Allosteric Modulation of Nuclear Receptor Function Open Access

Weikum, Emily Rye (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/12579t01f?locale=en
Published

Abstract

Nuclear receptors are a family of ligand-regulated transcription factors that control specific gene programs across numerous biological processes. The assembly of distinct transcriptional complexes drives regulatory specificity, each complex attuned to a particular gene-, cell- and physiologic-context. These distinct complexes are influenced by allosteric effectors, such as DNA sequence and ligands, which modulate nuclear receptor function. These collective works utilize structural biology and biochemistry to examine these allosteric effectors of nuclear receptor function. We explore the idea that different DNA sequences alter nuclear receptor structure. We show that the glucocorticoid receptor can interact directly with a sequence within inflammatory gene promoters. This finding represents a paradigm shift in our understanding of how the glucocorticoid receptor could repress transcription at these sites. We also show the first reported crystal structure of germ cell nuclear factor bound to its DNA response element. This nuclear receptor is critical in development and understanding the DNA binding properties of this protein can gleam insight into its function. In addition to DNA sequences, we also structurally characterize the glucocorticoid receptor ligand-binding domain in complex with a widely used and potent clinical ligand. As there are only a few GR ligand binding domain structures reported, this work provided structural mechanisms driving this highly stabilizing ligand. Furthermore, this work also reports the first glucocorticoid receptor structure in complex with a peptide from the atypical coregulator, small heterodimer partner. Collectively, this work reviews the idea that these allosteric modifications drive different NR surfaces that are read by coregulator proteins, resulting in alternative transcriptional programs.

Table of Contents

Abbreviations 1-5 Chapter 1: Introduction 7 Nuclear Receptor Superfamily 8

Classifications 9-10 Structural Insight Into Nuclear Receptor Action 11

Overall Architecture 11-13 Nuclear Receptor-Ligand Interactions 13-14 Nuclear Receptor-DNA Interactions 14 Nuclear Receptors can form monomers, dimers, or heterodimers 14-15 Nuclear Receptor-Coregulator Interactions 15-16

Nuclear Receptor Signaling 16 Nuclear Receptor Mechanism of Action 16-17 Transactivation and Transrepression 17-18

Nuclear Receptors as Critical Pharmaceutical Targets 18-19 Tables and Figures

Table 1.1: Nuclear Receptor Superfamily 20-22 Figure 1.1: Modular Domain Structure of Nuclear Receptors 23 Figure 1.2: Nuclear Receptor DNA Binding Domains 24 Figure 1.3: Nuclear Receptor Ligand Binding Domains 25

8

Figure Figure Figure Figure Figure

Figure

References

1.4: Nuclear Receptor-Ligand Interactions
1.5: Genomic Response Elements
1.6: Nuclear Receptor Dimerization Interfaces 1.7: Nuclear Receptor-Coregulator Interactions 1.8: Schematic of Nuclear Receptor

Signaling Mechanisms

1.9: Nuclear Receptors both Activate and Repress Transcription

26-27 28-29 30-31 32-33

34-35

36-37 38-53

54
56 57-59 59-64 60-62 63-64 65-67 67-76 68-70 70-71 72-75

Chapter 2: Glucocorticoid Receptor Control of Transcription: Precision and Plasticity via Allostery

Author Summary Abstract
Introduction GR-Genome Interactions

Direct and indirect sequence-specific binding in vitro

Context-specific genomic occupancy in vivo

GRE Context and Regulatory Logic Allosteric Effectors of GR

DNA binding sequences LBD-binding ligands Post-translational modifications

9

Composite GRE-bound non-GR TRFs

Coregulators as GR signaling readers

Basis of GR-coregulator interactions Functional classes of GR coregulators Structural and enzymatic complexes Chromatin remodeling complexes Methyltransferases

Histone acetyltransferases (HATs)

Histone deacetylases (HDACs)

Precision and Plasticity Via Allostery Conclusions and Perspectives Acknowledgements
Figures and Tables

Figure 2.1: Glucocorticoid receptor signaling and DNA binding

Figure 2.2: Modes of site-specific glucocorticoid receptor-genome interactions

Figure 2.3: Context-specific glucocorticoid receptor occupancy and gene regulation

Figure 2.4: Glucocorticoid receptor-ligand interactions

Figure 2.5: Sites of glucocorticoid receptor post-translational modifications

75-76 76-81 77 77-78 78-79 80

80 80-81 81 81-82 83-84 85

86-87 88-89

90-91 92-93

94

10

Figure 2.6: A model of transcriptional regulation - precision and plasticity of TRF function achieved via allostery

Table 2.1: Five functional classes of regulators reported to interact with GR

Supplemental Table 2.1: Methods to probe glucocorticoid receptor-DNA interactions

References

95-96 97

98-102

103-136

Chapter 3: The Glucocorticoid Receptor Binds Directly to a GR Half-Site Sequence Embedded Within AP-1 Recognition Motifs

Author Summary Abstract Introduction Results

GR is recruited to AP-1 target genes in a DNA-binding dependent manner

GR Ser425Gly mutation cannot be used to distinguish DNA-binding-dependent from independent mechanisms

