Kromatin-struktur - en oppfriskning

Kromatin-struktur - en oppfriskning

The super-family of nuclear receptors MBV4230 The super-family of nuclear receptors The larges TF family of metazoan trx regulators Signal responsivee factors mediate transcriptional response to complex extracellular signals Short signal transduction

pathway lipophilic signalling molecules TF transcriptional response classical steroid hormones secreted from endocrine cells transported by the blood target cell diffuse into the cells bind receptor activated modulate target genes MBV4230 Ligand responsiveeness lipophilic hormone ligands

Can pass the lipidlayer in the cell membrane activating ligand can be generated in three ways: Classical model: synthesized in a remote endocrine cell (e.g. thyroide hormone) Made in the target cell from an apohormone (e.g. 9-cis-retinoic acid) Metabolite synthesized intracellularly in the target cell (e.g. prostaglandines) hormone + inactive receptor allosteric change active receptor binds DNA and modulate transcription Ligand-binding domain - LBD

LBD - a molecular switch which upon ligand binding changes the receptor into a transcriptional active form MBV4230 Common design MBV4230 Common structures with multiple sub-domains A/B: variable, constitutive activator function AF-1 C: conserved DBD

with two C4-zinc fingers which also mediate dimerization D: hinge N-terminal domain variable in sequence/length having activator function (AF-1) variable hinge-region often carrying an NLS E: ligand binding domain LBD, ligand-dependent AF-2 conserved larger ligand-binding domain (LBD) functionally complex ligand-binding, hsp interaction, dimerization, NLS

F: variable C-term Variable C-terminal domain wihtout specific function MBV4230 Functional domains DBD mediate binding to a hormone responsivee element (HSE) 2 conserved zinc fingers MBV4230 DBD

MBV4230 TAD= AF1 and AF2 NR function = Activators and represssors TAD Ligand dep TAD Lig indep MBV4230 Classification - subfamilys Subfamilys Type

undergo nuclear translocation upon ligand activation bind as homodimers to inverted repeat DNA half sites, Type SHR SHR RXR ?R

II: RXR heterodimers often retained in the nucleus regardless of the presence of ligand usually bind as heterodimers with RXR to direct repeats. Type I: Steroid receptors III: orphan NRs dimeric orphan receptors Monomere orphan receptors Half receptors MBV4230 Altern.

class. Evolutionary analysis of the receptors has led to a subdivision in six different subfamilys MBV4230 Classification depending on source and type of ligand Type I: Steroid receptors MBV4230

1. Sub-family: Steroid hormone receptors First TFs (for RNAP-II) cloned: GR and ER Steroid hormones lipids with cholesterol-derived skeleton produced in adrenal cortex (ac - binyrebark), gonades (testis/ovaries), placenta known receptor ligands:

Aldosterone = mineralocorticoid with effect on salt metabolism/electrolyte balance [ac] Cortisol = glucocorticoid with effect on glucose metabolism [ac] Testosterone = androgen (male sex hormone) [testis] Estradiol = estrogen (female sex hormone) [ovaries] Progesterone [ovaries] MBV4230 Steroid hormone receptors Characteristic features of steroid receptors (SHR)

binds palindromic HSE (spacer 3 nt) 2x AGAACA - GR, MR, PR, AR 2x AGGTCA - ER binds as homodimers long A/B-domains Chaperone-complex in absence of ligand SHR

SHR - ligand: SHR associates with a multicomplex [8-10S] of chaperones (hsp-90,-70, -56) inactive, not DNA-binding, ligandreceptive conformation + ligand: hsp-complex dissociates 4S active complex able to dimerize, bind to DNA, transactivate induced transport to nucleus in specific cases chaperon-function: >inactivation MBV4230 Steroid hormone receptors The relative sizes of the human receptors indicated. The numbers above the bars indicate the percentage homology of the consensus

regions of the DNA- and ligand-binding domains MBV4230 Induced nuclear transport? Steroid import NR-Hsp90 complex In the nucleus Not general MBV4230 Induced nuclear transport?

Steroid import NR-Hsp90 complex When bound to the receptor (R) it induces a conformational change that allows it to bind the Hsp90 dimer, which acts as its chaperone. The NLS of the NR allows the R-Hsp90 complex to translocate into the nucleus. In the nucleus The steroid released from steroid-binding protein (SBP) is transported into the cytoplasm of the target cell.

ligandreceptor complex dissociates from Hsp90 and itself dimerizes. The removal of Hsp90 unmasks the DNA-binding site of the receptor,which allows it to interact with the target gene promoter. Not general Many NRs are localized in the target cell nucleus, often tightly bound to chromatin. Type II: RXR heterodimers MBV4230 2. Sub-family: RXR heterodimers Prototypiske members:

