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.