GR represses AP-1 target genes in the absence of tethering factors GR binds directly to TREs
GR recognizes TREs in a sequence specific manner
GR and AP-1 likely compete for the same binding site

Discussion
Materials and Methods

137
139 140-142

143-144

144-145 145-146 146-147 148-149 149-150 151-154 155-161

11

Reporter Gene Assays 155

ChIP-PCR 156-157

Protein Expression and Purification 158

Nucleic Acid Binding Assays 158-159

NMR Analysis 159-160

Structure Determination of GR-TRE Complexes 160

Generation of Lys442Ala/Arg447Ala "DNA Dead" and GR Ala458Thr/Ile634Ala (GR mon) mutants 161

TR-FRET Competition Assays 161 Acknowledgements 162 Figures and Tables 163-173

Figure Figure

Figure Figure

Figure

References 174-183

3.1: WT GR, but not GR Ser425Gly, is recruited to TREs
in the absence of tethering factors
163-164

3.2: WT GR, but not Lys442Ala/Arg447Ala, is able to
transrepress inflammatory genes
165-166

3.3: GR binds a variety of TRE sequences 167 3.4: Monomeric GR is preferred to repress inflammatory

genes 168-169 3.5: Crystal structures of GR DBD bound to the IL11 TRE,

VCAM1 TRE, and a GBS 170-171

3.6: GR and AP-1 compete for the same DNA binding site 172 Table 3.1: Data collection and refinement statistics 173

Figure

12

Chapter 4: Structural analysis of the glucocorticoid receptor ligand-binding domain in complex with triamcinolone acetonide and a fragment of the atypical coregulator, SHP

Author Summary 184 Abstract 186 Introduction 187-189 Materials and Methods 190-194

Protein expression and purification 190 Protein crystallization, data collection, and structure determination 190-191

Ligand binding and competition assays Differential scanning fluorimetry (DSF) ProSMART analysis
Molecular Dynamics Simulations

Results

191-192 192 192-193 193-194 195-200 195-196

GR LBD over Dex, similar to MF 196-197 TA increases intramolecular contacts within the LBD to drive affinity and

Structural analysis of AncGR2-Triamcinolone/SHP complex
The C-17 acetonide moiety on TA increases the affinity and stability for

stability
Recognition of the SHP NR Box1 LXXLL Motif

198-199 199-200 201-203 204

Discussion Acknowledgements

13

Figures and Tables

205-214 205

206 207-208 209-210 211-212

213
214 215-224

Figure Figure

Figure Figure Figure Figure

4.1: Structure of the AncGR2-TA-SHP NR Box 1 Complex

4.2: MD simulations support LBP-Hydrogen bonding interactions

4.3: TA readily competes Dex out of the binding pocket and highly stabilizes the LBD

4.4: Comparison of glucocorticoid ligands bound to AncGR2 LBD

4.5: Structural comparison of different GR-ligand complexes

4.6: AncGR2-TA complex is bound by atypical coregulator, SHP

Table 4.1: Data collection and refinement statistics

References

Chapter 5: Structural Investigation into Oct4 Regulation by Orphan Nuclear Receptors, Germ Cell Nuclear Factor (GCNF) and Liver Receptor Homolog-1 (LRH-1)

Author Summary 225 Abstract 227 Introduction 228-231 Results 232-236

GCNF and LRH-1 directly bind the mOct4 DR0 232 Structural analysis of GCNF and LRH-1 - Oct4 complexes 232-233

14

LRH-1 and GCNF differentially recognize the DR0 of the Oct4 proximal promoter

Discussion Methods

234-236 237-240 241-244

Protein expression and purification
Generation of GCNF DBD Gly149Arg/Pro151Arg and LRH-1 DBD

Arg160Gly/Arg162Pro 242 Sequence alignments and analysis 242 Nucleic acid binding analysis 243 Crystallization, data collection, and structure determination 243-244 Accession Numbers 244

Acknowledgements 245

Figures

Figure

246-254

5.1: Schematic representation of differential regulation of Oct4 by LRH-1 and GCNF 246

Figure
Figure
Figure
Figure
Figure
Table 5.1: Data collection and refinement statistics
254

5.2: GCNF and LRH-1 bind directly to the Oct4 DR0 247
5.3: Structural analysis of GCNF-mOct4 complex 248-249 5.4: Interactions between GCNF molecules 250
5.5: Structural analysis of LRH-1-mOct4 complex 251
5.6: GCNF and LRH-1 comparison 252-253

References 255-260

241-242

15

Chapter 6: Discussion

Conclusions

DNA as an allosteric modulator Ligands as allosteric modulators NRs as scaffolds

Remaining Questions and Future Directions

Can the Oligomeric state of GR distinguish transactivation from transrepression?

Are GR selective gene modulators possible?

What techniques are on the horizon for expanding our understanding of NRs?

Concluding Remarks Figures

Figure 6.1: G-C and A-T base pairs

Figure 6.2: Generation of GR DIMER

References

262-270 262-268 268-269 269-270 270-271

270-272 272

274

274 275-277 275 276-277 278-283

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified

Primary PDF

Supplemental Files