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DNA methylation basics
Acetylation
Acetyltransferases/Deacetylases
Transcription Regulation And Gene Expression in Eukaryotes
Cycle G2 (lecture 13709), P. Matthias, April 20th, 2016
Epigenetics & Chromatin
TFIIB
RNAPII
TFIIATFIID
TFIIH TFIIFTFIIE
The Players in Transcription Regulation
• DNA-binding transcription factors (upstream factors)• Chromatin regulators• Coactivators and corepressors: Mediator, etc..• Basal Machinery: RNA PolII, GTFs
Gene
BasalMachinery
DNA-binding TFs
Chromatin regulatorsMediator
Co-activator
Wikipedia
DNA methylation basics
DNA methylation is usually associated with gene inactivity
Methyl-C, the 5th base
B. Lewin, Genes VIII
B. Lewin, Genes VIII
Maintenance of DNA methylation
Distribution of methyl groups in the genome: CpG islands
Distribution of methyl groups in the genome: CpG islands
CpG islands are not methylated
Genes & Dev, 2: 1127 (1988)
Genes & Dev, 3: 612 (1989)
24/31 DECEMBER 2015 | VOL 528 | NATURE | 575
Cell, Vol. 64, 1123-1134, March 22, 1991,
DNA methylation and demethylation
TET: Ten eleven translocation
7th and 8th base
5-formylcytosine5-carboxylcytosine
3. Mai 2016
HDAC
HAT
Sparmann A et al., 2006
Chromatin structure and histone modifications in eukaryotes
NaButyrate-treated
Control
H3, H4 mononucleosomes can be acetylated
NaButyrate-treated
+DNAseI
NaButyrate-treated
H3, H4 mononucleosomes
Ratio H4-3/H4-0 = 0.9
Ratio H4-3/H4-0 = 1.9
TJ Richmond, Nature 1997 vol. 389 (6648) pp. 251-60
Reversible Lysine acetylation
The effects of Histone lysine acetylation
P. Jenö, Lecture 4610
In-gel assay
Histones BSA
Tetrahymena crude Macronuclear activity
Some of the chromatin-binding domains present in HAT complexes in yeast
HATs: Complexes and single proteins
More HATs: CIITA, TFIIB, CLOCK,…
Histone Acetyltransferases (HATs): GNAT family
Histone Acetyltransferases (HATs): MYST family
Some HAT inhibitors: anacardic acid, curcumin, garcinol…
Elp3: HAT of GNAT family
P. Jenö, Lecture 4610
Some HDAC inhibitors
SAHA/VorinostatZolinza®
TSA
RomidepsinIstodax®
HDAC inhibitors:
TSA, SAHA, VPA, LBH …
HDAC inhibitors as drugs
Cancer,
Leukemia,
PolyQ disorders (neurodegeneration),
Autoimmune and inflammatory diseases,
…
3. Mai 2016
Acetylation of histones, transcriptional regulators and other
targets
Cell cycle, Apoptosis, Autophagy,
M tilit
Hundreds of clinical trials
HDACs
MOA: “The mechanism of the antineoplastic effect of Vorinostat/ Romidepsinhas not been fully characterized”
“We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275.”
Widespread lysine acetylation
HDACis in the food: broccoli sproutsDietary compounds: Na-butyrate, diallyl disulphide, sulphoraphaneFound at high levels in cruciferous (Brassicaceae) vegetables
Human healthy volunteers
Human colon cells
Seminars in Cancer Biology 17 (2007) 363-369
Classical Zn-dependent HDACs
NAD-dependent HDACs: Sirtuins
Rajundran et al., JBB 2011, doi:10.1155/2011/368276
Ahringer (2000). NuRD and SIN3. Trends Genet. 16, 351-356.
NuRD and Sin3 Complexes
NuRD Sin3
HDAC1 and 2 complexes
• The PAH domains are suggested to be organized into two a-helices separated by a flexible spacer region
• They are among the most evolutionarily conserved regions of the large SIN3 proteins (100–170 kDa)
• These domains are important for Sin3 function through their independentassociations with various repressors and other associated proteins
• PAH2 is both necessary and sufficient for interaction with the Mad proteins
HDAC Interaction Domain
Paired Amphipathic Helix (PAH)Domains
Sin3 schematic structure
Sin3 schematic structure
Comprehensive HDAC interaction network.
Joshi P et al. Mol Syst Biol 2013;9:672
©2013 by European Molecular Biology Organization
Phenotypes of class I HDACs mouse knockouts
Phenotypes of class I HDACs mouse knockouts
Reichert, Choukrallah and Matthias, CMLS, 2012 DOI 10.1007/s00018-012-0921-9
ClassII HDACs as “signal transducers”
ClassII HDACs as “cytoplasmic regulator”: HDAC6
Cre transgenic mouse Cre
Cell type specific expression
HDAC1
HDAC2
482 aa
489 aa
Exon 6HDAC domain
HDAC1HDAC2
KYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYYFPGTGDLRKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYYFPGTGDLR
Hdac2 floxed mouseExon 14Exon 1
Hdac1 floxed mouseExon 6 Exon 15Exon 1
Essential for catalytic activity** *
Conditional inactivation of HDAC1 and HDAC2 in specific lineages
Residue specificity for HDAC1 and 2
p21 and p57 are upregulated in HDAC1 and HDAC2 deficient cells
HDAC1HDAC2
days0 0.5 1 2 3 4 5
HDAC2 Kd cells HDAC1/2 KO/Kd cells
p21
Days afterTamoxifen:
Actin
0
1
2
3
4
5
6
7
8
9
10
0 1 1 2 3 4 5-0,1
0,1
0,3
0,5
0,7
0,9
1,1
1,3
1,5
HDAC-1p21
Upregulation of p21 and p57 in MEFs lacking HDAC1 and 2
Yamaguchi et al., G&D 24: 455-469
Direct regulation of the p21 and p57 genes
Yamaguchi et al., G&D 24: 455-469
3. Mai 2016Orkin & Zon, Cell 132, 2008
Mb1-Cre
HDAC inactivation in the B cell lineage
B cells are almost lacking in vivo after deletion of HDAC1 and 2
Early B cell development
Bone Marrow Spleen, periphery
Plasma cell
V(D)J recombination
Pre-BCR BCR
VJ recombination
mb1-Cre
CD4
CD
8
T cells
T cells
Spleen cellsSpleen cells
WT
B220
IgM
Mature Bcells
HDAC1/2 DKO
Mature Bcells
Differentiation
Bone Marrow cells
B220
IgM
Pro-pre B cells
B cells
B cells
Pro-pre Bcells
HDAC1/2 dKO mice have strongly reduced B cell populations
B cells are almost lacking in vivo after deletion of HDAC1 and 2
Block at preB cell stage in absence of HDAC1 and 2
mb1-Cre
4x
Pre BI
100x
Pre BI Pre BIIPro B
Pre BI Pre BII
Cell cycle block at preB cell stage in absence of HDAC1 and 2
7-AAD
Brd
U
3. Mai 2016
Pre BII
WT
dKO
Pre BI
Yamaguchi et al., G&D 24: 455-469
Apoptosis at preB cell stage in absence of HDAC1 and 2
HDAC1/2 are associated with p21 and p57 promotersp21 and p57 are directly regulated by HDAC1 and 2
December 13, 2010
Normal mature resting B cell numbers in absence of HDAC1 and 2
cd23-Cre
Normal mature resting B cell numbers in absence of HDAC1 and 2
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