Post on 31-Aug-2020
transcript
Transcription Regulation And Gene Expression in
Eukaryotes
Cycle G2 (lecture 13709)
RG Clerc 11.04.2012
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
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 hormone
1925 - Kendall and Reichstein complete structural analysis of cortisol from adrenal cortex
1946 – Selye coins the term glucocorticoid, needed for survival and response to stress
1949 – Hench administers cortisone to arthritic patients with dramatic effects
1950 – Kendall, Hench and Reichstein get the Nobel Prize
1950-1985 – Classical model of steroid hormone action
1986 – 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 receptor
Androgen receptor
Mineralocorticoid receptor.
Progesteron receptor
Retinoic acid receptor (RAR)
Retinoid X receptor (RXR)
Vitamin D3 receptor (VD3R)
Thyroid hormone receptor (T3R)
Peroxisome proliferator activated
receptor (PPAR)
Liver X receptor (LXR)
Farnesol X receptor (FXR)
Pregnane activated X receptor (PXR)
Steroid+xenobiotic X receptor (SXR)
Benzoate X receptor (BXR)
NGFI-B
ELP
Nurr77
Localisation Cytoplasma – nuclear
(HSP90 complexation)
nuclear nuclear
Dimerisation homo hetero (RXR) hetero (RXR)
Binding IR 3 DR Single Repeat + extension
Mode of action Transactivation
“systemic”
Transactivation
AP-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. ERa
Cooperative 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
POL2
G TF‘s
GR
GR
GR GR
GR GR
coregulator
steroid
dissociation
dimerization
Transfer into nucleus
cytoplasm
nucleus
Membrane
receptor?
hsp90
hormone
aporeceptor complex
active receptor
molecular chaperones
Steroid Signaling Pathway
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/TESTOSTERONE GLUCOCORTICOID
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)
Fibrates
TZDs
Prostaglandins
PUFAs
Rexinoids
apo A-I, II
apo C-III
aco
P450
LPL
PPRE
PPAR RXR
RXR
POL2 PGC-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 Domain
N 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
PPAR
GR
PPAR
other NRs
PGC-1
txn
PGC-1
NR
Hormones
gluconeogenesis
fatty acid oxidation
mitochondrial
biogenesis
respiration
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
Remodelling Complexes
Mediator Complexes
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. ER
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 SERM
Tamoxifen
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
-b
•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 PPAR and - Isoforms
FIBRATES Gemfibrozil, Benzafibrate,
Fenofibrate
THIAZOLIDINEDIONES Rosiglitazone, Pioglitazone
Ciglitazone
POLYUNSATURATED FATTY ACIDS Docohexaenoic acid, eicosapentaenoic
acid, linoleic acid, linolenic acid and arachidonic acid
PPAR PPAR
HDL RAISE, LIPID CATABOLISM PEROXISOME PROLIFERATION CONTROL OF INFLAMMATION
GLUCOSE HOMEOSTASIS LIPID STORAGE
ADIPOCYTE DIFFERENTIATION CONTROL OF INFLAMMATION
Ligands and Functions of PPARb
DIFFERENTIATION of oligodendrocytes, epithelial cells, keratinocytes and adipocytes
LIPID METABOLISM IN THE BRAIN EMBRYO IMPLANTATION AND DECIDUALIZATION
TUMORIGENESIS IN THE COLON REVERSE CHOLESTEROL TRANSPORT
WOUND HEALING
PPARb/
GW501516
POLYUNSATURED FATTY ACIDS PROSTAGLANDINS
Synthetic PPAR’s Ligands
•thiazolidinediones (TZDs)
– treatment of Type II Diabetes
•PPAR alpha specific
– GW 7647
•PPAR beta specific
– GW 50-1516
EC50 (mM)
0.55
0.58
0.043
SOS
N
O
ON
O
with nM affinity
Correlations between PPAR-activity, insulin
sensitivity and adipogenesis:
• The relationship between PPAR-activation and
adipogenesis is linear, while bell-shaped with insulin
sensitivity
• Thus, neither PPAR 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
PPAR1 RXR
PPAR2 RXR
Ligand-dependent differential coactivator/corepressor
recruitment
ligand-specific expression of “LEAN” genes
ligand-specific expression of “FAT” genes
SRC1, PGC1 TIF2, PGC1,
DRIP/
TRAP
ligand-specific genomic
& 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
• PPAR - cardiac muscle/glucogenesis tissues, liver, intestine,
renal cortex
• PPARb - ubiquitous/skeleton muscle
• PPAR - adipose tissue, large intestine, immune cells
1 - predominant form in above tissues
2 - adipose tissue only
3 - macrophage and large intestine
note: 2 has a distinct N-terminal extension
Association of PPAR polymorphisms with the metabolic syndrome
PPAR Gene
Three mRNAs via two promoters
and alternative splicing
1and 3 encode the same protein,
2 is distinct
identical ligand binding domains
Tissue Distribution
PPAR1 adipose, intestine, kidney, liver, muscle
PPAR2 adipose - elevated in obese subjects
very low in muscle & liver
PPAR3 macrophage, adipose, colon epithelium
28 aa N-terminal addition in PPAR2
increases ligand-independent activation 5X
PPAR - critical role in adipogenesis
Dominant negative mutants and chemical inhibitors block
agonist-mediated adipocyte differentiation
Ectopic expression of PPAR converts fibroblasts to adipocytes
PPAR KO mouse: complete absence of adipose tissue
adipogenic
signals
Association of PPAR polymorphisms with the
metabolic syndrome ?
The Pro12Ala substitution in PPAR2 associated with decreased receptor
activity, lower body mass index and improved insulin sensitivity.
Proline to Alanine substitution at codon 12 of PPAR2
The codon Ala confers reduced activity compared to the Pro isoform
Auwerx J. and co-workers. 1998. Nat Genet.3:284
Association of PPAR 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.