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Transcription factors (TFs) bind to specific DNA sequences near target genes to precisely coordinate their regulation. Despite the central role of transcription factors in development and homeostasis, the mechanisms by which TFs have evolved to bind and regulate distinct DNA sequences are poorly understood. This dissertation details the highly collaborative work to determine the genetic, biochemical and biophysical mechanisms by which distinct DNA-binding specificities evolved in the steroid receptor (SR) family of transcription factors. Using ancestral protein reconstruction, we resurrected and functionally characterized the historical transition in DNA-binding specificity between ancient SR proteins. We found that DNA-binding specificity evolved by changes in the energetic components of binding; interactions at the protein-DNA interface were weakened while inter-protein cooperativity was greatly improved. We identified a group of fourteen historical substitutions that were sufficient to recapitulate the derived protein's binding function. Three of these substitutions, which we defined as function-switching, were sufficient to change DNA specificity; however, their introduction greatly decreased binding affinity and was deleterious for protein function. A group of eleven permissive substitutions, which had no effect on DNA specificity, allowed for the protein to tolerate the deleterious effects of the function-switching substitutions. They non-specifically increased binding affinity by improving interactions at the protein-DNA interface and increasing inter-protein cooperativity. We then dissected the functional role of individual substitutions in both the function-switching and permissive groups. We first determined the binding affinity of all possible combinations of function-switching substitutions for a library of DNA sequences. This allowed for us to functionally characterize the sequence space that separated the ancestral and derived DNA-binding specificities as well as identify the genetic determinants for DNA specificity. Lastly, we dissected the effects of the permissive substitutions on the energetics of DNA binding to determine the mechanisms by which they exerted their permissive effect. Together, this work provides insight into the molecular determinants of DNA specificity and identifies the molecular mechanisms by which these interactions changed during the evolution of novel specificity in an important transcription factor family. This dissertation includes previously published and unpublished co-authored material. / 2016-01-14

Lui, Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-171). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.v / Acknowledgements --- p.vii / Abbreviations --- p.ix / Table of Content --- p.x / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Overview and Endocrinology of hormones and hormone receptors --- p.1 / Chapter 1.2 --- Hormone receptors: membrane bounded receptors --- p.3 / Chapter 1.3 --- Hormone receptors: steroid nuclear receptors --- p.4 / Chapter 1.4 --- "Estrogen, estrogen receptor alpha and beta (ERa, ERβ) and prostate gland" --- p.6 / Chapter 1.5 --- Orphan nuclear receptors --- p.10 / Chapter 1.6 --- The first orphan receptors identified-estrogen receptor related receptors --- p.12 / Chapter 1.6.1 --- Estrogen receptor related receptor alpha (ERRα) --- p.13 / Chapter 1.6.2 --- Estrogen receptor related receptor alpha (ERRβ) --- p.17 / Chapter 1.6.3 --- Estrogen receptor related receptor alpha (ERRγ) --- p.19 / Chapter 1.7 --- Aim of study --- p.21 / Figure 1.1 Mechanism of activation of classical nuclear receptor by ligand --- p.23 / Figure 1.2 Distribution of ERa and ERβ in human body --- p.24 / Chapter Chapter 2. --- Methods and Materials / Chapter 2.1 --- Origin and supply of Noble rats --- p.25 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Cell lines and culture media --- p.26 / Chapter 2.2.2 --- Cell culture onto cover slips for immunohistochemistry --- p.27 / Chapter 2.3 --- RNA preparation / Chapter 2.3.1 --- Total RNA extraction --- p.27 / Chapter 2.3.2 --- mRNA extraction by Oligote´xёØ procedure --- p.29 / Chapter 2.3.3 --- mRNA extraction by Fast Track 2.0 procedure --- p.30 / Chapter 2.4 --- Molecular