Transcription Regulation And Gene Expression in … FS 2010/21APR2010...Transcription Regulation and...
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Transcription Regulation And Gene Expressionin Eukaryotes (Cycle G2 #13709-01)
NUCLEAR HORMONE RECEPTORS AND COREGULATORS
RG. Clerc_April 21, 2010 www.fmi.ch/training/teaching
Transcription Regulation and Gene Expression in Eukaryotes
NUCLEAR HORMONE RECEPTORS
RG. Clerc, May 31. 2006
Brief history of steroid signaling (100 years on)1902-1905 - Starling refers to bioactive chemicals extracted from glands as „hormones“1915 - Kendall crystallizes thyroid hormone1925 - Kendall and Reichstein complete structural analysis of cortisol from adrenal cortex1946 – Selye coins the term glucocorticoid, needed for survival and response to stress1949 – Hench administers cortisone to arthritic patients with dramatic effects1950 – Kendall, Hench and Reichstein get the Nobel Prize1950-1985 – Classical model of steroid hormone action1986 – today - Receptor identification : „reverse endocrinology“ GR, ER, TR, RAR, RXR …
R.G. Clerc 3
Nuclear Receptors and Small Lipophilic Ligands
The Nuclear Hormone Receptor Family. P. Chambon Academic Press 1991
The family of nuclear hormone receptors
Type I Type II Type III/orphanMembers Glucocorticoid receptor
Estrogen receptorAndrogen receptorMineralocorticoid receptor.Progesteron receptor
Retinoic acid receptor (RAR)Retinoid X receptor (RXR)Vitamin D3 receptor (VD3R)Thyroid hormone receptor (T3R)Peroxisome proliferator activatedreceptor (PPAR)Liver X receptor (LXR)Farnesol X receptor (FXR)Pregnane activated X receptor (PXR)Steroid+xenobiotic X receptor (SXR)Benzoate X receptor (BXR)
NGFI-BELPNurr77
Localisation Cytoplasma – nuclear(HSP90 complexation)
nuclear nuclear
Dimerisation homo hetero (RXR) hetero (RXR)Binding IR 3 DR Single Repeat + extensionMode of action Transactivation
“systemic”TransactivationAP-1 antagonism
?
SHP
DAX
LRH1
ERR
(48 in human; 230 in C. elegans; 21 in D. melanogaster)
steroid receptors adopted nuclear receptors orphan receptors
GR
Androstane receptor(CAR)
Nuclear Hormone Receptors are Modular in Nature (operationally defined from A-F)
Ligand independent trx
“AD”
AF-1 function
DNA binding
dimerization
Hinge
NLS
Hsp90
Ligand dependent trx “AD”
dimerization
AF-2 function
co-regulator recruitment
NLS Hsp90
C domain (DBD): 2 cys-cys Zinc Fingers eg. ERaCooperative Binding (homodimer/heterodimer)
Homodimer/heterodimer (NR/RXR) formation involves both the C and E domains
Nuclear Hormone Receptors are Transcriptonal Regulators
P. Chambon and co-workers. IGBMC. Illkirch France
(48 in human)The family of nuclear hormone receptor: unified nomenclature
(based on the two well conserved domains C and E)Laudet V. .J. Mol. Endo. 19:207 (1997) NHR Nomenclature Committee. Cell 97:161 (1999)
Induction of steroid responsive genes involves hormone dependent dissociation of the receptor from hsp90 (type I nuclear receptors)
eg. glucocorticoid responsive genes
GRE
hsp90
POL2G TF‘s
GR
GR
GRGR
GR GR
coregulator
steroid
dissociation
dimerization
Transfer into nucleus
cytoplasm
nucleus
Membrane receptor?
