Transcription Regulation AndGene Expression in Eukaryotes

FS 2016 Graduate Course G2

P. Matthias and RG ClercPharmazentrum Hörsaal 2 16h15-18h00

RG. Clerc April 27. 2016

NUCLEAR HORMOME RECEPTORS

• The nuclear receptor atlas• Structural and functional key features• Gene repertoire regulation in health

and disease

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 nuclear hormone receptors in H. sapiens, 284 in C. elegans, 21 in D. melanogaster )

steroid receptors adopted nuclear receptors orphan receptors

GR

Androstane receptor(CAR)

R.G. Clerc 3

Nuclear Receptors Atlas in Physiology

Bookout AL et al. Cell 126:789 (2006)

Steroid Signaling Discovery Timeline : 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 arthiritic patients with dramatic effects1950 – Kendall, Hench and Reichstein get the Nobel Prize1950-1985 – Classical model of steroid hormone action 1986 – today - Receptor identification : „reverse endocrinology“ RAR, RXR ..

Nuclear Receptors Discovery Timeline : What’s My Ligand ?

Evans RM and Mangelsdorf DJ (2014). Cell 157:255

Nuclear Receptors and Small Lipophilic Ligands

RXR

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

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

(48 in human)

The Family of Nuclear Hormone Receptors : Unified Nomenclature Based on the Well Conserved Domains C and E

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

HDAC6 dependent Hsp90 deacetylation required for GR Nuclear Translocation in Presence of Hormone

Zhang Y and Matthias P (2003) EMBO J 22 :1168-79.

Mammalian Histone deacetylases (HDACs)

HDAC6 1215CD1 CD2 ZnF

IIbHDAC10

IV. Class IV HDACHDAC11

III. Class III-SIR2-like HDAC: NAD+ dependentSIRT1SIRT2SIRT3SIRT4SIRT5SIRT6SIRT7

Subcellularlocalization

NucleusNucleus

Nucleus & cytoplasmNucleus & cytoplasm

Nucleus & cytoplasm

Nucleus & cytoplasm

Nucleus & cytoplasm

Nucleus & cytoplasm

mainly cytoplasmNucleus & cytoplasm

Nucleus & cytoplasm

NucleusCytoplasm

Mitochondria

I. Class I-RPD 3-like HDAC: Zn2+ dependent

II. Class II-HAD 1-like HDAC: Zn2+ dependent

HDAC1HDAC2HDAC3HDAC8

HDAC4

HDAC5

HDAC7

HDAC9

IIa

Catalytic domain

Zhang Y and Matthias P (2003) EMBO J 22 :1168-79.

C domain (DBD): 2 cys-cys Zinc Fingers eg. ERαCooperative Binding (homodimer/heterodimer)

Glucocorticoid Receptor-DNA Complex (PDB 1glu)

Zn finger

K461 V462 R466

5’A G A A C A NNN T G T T C T3’

T C T T G T NNN A C AA G A

Generation of DNA binding specificity mutants

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

The RXR “Big Bang”

9-cis RA

Evans RM and Mangelsdorf DJ (2014). Cell 157:255

Homodimer/heterodimer (NHR/RXR) formation involves both the C and E domains

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

NHR Ligand Binding “pocket” Delineated by 3 layers H3, H5, H7, H11, H12 α-Helices and β-Strands

Three Different Conformational Changes for RXRα

unliganded liganded liganded

The general problem

RNA

Transcription factor binding sites convey specificity to regulation

Nuclear Hormone Receptors are TranscriptonalRegulators

Corepressor vs. Coactivator Interfaces Structure Remodeling (eg,. RXR)

unliganded liganded

co-repressor binding co-activator binding

“mouse trap”

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

Nolte RT, Glass CK et al. (1998) Nature 395:137-143

“E471 / K301 charged clamp”

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

Estrogen : The Feminine Homone With Ambivalent

Functions

RALOXIFENosteoporosis

estradiol

estriol

estrone

Effects of Estrogen at Various Sites in the Body

Tissue Effect of Estrogen Stimulation

Clinical Effect of Stimulation

Clinical Effect of Absence of Stimulation

Bone Increased deposits of calcium into bone

Increased bone density Osteoporosis

Brain Blocks the release of ovarian estrogen

None Hot flashes, sleep disorders, mood changes, problems with memory? Alzheimer’s disease??

Breast Stimulates growth of breast tissue Bigger breasts,? Increased

risk of breast cancer, increased sensitivity of the breast,

Smaller breasts

Blood Clotting Increased risk of blood clots No change in clotting

Blood Fats Increased HDL, decreased LDL, decreased Cholesterol,

Decreased HDL, increased LDL, increased Cholesterol

Skin Increased fat deposits in skin Softer skin Thinner skin, liver spots, dry skin

Uterus Increased stimulation of uterine lining and muscle

Heavier cycles, increased risk of uterine cancer

No periods

Vagina Increased thickening of skin, better blood supply to tissue

Vaginal discharge, feelings of pelvic congestion

Dryness, vaginal infections, painful sex, incontinence of urine, pelvic weakness

Molecular Action of Estradiol

Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 . Cancer 2000; 89: 819.