RAR [retinoic acid receptor] vitamin A metabolite VDR [vitamin D receptor] TR [thyroid hormone receptor] PPAR [prostaglandine J2] several Orphan receptors with unknown ligand Characteristic feature of the RXR-heterodimers Broader chemical variation of ligands Not all ligands are endocrine hormones

ligand-independent activation mechanisms exist bind DNA also in absence of ligand bind often to direct repeats bind as heterodimers RXR ?R MBV4230 RXR - a common partner and main regulator mediator of receptor heterodimerization high affinity DNA-binding responsivee to 9-cis retinoic acid

three subtypes RXR(+ isoforms 1 2 subtypes - products of individual genes isoforms - products of alternative splicing, alternative promoters etc. MBV4230 RXR = active or silent partner May be a silent partner without ligand-binding and response as heterodimer with VDR, TR and RAR May be an active partner responsivee to 9-cis-retinoic acid

e.g. RXR-PPAR responsivee to both ligands (synergistic) RXR ?R Active partner RXR ?R Silent partner MBV4230 Specificity in DNA-binding: the 1-5 rule

The 1-5 rule direct repeats of AGGTCA with variable spacing (n) DRn where n determines partner DR1 - RXR-RXR DR2 - RXR-RAR DR3 - RXR-VDR

DR4 - RXR-TR DR5 - RXR-RAR RXR binds 1. half site, partner binds 2. half site Sequence context modulates also complex HSEs MBV4230 HSEs Hormone response elements MBV4230 Three DNA-bound dimers with RXR RXR RXR

RAR MBV4230 DBD structure Several 3D structures determined without DNA: RXR and RAR DBD (92,93) with DNA: RXR-TR-DR4 (95) C4 zinc fingers with major groove contact + 3. helix as a C-terminal extenstion (CTE) with minor groove contact

Head-to-tail arrangement dimer interphase different DBD alone is a monomer in solution cooperative dimer formation on DRn The structural implications of the 1-5 rule n+1 36o rotation + 3.4 translation RXR has to use different interphases for different partners (jfr tannhjul) Ligand-induced activation MBV4230 Ligand-binding domain Multiple functions

Solved 3D structures ligand binding Dimerization interphase hormone-dependent transactivation RXR LBD without ligand TR LBD with ligand (see picture) RAR, ER, PPAR, PR, VDR Common fold with 65% helices 12 helices (H1-H12) + a -turn is

arranged as a three-layered antiparallel sandwich, relatively similar in all MBV4230 Ligand-binding domain Activator function (AF-2 or c) Ligand completely buried in the inner parts of LBDs protruding amphipatic helix 12 without ligand

(RXR) closed over a ligand pocket with ligand-contact when bound Becomes an integrated part of a hydrophobic core Ligand-binding large allosteric changes From an open apo-form to a compact closed holo-form MBV4230 Three conformations of LBD Ligand-binding major structural changes

From an open apo-form to a compact closed holo-form Two distinct conformations for H12 positioned in two conserved hydrophobic grooves - agonist and antagonist grooves on the surface MBV4230 Ligand-binding domain LBDs are signal-responsivee regulatory modules adopting distinct conformations as aporeceptors, holo-agonist bound or holo-antagonist bound species MBV4230

Ligand-induced conformational change Ligand binding first acts through the rearrangement of H3, thus expelling H11, leading to repositioning of H12 H12 helix MBV4230 LBD conformational change

Left: the LBD from the crystal structure of the unliganded RXR. Right: the ligandbound LBD of the RAR. MBV4230 Model of intact heterodimers link DBD with LBD structures symmetric LBD + asymm

DBD 180o rotation Two-step model for binding in solution heterodimers are formed through LBD binds DNA with swivel flexibility On proper sites (DRn) the DBDs dimer interphases make contact Strong dimerization Swivel? Weak dimerization Coregulators

MBV4230 Several coactivators and corepressors implicated MBV4230 Coregulators Corepressors - interact with receptors without ligand SMRT - silencing mediator for retinoid and thyroid hormone receptors N-CoR - nuclear receptor corepressor

Liberated upon ligand-binding C-term: receptor-interaction, N-term: repressor motifs Act as an adaptor between NR without ligand and the Sin3complex with HDAC activity MBV4230 Coregulators Coactivators that bind ligand-bound receptors several coactivators that stimulate ligand-dependent activation, SRC/p160 family:

SRC-1/NCoA-1 family [steroid receptor coactivator 1 / nuclear receptor coactivator 1] TIF2/GRIP1/NCoA-2 [trx.intermed. factor /glucocort. recept. interact.prot / nuclear receptor coactivator 2] pCIP/ACTR/AIB1 [p300/CBP-cointegr. ass. prot. / activator TR and RAR / amplified in breast cancer] CBP/p300 and p/CAF also coactivator for NRs Swi/Snf complex Probably multi-coactivator complexes MBV4230 SRC/p160 family HAT enzyme and interphase to other complexes CoCoA coactivator,

coiled-coil coactivator NR contact CBP contact HMT contact HAT MBV4230 LxxLL-interactions with coactivators and corepressors Coactivators

g dL f b a e L SRC/p160 family Associate with NR through LxxLL motif Fig: PPAR LBD bound to a SRC-1 peptide with two LxxLL (yellow)