hsp90
hormoneaporeceptor complex
active receptor
molecular chaperones
Steroid Signaling Pathway
Regulation of Specific Gene Expression
Steroid/thyroid dependent gene regulation: gene expression induced denovo and gene expression regulated (rate of transcription) by the hormone
LBD of GR Mediates Translocation to the Nucleus in presence of Hormone
The adrenal cortex is responsible for production of 3 major classes of steroid hormones: glucocorticoids, which regulate carbohydrate metabolism; mineralocorticoids, which regulate the body levels of sodium and potassium; and androgens, whose actions are similar to that of
steroids produced by the male gonads
Steroids and the Adrenal Cortex
MINERALOCORTICOID ESTRADIOL/TESTOSTERONEGLUCOCORTICOID
NHR are the final effectors of a complex cytoplasmic/nuclear transduction cascade
• PPAR - Peroxisome Proliferator Activated Receptor• RXR - Rexinoid Receptor
PPAR/RXR-dependent nuclear signaling(type II nuclear receptors)
FibratesTZDsProstaglandinsPUFAs
Rexinoids
apo A-I, IIapo C-IIIacoP450LPL
PPRE
PPAR RXR
RXR
POL2PGC-1
coregulator
E domain: canonical structure of the LBD
•Characteristical sandwich architecture with 3 layers built in by 12 alpha antiparallel helices and 1 antiparallel beta sheet
•Structure-AA sequence relationship for NHR LBD’s
•Ligand binding dependent pocket remodeling
•Receptor dimerization surface•Co-regulator proteins interface
•Control of agonist vs. antagonist modes of action
Nuclear Hormone Receptor Superfamily: Well Conserved DBD, Poorly Conserved LBD
DNA-Binding Domain Ligand Binding DomainN C
N C
N C
N C
Progesterone Receptor
Thyroid Hormone Receptor
Retinoid X Receptor
NGF1B
100%
28%
35%
40%
100%
18%
18%
16%
well conserved at sequence level poorly conserved at sequence level
well conserved at structural level well conserved at structural level
Corepressor vs. Coactivator Interfaces Structure Remodeling
unliganded liganded
co-repressor binding co-activator binding
“mouse trap”
Protein:protein interaction in vivo screen: “two hybrid-screen”
S. Fields. Nature 340:245 (1989)
(b)
Coactivator CBP/p300, Corepresssor Ncor, etc
Combinatorial roles of multiple cofactor complexes are required to switch between transcriptional repression and activation functions
Coactivator Family PGC1a, 1b and PRC
J. Lin, C. Handschin, BM. Spiegelmann. Cell Met 1:361 (2005)
Structure and function of the PGC-1 family coregulators: binding to the HAT and TRAP/DRIP/Mediator complexes at the amino and carboxyl termini, respectively. SirT1 and p160 bind to the repression domain, which also contains three p38 MAP kinase phosphorylation sites
PPARα
GR
PPARγ
other NRsPGC-1
txnPGC-1
NR
Hormones
gluconeogenesis
fatty acid oxidation
mitochondrial biogenesisrespiration
TR/
/HNF4
PGC-1 Coactivator Functions in Maintenance of Glucose, Lipid and Energy Homeostasis
ERR/
FOXO1
REPRESSION
Deacetylase (HDAC)Corepressor Complexes
Coactivator and Corepressor Complexes with the Basal Machinery are Involved in the Regulation of NHR Transcriptional Activity
ACTIVATION
REPRESSION
RemodellingComplexes
MediatorComplexes
Acetylase (HAT)Complexes
Deacetylase (HDAC)Corepressor Complexes
Nuclear Receptor Coregulator
Interaction Motifs
Bookout AL, Evans RM, Mangelsdorf DJ. Cell 126 2006
Nuclear receptors and coregulators manifest in reproduction, development, central and basal metabolism and energy homeostasis
NURSA - Nuclear Receptor Signaling Atlas (www.nursa.org)
Molecular Bases for Agonism vs partial Agonist vs Antagonism: Selective Modulator Concept eg. ERα
Co-regulator box LXXLL
Estrogen : Hormone with Ambivalent Functions
Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 (Faslodex®), development of a novel, "pure" antiestrogen. Cancer 2000; 89: 819.
