Transcript Document

Epigenetics
Heritable alterations in chromatin structure can govern gene
expression without altering the DNA sequence.
Viterbo
Università degli Studi della Tuscia
Epigenetics denotes all those hereditary
phenomena in which the phenotype is not only
determined by the genotype (the DNA
sequence itself) but also by the
establishment over the genotype
(in greek “epi” means “over”) of an
imprint that modulates its
functional behavior
Epigenetic phenomena
Genic, chromosome and genomic imprinting
Heterochromatin formation
Eukaryotes
Centromere function
Mammals
Polycomb group proteins
Drosophila
RNA interference (PTGS)
Transvection
Paramutation
RIP e MIP (Quelling)
Eukaryotes
Drosophila
Plants
Fungi
Vertebrates,
Invertebrates and Plants
Genic, chromosome or genomic
IMPRINTING
Sciara coprofila
x A
x A
x
zygote
x
embryo
x x AA
maternal genome
paternal genome
x x
Differential
behavior of
homologous
chromosomes
embryo
x
AA
The chromosome which passes through the male germ
line aquires an imprint that results in behaviour exactly
opposite to the imprint
conferred on the same chromosome by the
female germ line
(H. Crouse, 1960)
M P
Nuclear transplantation in
mammals
zygote
M
P
P
M
P
M
P
androgenetic embryos
(two male pronuclei)
Poor development of the embryo proper
M
gynogenetic embryos
(two female pronuclei)
Poor development of extraembryonic
components
Angelman, Prader-Willi
syndromes
• Usually caused by large (megabase+)
deletions of 15q11-q13
• Delete maternal chromosome = AS
• Delete paternal chromosome = PWS
–Prader-Willi Syndrome - obesity, mental retardation,
short stature.
–Angelman Syndrome - uncontrollable laughter, jerky
movements, and other motor and
mental symptoms.
PWS
AS
PWS
Mouse
model
AS
Mouse
model
Imprinting cycle
establishment, maintenance and erasure
What Mendel (fortunately) didn’t find in his experiments with peas
1:1
Does the genomic imprinting falsifies the Mendel’s rules?
NO
Neither the segregation of single gene alleles, nor the indipendent
behavior of different genes are affected by the existence
of imprinting
What the imprinting may mask are the dominance relations
between alleles, and hence only the phenotypic output of a cross
HETEROCHROMATIN
NUCLEATION AND MAINTENANCE
 In 1928, Heitz defined the heterochromatin as regions of chromosomes that
do not undergo cyclical changes in condensation during cell
cycle as the
other chromosome regions (euchromatin) do.
 Heterochromatin is not only allocyclic but also very poor of active genes,
leading to define it as genetically inert (junk DNA).
 Heterochromatin can be subdivided into two classes: constitutive
heterochromatin and facultative heterochromatin.
 Constitutive heterochromatin indicates those chromatin regions that are
permanently heterochromatic. These regions occupy fixed sites on
the chromosomes of a given species, are present in both
homologous chromosomes, throughout the life cycle of the
individual.
 Facultative heterochromatization is a phenomenon leading to the
developmentally or tissue-specific co-ordinate reversible
inactivation of discrete chromosome regions, entire chromosomes
or whole haploid chromosome sets.
Position Effect Variegation (PEV)
W+
W-
Drosophila melanogaster X chromosome
White+
pericentric
heterochromatin
W+
Y
inversion
White+
Wm4
Wm4
WWm4
Y

In all cases an inversion or translocation changed the position of
the gene from a euchromatic to heterochromatic position
this results in variegation

Some rearrangements gave large patches of red facets adjacent to
large patches of white
Conclusion: Decision on expression of white is made early during
tissue development and maintained through multiple cell divisions

Gene is not mutated – movement of the rearranged allele away
from heterochromatin can restore expression

