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The Binding of Estrogen, Progesterone and Glucocorticoid

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The Binding of Estrogen, Progesterone and Glucocorticoid THE BINDING OF ESTROGEN, PROGESTERONE AND GLUCOCORTICOID RECEPTORS TO THEIR RECOGNITION SITES IN A NUCLEOSOME AND THE EFFECT OF HMGB1 ON THE BINDING AFFINITY Yaw Acheampong Sarpong A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 2006 Committee: William Scovell, Advisor Thomas Kinstle Scott Rogers ii ABSTRACT William M. Scovell, Advisor The eukaryotic genome is packaged in a chain of nucleosomes called chromatin. For transcription to take place in chromatin, RNA polymerase II, transcriptional activators and general transcription factors must interact with their binding sites in the regulatory region of specific genes. When these sites occur in nucleosome, the binding of the transcription factors (TF) is generally reduced drastically and in some cases, may not even bind to these regulatory sites. It therefore follows that, for the TFs to bind, the DNA must be remodeled or moved out of the nucleosome, or the contacts between the histone protein and DNA must be weakened by some mechanism. It has been previously reported that estrogen receptor (ER) binds to its recognition site when in a nucleosome in a dose dependent manner. We find, however, that ER does not bind even at the highest ER concentration of 160 nM. We show that estrogen receptor does not bind to estrogen response element (ERE) when DNA is reconstituted in a nucleosome. However, the ubiquitous protein, high mobility group box-1 (HMGB1) protein relieves ER binding repression and allows ER to bind with a dissociation constant (KD ) of about 60 nM. We also find that ER binds to 2 different translationally positioned ERE on the nucleosome with the same KD values in the presence of HMGB1 protein. We suggest that HMGB1 protein does not strip the nucleosome off the DNA but rather makes the contact between the histone and DNA weaker to allow ER to bind. We show that HMGB1 protein does not form a stable part of the complex formed when ER binds to DNA and (or nucleosome). Our data indicate that glucocorticoid receptor (GR) acts more like a “bubble gum” because it binds to all DNAs and nucleosomes that we tested, with a KD of iii approximately 2.5 nM. HMGB1 protein does not have any significant effect in enhancing the binding affinities for GR. PR binds to glucocorticoid response element (GRE) with a KD value of 2.5 nM. The binding affinity is enhanced in the presence of HMGB1 protein to a low KD of 0.9 nM. However, when GRE was reconstituted into a nucleosome, PR fails to bind even in the presence of HMGB1 protein. iv This work is dedicated my parents v ACKNOWLEDGEMENTS I would like to extend my gratitude to my thesis advisor, Dr. Scovell, for his advice and guidance throughout my stay at his lab. I thank Dr Kinstle and Dr Rogers for sitting in as committee members. Dr Peterson for making all the DNAs, you are appreciated. I would also like to thank all faculty and staff of the Department of Chemistry for giving the opportunity to pursue a graduate degree and to Dr Brecher especially, for the daily food for thought. My sincere thanks go to my teachers from University of Ghana, Dr Adjimani and Dr Okine. Lastly I would like to thank everyone who made this project happen. Thank you. Jah guidance. vi TABLE OF CONTENTS Page CHAPTER 1 1. Chromatin structure 1 2. HMGB-1 protein 5 3. Nucleosome positioning sequences 6 4. Rotational phasing and translational positioning of DNA within a nucleosome 7 5. Nucleosome and transcription 7 CHAPTER 2 MATERIALS AND METHOD 6. Materials 12 7. Isolation of erythrocytes from chicken blood 13 8. Isolation of nuclei from chicken erythrocytes 13 9. Micrococcal nuclease 14 10. Proteinase K 14 11. Preparation of samples for micrococcal nuclease digestion 14 12. Monitoring micrococcal nuclear digestion by % acid solubility 15 13. DNA extraction from micrococcal nuclease digested products 16 14. Sucrose gradient centrifugation 17 15. Isolation of H1 free oligonucleosomes 18 16. Sepharose CL-4B column preparation 19 vii 17. Preparation of DNA constructs with ERE and GRE at different translational positions 20 18. Labeling of DNA 27 19. Separation of end-labeled DNA from free ATP 27 20. Nucleosome reconstitution 28 21. Estrogen receptor 29 22. Progesterone receptor 30 23. Electrophoretic mobility shift assay 30 24. Gel preparation and electrophoresis 31 25. Preparation of ER samples 33 26. PAGE separation of