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Epigenetik:
Von der Genomforschung zur Epigenomforschung
4362
4355
Horn and Peterson, 2002
Exploring the hierarchy of epigenetic mechanisms
DNA methylation
Histone modificationsand chromatin
remodelling
Chromosome territories,chromatin fibers and domains,
interchromatin compartment,topology of active and silent genes
2050
0777
Metaphasechromomen aus einer diploiden, menschlichen Zelle in der Mitose
2531
2531
733
2520
Fluoreszenz in situ Hybridisierung (FISH)
2. Herstellung der DNA-Sonde 1. Herstellung von Metaphase-Präparaten aus Vollblutkulturen
3. Denaturierung der Sonden- und Ziel-DNA
4. Hybridisierung der Sonde an die komplementären Ziel-DNA Sequenzen; bei indirekter Markierung anschließende Immunodetektion
Markieren der Sonde mit Reportermolekülen (Biotin, etc.) bzw. Fluoreszenzfarbstoffen
Ziel-DNA Sonden
Ziel-DNA
Sonde
5. Visualisierung der Fluoreszenz-signale
3D map of chromosome territory arrangements in
postmitotic human fibroblast cell nuclei (G0)
“true color“ image (RGB)
classification(M-FISH 3D-program)
24 color painting of all chromosome territories in male
diploid human fibroblast nucleus
Bolzer et al, 2005
2192
the DNA sequence levelthe textbook story
• linear array of sequence elements– transcription units
– promoters, enhancers, silencers
• sequences guide the machineries for transcription
X
X
AACGAGATCGTTCGAA
AACGAGATCGTTCGAA
mm
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
shiftingof nucleosomes
K= Lysin, R= Arginin, S= Serin,
Modification sites at the N-termini of histone tails
Modifications above an amino acid are linked to transcriptionally active chromatinModifications below an amino acid are linked to transcriptionally inactive chromatin
Note that methylation can exist in a mono-, di- or trimethylated state
Mono-methyl H3-K9 Transcriptionally active (eu)chromatin
Mono-methyl H4-K20 Unknown (active chromatin?)
Tri-methyl H3-K27 Facultative Heterochromatin
Tri-methyl H3-K9Tri-methyl H4-K20
Transcriptionally repressed and Constitutive (hetero)chromatin
Acetyl-H4-K8 Transcriptionally active (eu)chromatin
H3/H4-multi-methyl lysine (MML) sites
Currently used antibodies against histone modification sites
Mostly repressed chromatin
Histone Acetylation Enzymes
HDAC(HistoneDeACetylase)
HAT (HistoneAcetyl-
Transferase)
Acetyl-CoA
CoA
O
N
C
C C
C
C
C
N
O
C
CUnchargedAcetyl-Lysinein histone
O
N
C
C C
C
C
CN+
OO
O
P
O
-
Lysinein histone:
DNAcell membrane
[3H]-Ac
O
C
C
O-
Jakob [email protected]
Function of Histone Acetylation
30 nm
11 nm
HIGH in histone H1 Often low in histone H1
acetylatedhistone tail
+chargedhistone
tail
Jakob [email protected]
