transcription regulation and gene expression in eukaryotes fs 2010/14apr2010_… · transcription...
TRANSCRIPT
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Transcription Regulation And Gene Expression in Eukaryotes
Cycle G2 (lecture 13709) P. Matthias, April 14th, 2010
Acetylation and Deacetylation
www.fmi.ch/training/teaching
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HDAC
HAT
Sparmann A et al., 2006
Chromatin structure and histone modifications in eukaryotes
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NaButyrate-treated
Control
H3, H4 mononucleosomes can beacetylated
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NaButyrate-treated
H3, H4 mononucleosomes
+DNAseI
NaButyrate-treated
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TJ Richmond, Nature 1997 vol. 389 (6648) pp. 251-60
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Reversible Lysine acetylation
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The effects of Histone lysine acetylation
P. Jenö, Lecture 4610
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In-gel assay
Histones BSA
Tetrahymena crude macronuclear activity
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Some of the chromatin-binding domains present in HAT complexes in yeast
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HATs: Complexes and single proteins
More HATs: CIITA, TFIIB, CLOCK,…
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Histone Acetyltransferases (HATs): GNAT family
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Histone Acetyltransferases (HATs): MYST family
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Elp3: HAT of GNAT family
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P. Jenö, Lecture 4610
Histone (Protein) deacetylases: HDACs
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Some HDAC inhibitors
SAHA/VorinostatZolinza®
TSA
RomidepsinIstodax®
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HDAC inhibitors:
TSA, SAHA, Valproic acid…
HDACs
Genomic effects
- Acetylation of histone and various kinds of transcriptional regulators
Non genomic effects
- Acetylation of HSP90, α-tublin...
Changes in gene expression Changes in protein activity, localization, stability...
Cell cycle arrest
Promoting apoptosis
Inducing differentiation
Other effects
HDAC inhibitors as multipotent drugs
Cancer,
Leukemia
PolyQ disorders (Htt,..)
Autoimmune and inflammatory diseases
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22
(H)DAC inhibitors approved as drugs
Zolinza® (Vorinostat): FDA-approved on 6 Oct. 2006 for cutaneous T cell leukemia (CTCL)
Mode of action: “The mechanism of the antineoplastic effect of Vorinostat has not been fully characterized”
Istodax® (Romidepsin): FDA-approved on 6 Nov. 2009 for cutaneous T cell leukemia (CTCL)
Mode of action: “The mechanism of the antineoplastic effect of Romidepsin observed in nonclinical and clinical studies has not been fully characterized”
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Classical Zn-dependent HDACs
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Minucci S, Pelicci PG.
Nat Rev Cancer. 2006
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“We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275.”
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Widespread lysine acetylation
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HDACis in the food: broccoli sprouts
Dietary compounds: Na-butyrate, diallyl disulphide, sulphoraphaneFound at high levels in cruciferous (Brassicaceae) vegetables
Human healthy volunteers
Human colon cells
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Classical Zn-dependent HDACs
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NAD-dependent HDACs: Sirtuins
314
747
389
399
310
355
P P
400
SIRT4
SIRT1
SIRT2
SIRT3
SIRT5
SIRT6
SIRT7
Zn
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Ahringer (2000). NuRD and SIN3. Trends Genet. 16, 351-356.
NuRD and Sin3 Complexes
NuRD Sin3
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Modiefied from: Knoepfler and Eisenman (1999). Sin meets NuRD and other tails of repression. Cell 99, 447-450.
Gene-specificTranscription Factors
Other Gene-specificTranscription Factors
HDAC recruitment: core complex
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RpAp46RpAp48
Modified from Adcock IM et al., 2006
NuRD complex Sin3 complex
Co-REST complex
- Lethal: embryos die before E10.5
HDAC1 KO mouse
Lagger G et al. 2002
Redundancy?
