5. Gene regulation by NRs

• CASC


  Gene Regulation by Nuclear Receptors
• Nuclear receptors


  Ligand dependent transcription factors
• Nuclear receptors


  • Modular Structure: DBD=DNA binding domain; LBD=ligand binding domain; AF= activation function
  • Zinc fingers of DNA binding domain & Response elements: NRs activate transcription by binding to regulatory response elements of target genes
  • Ligand binding: Induces coactivator recruitment and transcriptional activation
• Types of nuclear receptors

  • Type I : "steroid" receptors AR, ERa, ERb, PR, GR : Homodimeric (generally)
  • Type II : TR, RAR, PPARg; RXR. Act as repressors in absence of agonist : Heterodimeric with RXR
  • Type III : Orphan nuclear receptors: heterodimeric (ligands unknown to date) Heterodimeric with RXR
• Autocrine


  Affects the cell producing them e.g., growth factors
• Paracrine


  Diffuse short distance to affect cells nearby e.g., neurotransmitters
• Endocrine


  Acts on target cells distance from site of synthesis
• Hormone


  Convey information from endocrine cells to hormone-sensitive target cells
• Characteristics of a hormone

  • Chemical agents
  • Synthesized and secreted by glands
  • Circulate in the blood to other parts of the body
  • Stimulate specific tissues (target tissues)
• Lipid soluble hormones


  • Transported in blood by carrier proteins
  • Diffuse through plasma membrane
  • Alters expression of genes at level of nucleus
• Water soluble hormones


  • Easily travel in blood
  • Bind to receptors on the surface of the cell
  • Results in series of intracellular events
• Where hormones are produced

  • Pineal
  • Hypothalamus
  • Pituitary
  • Thyroid
  • Parathyroid
  • Adrenals
  • Pancreas (endocrine/exocrine)
  • Testis
  • Ovary
• Examples of Natural Nuclear Receptor ligands

  • Cortisol (Adrenal cortex)
  • Thyroid Hormone (T4) (Thyroid)
  • Vitamin D (cholecalciferol)
  • All-trans-retinoic acid (Vitamin A)
• Steroid (Type I) Nuclear Receptor Action: Homodimers

  1. Cytoplasmic Activation: Binding of ligand causes dissociation of HSPs and activation of receptor

  2. Homodimerisation: Activated receptors form homodimers

  3. Nuclear Translocation and DNA Binding: Homodimers translocate to nucleus and bind to response elements

  4. Recruitment of Coactivators and Chromatin Remodeling: Coactivators with HAT, methyltransferase, and demethylase activities remodel chromatin

  5. Mediator Complex and Transcription Initiation: Coactivator complex recruits Mediator complex to initiate transcription
• Repression by Unliganded Type II Nuclear Receptor Action: RXR Heterodimers


  1. Unliganded Receptor in the Nucleus: Type II receptors form heterodimers with RXR and repress transcription in absence of ligand

  2. Recruitment of Corepressors and Repressive Complexes: Corepressors, HDACs, repressive methyltransferases, and chromatin remodeling complexes induce transcriptional repression
• Activation of Type II Nuclear Receptor Action: RXR Heterodimers

  1. Ligand Binding Relieves Repression: Binding of ligands like ATRA or 9-cis-retinoic acid relieves repression

  2. Recruitment of Coactivators and Chromatin Remodeling: Coactivators with HAT, methyltransferase, and demethylase activities remodel chromatin to activate transcription

  3. Mediator Complex and Transcription Initiation: Coactivator complex recruits Mediator complex to initiate transcription
• Ligand Binding Domain


  • Key role in receptor dimerization and ligand-dependent transactivation
  • In Type I (steroid) receptors agonist binding induces: a. heat stress protein (HSP) dissociation, b. nuclear translocation, c. receptor dimerization, d. rearrangement of helix 12 rendering coactivator binding surfaces accessible, e. recruitment of coactivators and transcriptional activation
  • In type II/III heterodimeric receptors, ligand binding induces coactivator recruitment and transcriptional activation
• Nuclear Receptors exhibit a preference for selective response elements
• Pioneer Factors