Molecular Action of Estradiol and of SERMTamoxifen
Selective Estrogen Receptor Modulators
Estrogens
Anti Estrogens
SERMs
SERMs- designed to act in specific ways at each of the estrogen receptor sites in different tissues
ERDR
Phytoestrogens
The Liver-X-Receptor Genes
LXRα LXRβExxxxxxxxxx Lxxxxx Kxxxxx xxxxxxxxxx
Ixxxxxxxxx WATx
Kxx Rxxxx Cxxxxxxxxxx Cxxxxxxxxxx
Mxxxxxxxxx Mxxxxxxxxx
Kxx Mxxxxxxxxxx Ixxxxxxx RCT Ixxxxxxx RCT
Ixxxxxxx Bxxx Axxx
Sxxxxxxxx
Ixxxxxxx xxxxxxxxxxx TG xxxxxxxxx
Nxxxxxx Lxxxxxxx xxxxxxxxxx 24xSxxHCx 22xRxxHC
24xSxx 25xEC
Sxxxxxxxx Lxxxxxxx xxxxxxxx T0901317x T0314407
Functions of Key LXR/RXR-regulated genes
Cxxxxxxxxxx xxxxxxxxxxx
Lxxxx Mxxxxxxxxxx
ABCA1 Cxxxxxxxxxx xxxxxxxxxxxxxx xxxxxxxxxxx xxxxxxxxx
ABCG1 Cxxxxxxxxxx xxxxxxxxxxxxxx xxxxxxxxxxx xxxxxxxxx
Cxx7A1 Bxxx Axxx Sxxxxxxxxx xxxxxxxxxxxxx xxxx xx xxxxxxx
xxxxxxx xxxxxxxxxxx xxxxxxxx
CETP Cxxxxxxxxxx xxxxxxxx xxxxxxx xxxxxxxxxxx xxxxxxxxx
SREBPx1x Rxxxxxxxx xx Lxxxxxxxx ExxxxxxxTG xxxxxxxxx
Gxxx Fxxxxxxx
Molecular Function of the Liver-X-Receptors
LXRα xxx LXRβ xxxxxxxxxx xxxxxxxxxxxxx xxxx xxxxxxx xxxxxxx
xxxxxxxxx xxxxxxx xx xxxxxxx xxxxxxxx xxxxxxxxxxxx xxxx RRx
Txx xxxxxxx xxx DR4 xxxxxxxx xx xxx xxxxxxxxx xx xxxxx xxxxxxxx
xx xxxxxxxxxxx xxx xxxxx xxxxxxxxxxx
ABCA1 LXRE TTTGACCGATAGTAACCTCTGCGCAAACTGGCTATCATTGGAGACGCG
DR4
CYP7A1-LXRE CCTTTGGTCACTCAAGTTCAAGTGGGAAACCAGTGAGTTCAAGTTCAC
DR4
DR4 xxxxxxxx xx xxxx xxxxxxxxxx xxxxxx xxxxx
SREBP-1c LXRE GTCACTGGCGGTCATTGGGGTCAGTGACCGCCAGTAACCCCA
DR4
Rxxx xxx Mxxxxxxxxxxx 2000 Axxx Rxx Cxxx Dxx Bxxx 16x459x81
LXR
FXR
Role of Cyp7A1 in Bile Acid Synthesis
SHP
Reverse Cholesterol Transport and Bile Acid Metabolism
liver
gut
macrophages
SR-BI
LDL R
ABC1,8
BABA
BSEP
LXROxC
CYP7A1CYP8B1C
VLDL
HDLA-1A-1
FXRNTCP
OATP-1
LRH-1SHP
LXR
Enterohepaticcirculation
Reverse Cholesterol Transport and Bile Acid Metabolism
Cholesterol and Triglyceride Synthesis PathwaysLXR
LXRα Null Mice Shows Defects in Cholesterol Disposal
Pxxx xx xxx 1998 Cxxx 93x 693x704
Gxxxx xxxxxxxxxx xx xxxxxx xx LXRα xx xx xxx Mxxx xx 2x xxxxxxxxxxx xxxxx
Cxxxxxx xx Bxxx Axxx Cxxxxxxxxxx xx
LXRα xx xx xxx Mxxx xx 2x xxxxxxxxxxx xxxxx
Axxxxxx xxx xx xxxxxxx xx
xxxxxxxxxx Cxx7A1 xxxxxxxxxx
Integrated Physiology by PPAR Isoforms
−β
•Inducing the proliferation of peroxisomes in rodents • Intimately connected to the cellular metabolism and cell differentiation
Peroxisome proliferator-activated receptors PPAR Isoforms
Ligands and Functions of the PPARα and -γ Isoforms
FIBRATESGemfibrozil, Benzafibrate,
Fenofibrate
THIAZOLIDINEDIONESRosiglitazone, Pioglitazone
Ciglitazone
POLYUNSATURATED FATTY ACIDSDocohexaenoic acid, eicosapentaenoic
acid, linoleic acid, linolenic acid and arachidonic acid
PPARγPPARα
HDL RAISE, LIPID CATABOLISMPEROXISOME PROLIFERATIONCONTROL OF INFLAMMATION
GLUCOSE HOMEOSTASISLIPID STORAGE
ADIPOCYTE DIFFERENTIATIONCONTROL OF INFLAMMATION
Ligands and Functions of PPARβ
DIFFERENTIATION of oligodendrocytes, epithelial cells, keratinocytes and adipocytes
LIPID METABOLISM IN THE BRAINEMBRYO IMPLANTATION AND DECIDUALIZATION
TUMORIGENESIS IN THE COLON REVERSE CHOLESTEROL TRANSPORT
WOUND HEALING
PPARβ/δ
GW501516
POLYUNSATURED FATTY ACIDSPROSTAGLANDINS
Synthetic PPAR’s Lxxxxxx
•thiazolidinediones (TZDs)– treatment of Type II Diabetes
•PPAR alpha specific– GW 7647
•PPAR beta specific– GW 50-1516