Specificity
HDAC1 and 2 complexes
Mi-2RpAp46RpAp48
MBD3MTA-2HDAC1/2
SDS3
Sap30
HDAC1/2
Sin3 Sap18
Co-REST
ZNF217 p40
p80p110b
HDAC1/2
HDAC1/2
HDAC1/2
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• The PAH domains are suggested to be organized into two α-helices separatedby 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
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• Thyroid hormone receptors (THRs) are gene-specific activators in the presence of ligand
• In absence of ligand, THRs recruit two large multidomain proteins, N-CoR and SMRT, which in turn bind SIN3 to turn the THR from an activator into a repressor
Sin3 complex in action with the THR
HDAC2
THR
Activation
Thyroid Hormone
THR
Repression
N-CoR SMRT
SIN3 HDAC1
HAT
Hyperacetylation Hypoacetylation
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ClassII HDACs as “signal transducers”
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ClassII HDACs as “cytoplasmic regulator”: HDAC6
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Cre transgenic mouse Cre
Cell type specific expression
HDAC1
HDAC2
482 aa
489 aa
Exon 6HDAC domain
HDAC1
HDAC2
KYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYYFPGTGDLRKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYYFPGTGDLR
Hdac2 floxed mouse
Exon 14Exon 1
Hdac1 floxed mouseExon 6 Exon 15Exon 1
Essential for catalytic activity** *
Conditional inactivation of HDAC1 and HDAC2 in specific lineages
Yamaguchi et al.
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Ablation of HDAC1 and 2 in primary MEFs
Yamaguchi, Cubizolles et al.,
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Residue specificity for HDAC1 and 2
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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
HDAC1
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Direct regulation of the p21 and p57 genes
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The hematopoietic system: from HSCs to differentiated cells
Common Myeloid Progenitors (CMP)
Common Lymphoid Progenitors (CLP)
B-cell progenitor
Mature B-cell
T-cellprogenitor
Mature T-cell
Basophil
Eosinophil
Neutrophil
MonocytesMacrophageGM-progenitor
Megakaryocytes Platelets
Erythroblast ErythrocyteME-progenitor
Th2
Th1
Plasma cell
HaematopoieticStem cells (HSC)
Multipotent progenitors
NK cells
Vav-iCre mouse
HDAC inactivation in the hematopoietic cell lineage
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HDAC1 and 2 double KO mice show embryonic lethality due to anaemia
Control
E13.5 E15.5 E17.5
HDAC1 f/fHDAC2 f/fVav iCre
Vav-iCre activity
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HDAC1 and 2 double KO mice show embryonic lethality due to anaemia
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Common Myeloid Progenitors (CMP)
Common Lymphoid Progenitors (CLP)
B-cell progenitor
Mature B-cell
T-cellprogenitor
Mature T-cell
Basophil
Eosinophil
Neutrophil
MonocytesMacrophageGM-progenitor
Megakaryocytes Platelets
Erythroblast ErythrocyteME-progenitor
Th2
Th1
Plasma cell
HaematopoieticStem cells (HSC)
Multipotent progenitors
NK cells
mb1-Cre mouse
HDAC inactivation in the B cell lineage
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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
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CD4
CD8
T cells
T cells
Spleen cellsSpleen cells
WT
B220
IgM
Mature B cells
HDAC1/2 DKO
Mature B cells
Differentiation
Bone Marrow cells
B220Ig
M
Pro-pre B cells
B cells
B cells
Pro-pre Bcells
HDAC1/2 dKO mice have strongly reduced B cell populations
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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
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Pro B cell
Pre BI Pre BII
Cell cycle block at preB cell stage in absence of HDAC1 and 2
7-AAD
BrdU
WT
dKO
Pre BI Pre BII
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Apoptosis at preB cell stage in absence of HDAC1 and 2
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Normal mature resting B cell numbers in absence of HDAC1 and 2
cd23-Cre
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Normal mature resting B cell numbers in absence of HDAC1 and 2
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Immunoglobulin heavy chain gene
V segments D segments J segments
CµVHJ558 VH7183 DH JH3
Analysis of VDJ rearrangements in preBI cells
Eµ
VDJ recombination RAG
Pre B cellCµVDJ
Cµ
Cγ3 Cγ1 CεEµ
Cµ Cγ3 Cγ1 Cε
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WT DKOWT dKO
IgH gene (3Mb)
V segments D segments J segments C
Distal VH probe CH probe
Loss of a large scale structural change?
IgH contraction
2 probes together
D-J recombination
Impaired locus contraction in preBI cells