  Guide nuclear receptors to their response elements
• LBD


  Ligand Binding Domain
• LBD facilitates receptor dimerization

  1. In absence of agonist, or presence of antagonist, Helix 12 interacts with corepressor complexes

  2. Agonist binding induces formation of Helix 11 and realignment of Helix 12 which creates a surface accessible to coactivator binding
• Nuclear Receptors : Ligand Binding Domain helix 12


  The key determinant of coregulator interactions
• Response elements


  • ERE (DR3)
  • AG GT CAnnnTG AC CT TC CA GTnnnAC TG GA
  • ARE (DR3)
  • AG AA CAnnnTG TT CT TC TT GTnnnAC AA GA
  • GRE (DR3)
  • AG AA CAnnnTG TT CT TC TT GTnnnAC AA GA
  • RARE (DR5)
  • AG GT CAnnnnnAG AC CA TC CA GTnnnnnTC TG GT
• How do Nuclear Receptors find their response elements? They are guided to the response elements by Pioneer Factors
• Ligand Binding Domain : Functions

  • Key role in receptor dimerization and ligand-dependent transactivation
  • In Type I (steroid) receptors agonist binding induces: a. heat stress protein (HSP) dissociation, b. nuclear translocation (steroid receptors), c. receptor dimerization, d. rearrangement of helix 12 rendering coactivator binding surfaces accessible, e. recruitment of coactivators and transcriptional activation
  • In type II/III heterodimeric receptors agonist binding induces: corepressor dissociation, coactivator recruitment
• NRs regulate transcription at hormone response elements (DNA) by influencing the rate of assembly and recruitment of general transcription factors
• A series of events facilitate cycles of transactivation and transcriptional termination

  1. Nucleosomes (packaged DNA) must be re-modelled in preparation for transcription

  2. Coactivators must dock with the ligand activated NRs

  3. Coactivators and scaffolding proteins recruit basal transcriptional machinery including TBP (TATA binding protein) and TAF IIs (TBP-associated factors) to the promoter

  4. In the absence of agonist transcription is terminated

  5. Corepressor complexes are recruited to silence the promoter
• Histones


  Small basic proteins, core histone H2A, H2B, H3 and H4
• Nucleosome


  ~147 base pairs DNA wrapped 1.7 times around a histone octamer, two copies each of H2A, H2B assoc. with an H3H4 tetramer
• Heterochromatin


  • Condensed DNA, Transcriptionally silenced, Hypo-acetylated histones, Hyper-methylated histones
• Euchromatin


  • Transcriptionally active, Hyper-acetylated histones, Hypo-methylated and hyper-phosphorylated
• Chromatin status influences gene expression
• Histone Tails are subject to numerous reversible covalent modifications


  • Lysine acetylation
  • Lysine methylation (mono, di and tri methylation)
  • Lysine lactylation
  • Lysine crotonylation
  • Lysine acylation
  • Arginine methylation
  • Serine phosphorylation
  • Threonine phospohylation
  • Glutamine serotonylation
• Histone Tails are subject to multiple and in some cases mutually exclusive Covalent Modifications
• Histone H3 Modifications


  • Transcriptional activation: Histone H3-serine 10 phosphorylation, H3-lysine14-acetylation, H3-lysine4/36/79-methylation
  • Transcriptional repression: Histone H3 and H4 lysine deacetylation, H3-lysine 9 methylation (mono-, di- and tri-) mediated by G9a and Suv39H recognized by HP1 (heterochromatin protein 1) associated repressive complex, H3-lysine 27 methylation (recognized by polycomb repressive complex)
• Histone Modifications = transcriptional cues
• Lysine acetylation causes chromatin to open up / decondense

  1. Acetylation masks histone charges resulting in charge-charge repulsion between the octamer and DNA backbone

  2. Acetylated H3/H4 decrease the overall positive charge associated with the histone octamer

  3. And this decreases the electrostatic interaction between the histone complex and the negatively charged DNA backbone
• KMTs


  Lysine Methyl Transferases, Examples: MLL / EZH2 / G9a / SET
• Epigenetic patterns flanking gene transcriptional start sites often are cell type specific (ENCODE)
• Everything is mechanistically linked! Epigenetics and Epitranscriptomics