PEV is not limited to Drosophila: see telomeric silencing in yeast
X chromosome inactivation
The Barr body
XY
XX
QuickTime™ e un
decompressore TIFF (LZW)
sono necessari per visualizzare quest'immagine.
XXXXY
XXXXX
XXX
In mammals the
dosage compensation
of the X chromosome
products, between XX
females and XY males
is achieved by
inactivating one of the
two Xs in each cell of a
female (Mary Lyon,
1961)
Genotype is Xyellow/Xblack
Yellow patches: black allele is inactive Xyellow/Xblack
Black patches: yellow allele is inactive Xyellow/Xblack
Coccid chromosome system
paternal chromosomes
maternal chromosomes
zygote
imprinted facultative
heterochromatization
embryo
Planococcus citri (2n=10)
embryo
Female and male cells from P.citri
PARAMUTATION
Alexander Brink
x
B-I
B’
x
B’/B-I*
B’/B-I
B-I
B-I*/B-I
MOLECULAR MECHANISMS
OF EPIGENETICS
The chromatin
nucleosomes
Histone protein
modifications
DNA
histones
DNA
modifications
HISTONE PROTEIN
MODIFICATIONS
Acetylation Phosforylation Methylation Ubiquitination
H3
H4
Me
…4K
Me
…9K
Me
…16KAc …20K
H2A
chromatin
H2B
Me
Me
16KAc
Me
4K
16KAc
16KAc
euchromatin
Me
Me
20K
9K
9K
Me
20K
4K
16KAc
Me
4K
Me
9K
Me
20K
heterochromatin
Me
9K
HP1 and modified histone tails interactions during
heterochromatin formation
chromatin
non histone chromatin
proteins: HP1
Me
9K
euchromatin
Me
9K
Me
9K
heterochromatin
Histone Code and Transcriptional Silencing
Epigenetic modifications leading to gene silencing.
(A) Gene repression through histone methylation.
Histone deacetylase deacetylates lysine 9 in H3,
which can then be methylated by HMTs. Methylated
lysine 9 in H3 is recognised by HP1, resulting in
maintenance of gene silencing.
B) Gene repression involving DNA methylation. DNA
methyltransferases methylate DNA by converting SAM
to SAH, a mechanism that can be inhibited by DNMT
inhibitors (DNMTi). MBPs recognise methylated DNA
and recruit HDACs, which deacetylate lysines in the
histone tails, leading to a repressive state.
(C) Interplay between DNMTs and HMTs results in
methylation of DNA and lysine 9 in H3, and consequent
local heterochromatin formation. The exact mechanism
of this cooperation is still poorly understood.
Histone Code and Transcriptional Activation
Epigenetic modifications leading to gene activation.
(A) Setting 'ON' marks in histone H3 to activate gene
transcription. Lysine 4 in H3 is methylated by HMT (for
example MLL) and lysine 9 is acetylated by HAT, allowing
genes to be transcribed. It is not known, if HMTs and HATs
have a direct connection to each other.
(B) In the postulated 'switch' hypothesis, phosphorylation
of serines or threonines adjacent to lysines displaces
histone methyl-binding proteins, accomplishing a binding
platform for other proteins with different enzymatic
activities. For example, phosphorylation of serine 10 in
H3 may prevent HP1 from binding to the methyl mark on
lysine 9. Other lysines in H3 may be acetylated by HATs,
therefore overwriting the repressive lysine 9 methyl mark
and allowing activation.
(C) Although there is no HDM identified to date, one can
speculate that, if this enzyme exists, serine 10 phosphorylation
in H3, for example, by Aurora kinases, can lead to recruitment
of HDMs that in turn demethylate lysine 9 in H3. Histone
acetyltransferases might then acetylate lysine 9 and HMTs
methylate lysine 4, resulting in the loosening of the chromatin
structure and allowing gene transcription.
Histone Modification Cassettes
Methylation of Lys-9 by DIM-5 (SUVAR39H1) recruits HP1 via its chromodomain.
In turn, HP1 can recruit additional SUVAR39H1 and other silencing proteins to
establish heterochromatin.
Phosphorylation of Ser-10 abolishes methylation of Lys9 by DIM-5 (SUVAR39H1) and
binding of the HP1, thereby blocking heterochromatin formation.
Phosphorylation of Ser-10 can modestly stimulate acetylation of Lys14 by GCN5,
thus promoting transcription.
Lys-9 and Ser-10 have been referred to as a methyl/phos switch:
Fischle W, Wang Y, Allis CD. Nature. 2003;425:475-9.
DNA MODIFICATIONS
Imprinting cycle/DNA metylation cycle
establishment, maintenance and erasure
Maternal genome
Paternal genome
zygote
m
m
m
maintenance
m
embryonic divisions
maintenance methylase
m
m
m
m
reversion
somatic cells
gametogenesis
demethylase
de novo methylase
de novo
establishment
m
m
m
m
gametes
Heterochromatin, HP1 and
histone tail modifications
dapi
HP1
Histone H3 lysine 9 methylation
m9KH3
Histone H4 lysine 20 methylation
dapi
m9KH3
HP1
merge
merge