nuclear hormone receptors - fmi · 2014. 5. 6. · transcription regulation and gene expression in...
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Transcription Regulation And Gene Expression in Eukaryotes
Cycle G2 (lecture 13709) FS 2014P.Matthias and RG Clerc
Roger G. Clerc 30.04.2014
NUCLEAR HORMONE RECEPTORS
•Classification, type I, II, III
•DNA and ligand binding
•Co-factor recruitment
•NHR in health and disease www.fmi.ch/training/teaching
R.G. Clerc 2
Nuclear Receptors are Transcription Factors that Utilize Small Lipophilic Ligands to Mediate their Function
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)
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 …
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
Liganded vs Unliganded Transcription FactorsCorepressor 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
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 Ligands
•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
Sirt1 reduces fat accumulation by repressing PPAR-γactivity through Sirt1–NCoR interactions (1)
Picard et al. (2004) Nature 429:771
Sirt1 reduces fat accumulation by repressing PPAR-γactivity through Sirt1–NCoR interactions (2)
Picard et al. (2004) Nature 429:771
Sirtuins are a class of deacetylases involved in reduction fat accumulation by repressing PPAR-γactivity through Sirt1–NCoR interactions
Sirtuins are potential target for the metabolic syndrome Resveratrol activates Sirt1, promoting
calorie restriction - longevity pill ??L. Guarente Nature 444:866 (2006)
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)
PPAR subtypes and expression patterns
• PPARα - cardiac muscle/glucogenesis tissues, liver, intestine, renal cortex
• PPARβ - ubiquitous/skeleton muscle• PPARγ - adipose tissue, large intestine, immune cells
γ1 - predominant form in above tissuesγ2 - adipose tissue onlyγ3 - macrophage and large intestinenote: γ2 has a distinct N-terminal extension
Association of PPARγ polymorphisms with the metabolic syndrome
PPARγ Gene
Three mRNAs via two promoters and alternative splicing
γ1and γ3 encode the same protein, γ2 is distinct
identical ligand binding domains
Tissue DistributionPPARγ1 adipose, intestine, kidney, liver, muscle
PPARγ2 adipose - elevated in obese subjectsvery low in muscle & liver
PPARγ3 macrophage, adipose, colon epithelium
28 aa N-terminal addition in PPARγ2increases ligand-independent activation 5X
PPARγ - critical role in adipogenesis
Dominant negative mutants and chemical inhibitors block agonist-mediated adipocyte differentiation
Ectopic expression of PPARγ converts fibroblasts to adipocytes
PPARγ KO mouse: complete absence of adipose tissue
adipogenic signals
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.