Transcript Epigenetik: Von der Genomforschung zur Epigenomforschung

Epigenetik:
Von der Genomforschung zur
Epigenomforschung
4362
4355
Exploring the hierarchy of epigenetic mechanisms
Chromosome territories,chromatin fibers and domains,
interchromatin compartment,
topology of active and silent genes
Histone modifications
and chromatin remodelling
DNA methylation
Horn and Peterson, 2002
2050
Metaphasechromomen aus einer diploiden,
menschlichen Zelle in der Mitose
0777
2531
2531
733
2520
Fluoreszenz in situ Hybridisierung (FISH)
1. Herstellung von Metaphase-Präparaten aus
Vollblutkulturen
2. Herstellung der DNA-Sonde
Markieren der Sonde mit Reportermolekülen
(Biotin, etc.) bzw. Fluoreszenzfarbstoffen
3. Denaturierung der Sonden- und Ziel-DNA
ZielDNA
Sonden
4. Hybridisierung der Sonde an die komplementären
Ziel-DNA Sequenzen; bei indirekter Markierung
anschließende Immunodetektion
Sonde
ZielDNA
5. Visualisierung
der Fluoreszenzsignale
3D map of chromosome territory arrangements in
postmitotic human fibroblast cell nuclei (G0)
24 color painting
of all chromosome
territories in male
diploid human
fibroblast nucleus
“true color“
image (RGB)
classification
(M-FISH
3D-program)
Bolzer et al, 2005
2192
the DNA sequence level
the textbook story
• linear array of sequence
elements
– transcription units
– promoters, enhancers,
silencers
• sequences guide the
machineries for
transcription
X
AACGAGATCGTTCGAA
X
AACGAGATCGTTCGAA
Heinrich Leonhardt
DNA Methylierung
Heinrich Leonhardt
Heinrich Leonhardt
de novo methylation
Heinrich Leonhardt
Transcriptional repression through DNA methylation
Heinrich Leonhardt
the chromatin
level
histone modifications
• methylation,
acetylation,
phosphorylation,
ubiquitinylation
chromatin remodelling
• ATP-dependent
shifting
of nucleosomes
Modification sites at the N-termini of histone tails
Modifications above an amino acid are linked to transcriptionally active chromatin
Modifications below an amino acid are linked to transcriptionally inactive chromatin
K= Lysin, R= Arginin, S= Serin,
Note that methylation can exist in a
mono-, di- or trimethylated state
Currently used antibodies against histone modification sites
Mono-methyl H3-K9
Transcriptionally
active (eu)chromatin
Mono-methyl H4-K20
Unknown (active chromatin?)
Acetyl-H4-K8
Transcriptionally
active (eu)chromatin
Tri-methyl H3-K27
Facultative Heterochromatin
Tri-methyl H3-K9
Tri-methyl H4-K20
Transcriptionally repressed and
Constitutive (hetero)chromatin
H3/H4-multi-methyl
lysine (MML) sites
Mostly repressed chromatin
Histone Acetylation Enzymes
Lysine
in histone:
cell membrane
N
O
C C g
a b
C
C
C
d
O
O
e
C
N+
O
[3H]-Ac
C
O
O
Acetyl-CoA
HAT (Histone
HDAC
AcetylTransferase)
C
-
DNA
P
O
(Histone
DeACetylase)
CoA
N
Uncharged
Acetyl-Lysine
in histone
C
O
C
C
C
C
e
C
C
N
C
O
Jakob Waterborg
[email protected]
Function of Histone Acetylation
30 nm
11 nm
HIGH in histone H1
Often low in histone H1
+
charged
histone
tail
acetylated
histone tail
Jakob Waterborg
[email protected]