EC50 (µM)
0.55
0.58
0.043
SO SN
O
ON
O
with nM affinity
Correlations between PPARγ-activity, insulin sensitivity and adipogenesis:
• The relationship between PPARγ-activation and adipogenesis is linear, while bell-shaped with insulin sensitivity
• Thus, neither PPARγ antagonism (lipodystrophic state) nor full agonism (obese state) results in optimal insulin sensitization (mouse/rat/human genetic models)
Selective modulator concept: SPPARMs
agonists < partial > antagonists
PPARγ1 RXR PPARγ2 RXR
Ligand-dependentdifferential coactivator/corepressor
recruitment
ligand-specific expression of“LEAN” genes
ligand-specific expression of“FAT” genes
SRC1, PGC1 TIF2, PGC1, DRIP/TRAP
ligand-specificgenomic
& non-genomic
effects
Selective Modulator Concept
• Gave boost to the continued research for SERMs.• Concept of profiling selective modulators has
been established since then eg. (SFXRM‘s, SPPARM‘s, SLXRM‘s)
• Ligand dependent selective co-regulator recruitment likely to elicit specific target gene repertoire linked to desirable pharmacological effects, (eg: LXR ligands which promote reverse cholesterol transport but do not induce hypertriglyceridaemia (SREBP1c gene pathway activation)
Association of PPARγ polymorphisms with the metabolic syndrome ?
The Pro12Ala substitution in PPARγ2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity.Proline to Alanine substitution at codon 12 of PPARγ2The codon Ala confers reduced activity compared to the Pro isoform
Auwerx J. and co-workers. 1998. Nat Genet.3:284
Association of PPARγ polymorphisms with the metabolic syndrome ?
Haplotype analysis of the PPARgamma Pro12Ala and C1431T variants reveals opposing associations with BMI
Doney et al. 2002. BMC Genetics 3:1-8.
Total by Gene Family
Other: (single member families) 56
monoamine oxidase 2
short-chain dehydrogenases/reductases 2
serine proteinase - S1(trypsin-like) 3
sodium-neurotransmitter symporter 3
cyclooxygenase 3
phosphodiesterase 3
dihydrofolate reductase 3
topoisomerase II 4serine proteinase - S11 3
cytokine receptor - type II 2
cytochrome P450 4
ligand-gated ion channel 4
penicillin-binding protein 1a; 2; 2B homolog 4
nuclear hormone receptor 14
voltage gated calcium channel 2
rhodopsin-like GPCR 37
cytokine receptor - type I 6cation transport ATPase 2
SLC12A family 2oxidoreductase 2
23%
9%
4%
35%
NHR are the second Most Precedented Drug Target Family
Competition Radioligand Binding Screening by Scintillation Proximity Detection
SPA BEAD
GST
3H-T0901317
Ant-GST antibody
LXR
GST LXRLightEmission
3H-T0901317
SPA BEAD
GST
Ant-GST antibody
LXR
GST LXRLightEmission
3H-T0901317
3H-T0901317
3H-T0901317
3H-T0901317
3H-T0901317
3H-T0901317
Non-competing compound X
Competing compound Y
NON competitor
GOOD competitor
R.G. Clerc 50
Principle of Transactivation Assay
receptor plasmid reporter plasmidHR reporterHREP
mRNA
HR
R.G. Clerc 51
Principle of Transactivation Assay II
receptor plasmid reporter plasmidHR reporterHREP
mRNA
HR
+ ligand Protein
mRNAligand
R.G. Clerc 52
Reporter Gene Transactivation Assay for NHR’s
ß-actinpromoter
NHR RE FF Luciferase+
Pool Transient transfection
Assay for Luciferases activity (Lumistar)
NHR
Receptor Reporter 1
Adh basal promoter
Reporter 2
CMV R luciferase
Seed 96 well format / DRC Ligands
Nuclear Hormone Receptors, Natural and Synthetic PPAR ligands