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

Selective Estrogen Receptor Modulator

The ideal SERM is one that prevents bone loss, has no risk of uterine or breast cancer, no adversed effect on lipids & cardiovascular system, relieves PMS and maintains cognitive function of the brain

Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780. Cancer 2000; 89: 819.

Molecular Action of Tamoxifen (SERM)About 1 in 8 women (about 12%) will develop invasive breast cancer over the course of her lifetimeIn 2016, an estimated 246,660 new cases of invasive breast cancer are expected to be diagnosed

Only patients that express nuclear receptor (ER-positive tumors) benefit from Tamoxifen. Other breast cancers are either ER-negative HER2 positive and so-called triple negative

Molecular Bases for Agonism vs, Antagonism: ERα

Co-regulator box LXXLLDES : diethylstilbestrol TAM : tamoxifen

Adapted from Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182, 780 (Faslodex®), development of a novel, "pure" antiestrogen. Cancer2000; 89: 819.

Molecular Action of Tamoxifen (SERM) as a prodrugKaplan Meier - metabolizer status

Goetz et al. 2008. Clin Pharm Ther 83:160

Regulation of ERBB2 by ER-PAX2 determinesTamoxifen response (Hurtado et al. (2008 )Nature 456:663-667)

Regulation of ERBB2 by ER-PAX2 determinesTamoxifen response (Hurtado et al. (2008 )Nature 456:663-667)

Regulation of ERBB2 by ER-PAX2 determinesTamoxifen response

Deteriorated

Impaired

Impaired

Lipid profile

Insulin sensitivityInsulinemiaGlycemia

Susceptibilityto thrombosis

Inflammationmarkers

Endothelialfunction

CHD Risk LowHigh

AbdominallyobeseHigh waist

Improved

Improved

Improved

Improved

Improved

ReducedobeseLow waist

Visceraladiposetissue

Visceraladiposetissue

Subcutaneous ATSubcutaneous AT

DietPhysical activity

10% Weight loss~ 30 % Visceral AT loss

Clinical Endpoint: Metabolic Syndrome

Adapted from Després et al. BMJ (2001) 322:716-720

Defective insulin secretion

Increased glucose output

Decreased glucose disposal

Insulin resistance

HYPERGLYCEMIA

Adipose tissue

Dyslipidemia

Obesity

Metabolic Syndrome and Tissue-Tissue Cross Talk

Integrated Physiology by PPAR Isoforms

−β

Originally Discovered as PeroxysomeProliferator-Activated Receptor (PPARs)

W.Wahli et al. (1992) Cell 68: 879-887

catabolism of long chain FA, reduction of ROS, etc

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

•Inducing the proliferation of peroxisomes in rodents • Intimately connected to the cellular metabolism and cell differentiation

Peroxisome proliferator-activated receptors PPAR Isoforms

Natural PPAR’s Ligands

– polyunsaturated fatty acids

– 15-lipoxygenase metabolites

– J series prostaglandins

“only” µM affinity

Synthetic PPAR’s Ligands : Transactivation Assays, IC50, EC50 etc

•thiazolidinediones (TZDs)(pioglitazone, rosiglitazone, troglitazone)

– 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

Ancient dogma

Roche and Millenium unravel the first adipokine: leptin

AT is a very efficient energy storehouse (9,3kcal/g)

FA's come from 3 main sources: dietary fats, TG's produced in the liver, and

excess blood glucose

1994-1996 TODAY

Selected AdipokinesVisfatin AdiponectinResistinTNFalphaIL-6LeptinANGPTL-4

Adipocytes as endocrine tissue regulating glucose and lipid metabolism: an emerging concept

PPARγ & Adipocyte Differentiation

pre-adipocyte

small

insulin sensitive

hypertrophichigh insulin resistance

largereduced

insulin resistance

High Fat Diet

+/+

+/-

PPARγ

differentiation

less TNFα less FFA more leptin less insulin resistance Activation of PPARα

Cell Size-/-

no adipose no leptin fatty liver high insulin resistance

PPARγ – C/EBPs Critical Roles 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

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

hPPARγ Isoforms

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

hPPARγ Isoforms

Association of PPARγ polymorphisms with the metabolic syndrome ?

Haplotype analysis of the PPARgamma Pro12Ala and C1431T variants reveals opposing associations with BMIDoney et al. 2002. BMC Genetics 3:1-8.

Association of PPARγ polymorphisms with the metabolic syndrome ?

Is the Ala12 variant of the PPARgamma gene an „unthrifty“ allele ?

(subject to negative selection during human evolution)

Ruiz-Narvaez et al. 2005. J. Med Genet 42:547

The Ala allele was originally associated with a protective effet against type 2 diabetes.

Increased weight and body mass index, which themselves predispose to type 2 diabetes have however been inconsistently associated with the Pro12Ala polymorphism

studies indicate that the Ala allele is associated with a higher BMI, or a lower BMI or no change at all !

See Doney et al. 2002. BMC Genetics 3:1-8.

Association of PPARγ polymorphisms with the metabolic syndrome ?

The Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity.Proline to Alanine substitution at codon 12 of PPARgamma2The codon Ala confers reduced activity compared to the Pro isoform

Auwerx J. and co-workers. 1998. Nat Genet.3:284