EpigeneticsHeritable alterations in chromatin structure can govern gene

expression without altering the DNA sequence.

ViterboUniversità 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

Centromere function

RNA interference (PTGS)

Paramutation

RIP e MIP (Quelling)

Polycomb group proteins

Transvection

Plants

Vertebrates,

Invertebrates and PlantsEukaryotes

Mammals

Drosophila

Drosophila

Fungi

Eukaryotes

Genic, chromosome or genomic

IMPRINTING

Differentialbehavior of homologous chromosome

s

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)

embryo

x AAx

x xx

maternal genomepaternal genome

zygote

xxAA

xSciara coprofila

embryo

x AA

androgenetic embryos(two male pronuclei)

Poor development of the embryo proper

MM

PP

M P

zygote

gynogenetic embryos(two female pronuclei)

Poor development of extraembryoniccomponents

PM

M

P

Nuclear transplantation in

mammals

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

PWSMousemodel

ASMousemodel

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?

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

NO

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)

inversion

White+ Wm4

Wm4

W-

Wm4

Y

White+

pericentricheterochromatin

Drosophila melanogaster X chromosome W+

W-

W+

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

QuickTime™ e undecompressore TIFF (LZW)

sono necessari per visualizzare quest'immagine.

XY XX XXX

XXXXY XXXXX

The Barr body

X chromosome inactivation

Genotype is Xyellow/Xblack

Yellow patches: black allele is inactive Black patches: yellow allele is inactive

Xyellow/Xblack

Xyellow/Xblack

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)

imprinted facultative heterochromatization

Coccid chromosome system

embryo

zygote

maternal chromosomes paternal chromosomes

embryo

Planococcus citri (2n=10)

Female and male cells from P.citri

B-IB’

B’/B-I* B-I

x

x

B’/B-I B-I*/B-I

PARAMUTATIONAlexander Brink

MOLECULAR MECHANISMS

OF EPIGENETICS

The chromatin

DNA

histones

nucleosomes

DNA modifications

Histone protein modifications

HISTONE PROTEIN

MODIFICATIONS

Acetylation Phosforylation Methylation Ubiquitination

H3

H4

H2A

H2B

euchromatin heterochromatin

chromatin

…20KMe

20KMe

20KMe

20KMe

4KMe

4KMe

4KMe

…4KMe…9K

Me

9KMe

9KMe

9KMe

9KMe

…16KAc

16KAc

16KAc16KAc

16KAc

chromatin

HP1 and modified histone tails interactions during heterochromatin formation

euchromatin heterochromatin

9KMe

9KMe

9KMe

non histone chromatin proteins: HP1

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 Silencing

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 Code and Transcriptional Activation

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 toestablish heterochromatin.

Phosphorylation of Ser-10 abolishes methylation of Lys9 by DIM-5 (SUVAR39H1) andbinding 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 cycleestablishment, maintenance and erasure

somatic cells

maintenance embryonic divisions

Maternal genome Paternal genome

zygote

gametes

gametogenesisreversion

de novo establishment

mm

maintenance methylase

mm

m mmm

m m

demethylasede novo methylase

m m

dapi

m9KH3

HP1

merge

Heterochromatin, HP1 and histone tail modifications

Histone H3 lysine 9 methylation

dapi

m9KH3

HP1

merge

Histone H4 lysine 20 methylation