic phenomena
X-inactivation
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The repressed X-chromosome
condenses to form a Barr body
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In fact, 10-15% of repressed
alleles are active – to
compensate for recessive alleles
of sex-linked disorders including
haemophilia and colour blindness
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How does the cell “count” X chromosomes?
Epigenetics and Disease
Prader-Willi Syndrome
Angelman Syndrome
Beckwith-Wiedemann Syndrome
del-15q11.2-13
del-15q11.2-13
del-11p15.5
• Prader-Willi:
obesity, muscular hypotonia, mental retardation, short stature,
hypogonadism, small limbs
• Up to 4Mb deleted, primary genes affected are:
• SNRPN
(small nuclear ribonucleoprotein polypeptide N)
• NDN
• MKRN3
• IPW
(necdin)
(makorin)
(imprinted in Prader-Willi syndrome) (!!!)
but
• Deletions account for ~70%, and always of the paternal chromosome
• 28% are uniparental disomy*, always maternal, with NO genomic deletion
• <2% are small mutations on the paternal side, affecting the whole region
*usually follows trisomic rescue – 47 chromosomes in fertilised ovum, one lost on cell division.
(correction by two mistakes)
Epigenetics and Disease
Prader-Willi:
Independent of primary gene sequence, there are genomic modifications that
can be passed on from the parental environment – “Epigenetic marks”
Epigenetic marks
Modification of primary sequence
• CpG methylation
Modification of amino terminal of histones:
• Methylation of lysines/arginines
• Acetylation/ubiquitination of lysines
• Serine phosphorylation
DNA methylation
CpG – Cytosine phosphate Guanine
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Strongly represented in repetitive sequence associated with retroviralderived sequence
Can be methylated to generate 5-methylcytosine
Spontaneously deaminates to form thymine
Poorly recognised by DNA repair systems thus:
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CG→TG mutation is propagated
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CpG levels are less frequent than predicted 1/16
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May contribute to relative inactivity of retroelements
DNA methylation
CpG–islands
CpG frequency
CpG islands in Rb gene
(180kb)
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low frequency
70-80% methylated
promoter-related CpG are usually unmethylated
methylation-free state is essential for transcription of the
associated gene
DNA methylation
3 human DNA methyltransferases
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DNMT1
DNMT3A
DNMT3B
maintenance methyltransferases
de novo methyltransferases – highly expressed at embryo
implantation when waves of de novo methylation are occurring in
the genome
◄daughter strand
◄daughter strand
DNA methylation
CpG–island methylation – how does it affect transcription?
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methylated-DNA binding proteins (MECP2, methyl CpG
binding protein 2 ) bind to DNA
this recruits a complex of histone deacetylases and SIN3A
induces a closed chromatin structure → gene silencing
in contrast to usual deacetylation-related silencing, when
methylation is involved, it’s (almost) irreversible
gene
Histone modification
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active silenced
Histone modification
Mechanism:
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Acetylation of H3 or H4 leads to unfolding and accessibility of
chromatin (histone acetyltransferases)
Methylation of K4 of H3 = active gene expression
Methylation of K9 of H3 = gene silencing
Prader-Willi revisited….
• Presentation with no genomic deletion
• Uniparental maternal disomy
◄ paternal
◄ maternal
Imprinting centre
◄ maternal
◄ maternal
▼
◄ paternal
◄ maternal
and Angelman syndrome
• Presentation with no genomic deletion
• Uniparental paternal disomy
• Imprinting center defect
• leading to loss of maternal UBE3A function
• UBE3A paternal allele silenced in specific brain regions, maternal
allele active almost exclusively in hippocampus and cerebellum
▲
Epigenetics in embryonic development
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In each generation, the imprint inherited from the parent of the
opposite sex must be erased and re-established in developing germ
cells so that maternal or paternal imprint is appropriate for the sex of
the individual
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During early embryogenesis, a wave of demethylation followed by a
wave of sex-appropriate remethylation (“resetting”)
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Is “resetting” universal? And when does it occur?
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In spermatazoa, all potential methylation sites for SNRPN
and NDN are unmethylated
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In oocytes, near complete methylation of these genes at the
germinal stage and metaphases I and II
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~50% methylation in the pre-implantation embryo
Epigenetics in embryonic development
Implications for assisted reproductive technology (ART)?
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in vitro fertilisation by intracytoplasmic sperm injection: girls
maintained paternal imprint
New England Journal of Medicine 346: 725-730 (2002)
Hansen M, Kurinczuk JJ, Bower C, Webb S
The risk of major birth defects after intracytoplasmic sperm injection and in vitro
fertilization
Following
and
ICS, 26/301 (8.6%)
IVF, 75/837 (9.0%) have major birth defects
compared with 168/4000 naturally-conceived infants (4.2%)
ART often required for sperm malfunction, but Angelman (for example) which
has increased incidence following IVF/ICS is a result of loss of maternal
methylation? Increased risk more a reflection of in vitro culture effects?
X-inactivation revisited
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How does the cell “know” it has more than one X-chromosome, and
needs to inactivate one?
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At the four cell stage, the paternally-derived X chromosome is
inactivated. The extra-embryonic tissue ( →placenta etc.) retain this
imprinting i.e. only the maternal X is active
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In the early blastocyst, this initial imprinted paternal X-inactivation is
reversed (a wave of demethylation) and both X are active
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Random inactivation, maintained in all descendants of that cell. Thus
mosaicism in calico cats, reflecting X-linked heterozygosity
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In the female germline, all X-inactivation is reversed
X-inactivation revisited (continued)
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Initially hypothesised that there were limiting levels of a blocker –
enough to block one X but not enough to block more than this.
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But this cannot be so since in cells with more than two X
chromosomes, all except one were blocked
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XIC – the X inactivation centre
• required for X-inactivation
• introduction of this region to ANY chromosome leads to silencing
• encodes two genes: XIST, and TSIX
• XIST: X (inactive)-specific transcript
• TSIX: X (inactive)-specific transcript-antisense
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No XIST, no inactivation – encodes a large RNA, not protein
X-inactivation revisited (continued)
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XIST is the only gene expressed from the inactive X (Xi) and not
expressed from the active X (Xa)
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Initially, both X weakly express XIST, then on the future Xi, dramatic
upregulation, and on future Xa, no expression
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XIST coats the Xi chromosome, moving out from the XIC
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The activity of TSIX is reciprocal to that of XIST, expressed by Xa it
suppresses XIST – alleles bearing a deletion of TSIX are much more
likely to be inactivated
XIST: X (inactive)-specific transcript ►
◄TSIX: (inactive)-specific transcript-antisense