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