cloning by Rapid Amplification of cDNA Ends (RACE) / Chapter 2.4.1 --- Molecular cloning of rERRα --- p.31 / Chapter 2.4.2 --- Molecular cloning of rERRβ --- p.36 / Chapter 2.4.3 --- Molecular cloning of rERRγ --- p.42 / Chapter 2.5 --- Molecular cloning into pCRII TOPO cloning vector --- p.47 / Chapter 2.6 --- Sequencing analysis of DNA sequence by dRodamine® or BigDye® --- p.47 / Chapter 2.7 --- DNA sequence analysis --- p.49 / Chapter 2.8 --- Reverse transcription and RT-PCR --- p.49 / Chapter 2.9 --- Southern blotting analysis / Chapter 2.9.1 --- Preparation of DNA blot membrane --- p.51 / Chapter 2.9.2 --- Purification of DNA fragment from agarose gel for DIG-DNA labeling --- p.52 / Chapter 2.9.3 --- Preparation of the DIG-labeled DNA probe --- p.53 / Chapter 2.9.4 --- Membrane hybridization and colorimetric detection --- p.53 / Chapter 2.10 --- In-situ hybridization histochemistry / Chapter 2.10.1 --- Linearization of DNA plasmid --- p.55 / Chapter 2.10.2 --- Synthesis of riboprobe --- p.56 / Chapter 2.10.3 --- Hybridization and detection --- p.56 / Chapter 2.11 --- Western blotting analysis / Chapter 2.11.1 --- Protein extraction --- p.59 / Chapter 2.11.2 --- Casting of SDS-PAGE electrophoresis --- p.59 / Chapter 2.11.3 --- Polyacrylamide gel electrophoresis --- p.61 / Chapter 2.11.4 --- Protein blotting analysis --- p.61 / Chapter 2.12.1 --- Immunohistochemistry / Chapter 2.12.1 --- Histological preparation --- p.63 / Chapter 2.12.2 --- Immunohistochemistry --- p.64 / Table 1. List of culture media --- p.66 / Table 2. Primer sequences for RACE-PCR --- p.67 / Table 3. PCR conditions for RT-PCR --- p.68 / Table 4. Primer sequences for RT-PCR --- p.68 / Table 5. Reagent mixtures for linearization of the plasmid DNA --- p.69 / Table 6. Riboprobe synthesis by in-vitro transcription --- p.70 / Chapter Chapter 3. --- Results / Chapter 3.1 --- Cloning of full-length cDNA of rERRs by RACE-PCR --- p.71 / Chapter 3.2 --- Cloning of full-length cDNA of rERRα from rat ovary cDNA library --- p.72 / Chapter 3.3 --- Cloning of full-length cDNA of rERRβ from rat ventral prostate --- p.76 / Chapter 3.4 --- Cloning of full-length cDNA of rERRγ from rat prostate --- p.80 / Chapter 3.5 --- Expression distribution of ERRs detected by RT-PCR --- p.83 / Chapter 3.6 --- mRNA expression of ERRs detected by in-situ hybridization --- p.86 / Chapter 3.7 --- Protein expression of ERRa and ERRγ detected by western blotting --- p.87 / Chapter 3.8 --- Expression of ERRa and ERRγ detected by immunohistochemistry --- p.88 / Figure 3.1 Full-length DNA sequence of rERRα --- p.92 / Figure 3.2 Predicted amino acid sequence of rERRα --- p.93 / "Figure 3.3 DNA sequence alignment of rat, mouse and human ERRα" --- p.94 / "Figure 3.4 Amino acid sequence alignment analysis of rat, mouse and human ERRα" --- p.95 / Figure 3.5 Full-length DNA sequence of rERRβ --- p.96 / Figure 3.6 Predicted amino acid sequence of rERRβ --- p.97 / "Figure 3.7 DNA sequence alignment of rat, mouse and human ERRβ" --- p.98 / "Figure 3.8 Amino acid sequence alignment analysis of rat, mouse and human ERRβ" --- p.99 / Figure 3.9 Full-length DNA sequence of rERRγ --- p.100 / Figure 3.10 Predicted amino acid sequence of rERRγ --- p.101 / "Figure 3.11 DNA sequence alignment of rat, mouse and human ERRγ" --- p.102 / "Figure 3.12 Amino acid sequence alignment analysis of rat, mouse and human ERRγ" --- p.103 / Figure 3.13 Restriction enzyme cutting of full-length plasmids --- p.104 / Figure 3.14 Expression pattern of rERRα in male sex accessory sex glands by RT-PCR --- p.105 / Figure 3.15 Expression pattern of rERRα in urinary system and female sex organs by RT-PCR --- p.106 / Figure 3.16 Tissue expression of rERRα by RT-PCR --- p.107 / Figure 3.17 In-situ hybridization of ERRα in ovary --- p.108 / Figure 3.18 Western blotting of ERRα --- p.109 / Figure 3.19 Immunohistochemistry of ERRα in ovary --- p.110 / Figure 3.20 Expression pattern of rERRβ in male sex accessory sex glands by RT-PCR --- p.111 / Figure 3.21 Expression pattern of rERRβ in urinary system and female sex organs by RT-PCR --- p.112 / Figure 3.22 Tissue expression of rERRβ by RT-PCR --- p.113 / Figure 3.23 In-situ hybridization of ERRβ in rat prostate --- p.114 / Figure 3.24 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.115 / Figure 3.25 