DNA labeled at only one end 39 27. Separation of DNA fragments from polyacrylamide gel 39 28. Preparation of G/A ladder for DNA sequencing gel 40 29. Quantification of radioactivity in gel bands to determine KD values 41 CHAPTER 3 RESULTS 30. Preliminary studies to characterize the digestion profile of nuclei with micrococcal nuclease 43 CHAPTER 4 DISCUSSION 31. Binding of nuclear receptors to free DNA and the effect of HMGB1 on the binding 95 32. Binding of nuclear receptors to its recognition site in a nucleosome 97 33. The effect of HMGB1 on the integrity of the nucleosome 104 viii TABLE OF FIGURES Page 1. The crystal structure of the nucleosome core particle at 2.8 Å resolution 3 2. Diagram showing histone interaction between H3 & H4, H2A &H2B 4 3. Domain structure of HMGB1 6 4. A sketch represents 2Eo2 or 2Go2 DNAs 20 5. A sketch represents 4Eo or 4Go DNAs 21 6. A sketch representing the A5 DNA 21 7. (a) Nucleotide sequence of DNA p2E02-pGEM-Q2 with consensus ERE between two segments of nucleosome-positioning sequence 22 (b) Nucleotide sequence of DNA p2G02-pGEM-Q2 with consensus GRE between two segments of nucleosome-positioning sequence 23 (c) Nucleotide sequence of DNA p4E0-pGEM-Q2 with consensus ERE near one end the of nucleosome-positioning segment. 24 (d) Nucleotide sequence of DNA p4G0-pGEM-Q2 with consensus GRE near one end the of nucleosome-positioning segment. 25 8. Preparation of DNA labeled on one strand only 35 9. Micrococcal nuclease digestion profile on chicken erythrocyte nuclei 44 10. Gel electrophoresis of nuclei digested to 12 % acid soluble products and fractionated on sucrose gradients 45 11. Sepharose (CL-4B) Chromatography of oligonucleosomes used as donor chromatin. 46 12. SDS-PAGE (18 %) of nucleosome fractions collected from the Sepharose CL-4B column for MNase digestion of chicken erythrocytes 47 ix Page 13. Sucrose gradients profile of free DNA and nucleosome DNA 48 14. A 4% polyacrylamide gel of the incorporated DNA from the sucrose gradient fractions 49 15. ER binding to free DNA which has the ERE placed at the center of 161 bp long DNA (2Eo2) 52 16. Equilibrium binding profile of ER to 2Eo2 in the absence of HMGB1 53 17. ER binding to free DNA which has the ERE placed at the center of 161 bp long DNA (2Eo2) in the presence of HMGB1 54 18. Equilibrium binding profile of ER to 2Eo2 in the presence of HMGB1 55 19. ER binding to DNA which has the ERE placed at 40 bp of the center of 161 bp DNA (4Eo) 56 20. Equilibrium binding profile of ER to 4Eo in the absence of HMGB1 57 21. ER binding to free DNA which has the ERE placed at the center of 161 bp long DNA (2Eo2) in the presence of HMGB1 58 22. Equilibrium binding profile of ER to 4Eo in the presence of HMGB1 59 23. The binding of ER to free DNA which has the GRE placed at the center of 161 bp DNA (2Go2). 61 24. Equilibrium binding profile of ER to 2Go2 in the absence of HMGB1 62 25. Binding of ER to 2Go2 in the Absence and in the Presence of HMGB1 63 26. The binding of ER to free DNA which has the GRE placed 40 bp off the center of 165 bp DNA (4Go) 65 27. Equilibrium binding profile of ER to 4Go in the absence of HMGB1 66 x Page 28. Binding of ER to 4Go in the Absence and in the Presence of HMGB1 67 29. Binding of ER to n2Eo2 in the absence and in the presence of HMGB1 69 30. Binding of ER to n2Eo2 in the presence of HMGB1 70 31. Equilibrium binding profile of ER to n2Eo2 in the presence of HMGB1 71 32. Binding of ER to n4Eo in the absence and presence of HMGB1 73 33. Binding of ER to n4Eo in the presence of HMGB1 74 34. Equilibrium binding profile of ER to n4Eo in the presence of HMGB1 75 35. Binding of ER to n2Go2 in the absence and presence of HMGB1 78 36. Binding of ER to n2Go2 in the presence of HMGB1 79 37. Equilibrium binding profile of ER to n2Go2 in the presence of HMGB1 80 38. Binding of PR to 2Go2 DNA in the absence of HMGB1 82 39. Binding of PR to 2Go2 DNA in the presence of HMGB1 83 40. Equilibrium binding profile of PR to 2Go2 in the absence and presence of HMGB1 84 41. Binding of PR to 4Go in the absence of HMGB1 85 42. Binding of PR to 4Go in the presence of HMGB1 86 43. Equilibrium binding profile of PR to 4Go in the absence and presence of HMGB1 87 44. Binding of PR to 2Go2 and n2Go2 in the absence and presence 89 45. EMSA of interaction of HMGB1 with 2Eo2 and in nucleosome (n2Eo2) and effect of anti-HMGB1 antibody on HMGB1 92 46. Binding of ER, PR and GR to A5 DNA. 106 xi Page 47. GR binding to n2Eo2 in the absence and presence of HMGB1 107 48.
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