Corepressors c SMRT and N-CoR C-term: receptor-interaction, N-term: repressor motifs Switch absence of ligand allows binding of NCoR through two LxxxIxxxI/L motifs. Binding of agonist changes LDBconformation such that a coactivator can be recruited through the LxxLL motif.

agonist antagonist L MBV4230 Ligand-activation - a switch from an active repressor to a full activator Active repression Activation MBV4230 Even more complexes - Mediator Several coactivators for nuclear receptors have been

found to be more general than first assumed and are probably identical with or variants of a Mediator-complex TRAP - TR-associated proteins DRIP - vit.D receptor-interacting proteins Isolated as coactivator for SREBP-1a and Sp1, also coactivator for VP16, NFkB Identical with DRIP

Human Mediator Isolated as coactivator for vitamin-D receptor (VDR) Very similar to TRAP in composition ARC - activator-recruited cofactor Isolated as coactivator for the thyroid receptor (TR) Isolated as E1A-interacting multicomplex with 30 polypeptides that binds the activatordomains of E1A and VP16 (Boyer et al. 1999) CRSP, NAT and SMCC Comparison reveals many of the same subunits in many of these complexes

MBV4230 Coregulators In addition: holoenzyme interaction through DRIP/TRAP other RNA coactivators Ubiquitin-ligase E6 AP MBV4230 Coactivators: both HAT-complexes and Mediator MBV4230

CARM1 = coactivator associated arginine methyltransferase Two-hybrid approach CARM1 = H3 specific HMT bait CARM1 MBV4230 Recruitment of two types of activating histone modification enzymes NR p160 acetyl + methylation Me

CARM AD1 AD2 Me Me Ac Ac Ac Similar recruitment of PRMT1 = H4-specific HMT MBV4230 Coregulators = targets for signaling tissue/promoter-specific effects?

Coregulator function modulated by distinct signaling pathways Hypothesis: phosphorylation codes determine the functional specificity of the coregulator for distinct NRs and promoters. Specificity? MBV4230 Chain of events

Ending repression Chromatin opening Kinase-mediated signaling pathways TRAP/DRIP directly contacts basal trx machinery initiation Variability MBV4230 Chain of events Ending repression

Chromatin opening may communicate directly with NR-regulated promoters. AF-1 phosphorylation might serve to further consolidate ligand-dependent NR-SRC interactions or to recruit SRCs directly to the promoter in the absence of ligand. TRAP/DRIP directly contacts components of the basal trx machinery to effect initiation Binding of SRCs and CBP local HAT activity and disruption of nucleosomal structure. Kinase-mediated signaling pathways

Binding of ligand dissociation of corepressors recruitment of SWI/SNF remodeling machines certain TAFs may afford some additional input into promoter-specific NR trx. Variability Local coactivator requirements may varyfor example, a promoter in a readily accessible chromatin context may not require significant chromatin remodeling or HAT activity for assembly of PIC. Type III: Orphan receptors MBV4230 3. subfamily: dimeric orphan receptors

Definition orphan receptor large family with over 30 subfamilys dimeric OR binds both DRn and IR Typically strong constitutive activators/repressors May function independent of ligand Eks 1: hepatocyte nuclear factor 4 (HNF4)

Based on homology = nuclear receptor ligand unknown homodimer Strong constitutive activator Eks 2: COUPs dominant repressors competitive binding + repressor domain + inactive RXR-dimers Function may be to keep target genes turned off in absence of ligand? MBV4230

The hunt for unknown ligands Number of OR > number probable ligands probably several ligand-independent ORs Seminar Nurr1 active without a ligand - its LBD resembles an agonist-bound, trx active LBDs in other NRs Nurr1 LBD contains no cavity as a result of the tight packing Nurr1 lacks a 'classical' binding site for coactivators. Those forming RXR-heterodimers are candidates for ligand-dependent NRs

MBV4230 Variant subfamilys: Monomeric orphan receptors binds a half site AGGTCA + 5-extension through CTE/minor groove Eks steroidogenic factor SF-1 expressed in adrenals, gonades, placenta controls production of other nuclear receptors Half receptors

Only LBD (e.g. DAX1) Only DBD (e.g. Knirps) MBV4230 Monomer orphan receptors CTE: Minor Groove contact Major Groove contact NR and the diet MBV4230 Bioactive lipids and

their nuclear receptors Cholesterol, fatty acids, fat-soluble vitamins, and other lipids present in our diets are not only nutritionally important but serve as precursors for ligands that bind to receptors in the nucleus. MBV4230 Bioactive lipids and their nuclear receptors

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