Expression pattern of rERRγ in male sex accessory sex glands by RT-PCR --- p.116 / Figure 3.26 Expression pattern of rERRy in urinary system and female sex organs by RT-PCR --- p.117 / Figure 3.27 Tissue expression of rERRγ by RT-PCR --- p.118 / Figure 3.28 Expression pattern of rERRγ in different prostatic cancer cell lines and xenografts by RT-PCR --- p.119 / Figure 3.29 In-situ hybridization of ERRγ in rat prostate --- p.120 / Figure 3.30 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.121 / Figure 3.31 Western blotting of ERRγ --- p.122 / Figure 3.32 Immunohistochemistry of ERRγ in ERRy-transfected MCF-7 cells --- p.123 / Figure 3.33 Immunohistochemistry of ERRγ in ventral prostate of rat --- p.124 / Figure 3.34 Immunohistochemistry of ERRγ in lateral prostate of rat --- p.125 / Figure 3.35 Immunohistochemistry of ERRγ in dorsal prostate of rat --- p.126 / Figure 3.36 Immunohistochemistry of ERRγ in testis of rat --- p.127 / Figure 3.37 Immunohistochemistry of ERRγ in epididymis of rat --- p.128 / Figure 3.38 Immunohistochemistry of ERRγ in brown adipose tissues of rat --- p.129 / Figure 3.39 Immunohistochemistry of ERRγ in brain of rat --- p.130 / Figure 3.40 Immunohistochemistry of ERRγ in brain of rat --- p.131 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- Sequence analysis of the full-length cDNA sequences of the rat estrogen receptor-related receptors (ERRs) --- p.132 / Chapter 4.2 --- Ligand independence and constitutive self-activation of estrogen receptor-related receptors --- p.133 / Chapter 4.3 --- Board expression pattern of estrogen receptor-related receptors --- p.138 / Chapter 4.3.1 --- Board expression pattern of estrogen receptor-related receptor alpha --- p.138 / Chapter 4.3.2 --- Board expression pattern of estrogen receptor-related receptor beta --- p.140 / Chapter 4.3.3 --- Board expression pattern of estrogen receptor-related receptor gamma --- p.141 / Chapter 4.4 --- Expression of ERRs in the prostate gland --- p.143 / Chapter 4.5 --- Expression of ERRs in the prostatic cell lines and cancer xenografts --- p.147 / Chapter 4.6 --- Expression of ERRs in the ERRγ-transfected MCF-7 cells --- p.149 / Chapter 4.7 --- Expression of ERRs in the testis and epididymis --- p.149 / Chapter 4.8 --- Expression of ERRs in the adipose tissue --- p.150 / Chapter 4.9 --- Expression of ERRs in the ovary --- p.151 / Chapter 4.10 --- Expression of ERRs in the brain --- p.153 / Figure 5.1 Map of full-length clone of rERRα --- p.155 / Figure 5.2 Map of full-length clone of rERRβ --- p.156 / Figure 5.3 Map of full-length clone of rERRα --- p.157 / Figure 5.4 Comparison of the homology of amino acid sequences amongst ERs and ERRs --- p.158 / Figure 5.5 Phylogeny tree of nuclear receptors --- p.159 / Figure 5.6 Relationship of different prostatic cell lines and xenografts --- p.160 / Chapter Chapter 5. --- Summary --- p.161 / References --- p.163-171

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 186-201). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 103-113).

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 190-204). Available also in a digital version from Dissertation Abstracts.

The mouse node is the structural equivalent of Spemann's Organizer. The transcription factor Foxa2 (HNF3beta) is required for node and notochord specification and its enhancer has been characterized to a 500bp element that drives the expression of a reporter gene to the node and notochord in transgenic embryos. / The aim of the study was to identify sequence elements, within this enhancer, required to drive node/notochord specific-expression. Since, in the Xenopus organizer, Siamois activates organizer-specific gene expression, it was tested whether this factor could activate expression from the Foxa2 node-specific enhancer. Using deletion analysis, the response element was mapped to a 33bp region and it was shown that this element was both necessary and sufficient for reporter gene activation by X-Siamois . Furthermore, it was shown that X-Siamois can bind this DNA element and two sequence motifs required for binding and transactivation by X-Siamois were identified. Preliminary results suggest that the 33bp element, within the 500bp enhancer, is required for the maintenance of expression in the notochord of transgenic mice.