Regulation of YY1, a Multifunctional Transcription Factor
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University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 5-2001 Regulation of YY1, a Multifunctional Transcription Factor Ya-Li Yao University of South Florida Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Yao, Ya-Li, "Regulation of YY1, a Multifunctional Transcription Factor" (2001). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/1544 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Office of Graduate Studies University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL __________________ Ph.D. Dissertation __________________ This is to certify that the Ph.D. Dissertation of YA-LI YAO with a major in Medical Sciences has been approved for the dissertation requirement on April 9, 2001 for the Doctor of Philosophy degree. Examining Committee: _________________________________________________ Major Professor : Edward Seto, Ph.D. _________________________________________________ Member : Burt Anderson, Ph.D. _________________________________________________ Member: Peter Medveczky, M.D. _________________________________________________ Member: Richard Jove, Ph.D. REGULATION OF YY1, A MULTIFUNCTIONAL TRANSCIPTION FACTOR by YA-LI YAO A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medical Microbiology and Immunology College of Medicine University of South Florida May 2001 Major Professor: Edward Seto, Ph.D. DEDICATION To my parents, Ta-Chung (Righteous and Magnanimous) and Shiao-Hung (Little Rainbow). ACKNOWLEDGEMENTS I am eternally grateful to my Lord, Who gave me the chance to fulfill my dream of earning a Ph.D. degree. I would like to thank my mentor, Dr. Edward Seto, for his endless support and guidance. Without him I would never have accomplished my goal. My gratitude also extends to my committee members, who gave me invaluable advice and helped me meet the requirements of the department. My special acknowledgement goes to my husband, Dr. Wen-Ming Yang, whose help and encouragement accompanied me when I needed them the most. I thank Dr. Nancy Olashaw for critical reading and the core facilities at the Moffitt Cancer Center as well as the Protein Chemistry Core at the University of Florida for technical support. I would also like to express my gratitude to Kathy (Mom), Linda, Susan, Cheryl, Sally, and B.J. They made my life as a graduate student much easier and happier. TABLE OF CONTENTS LIST OF FIGURES iv ABSTRACT viii INTRODUCTION 1 Transcription is an important step in gene regulation. 1 Transcription in prokaryotes. 2 Transcription in eukaryotes. 3 RNA polymerase II and the eukaryotic promoter. 4 Activators and transcriptional activation. 6 Repressors and transcriptional repression. 8 Chromatin and transcription. 9 Chromatin remodeling. 10 Histone acetyltransferases as co-activators. 11 Histone deacetylases as co-repressors. 13 YY1 is a transcriptional regulator. 13 General characteristics of YY1. 14 YY1 and transcriptional initiation. 15 OBJECTIVES 17 MATERIALS AND METHODS 20 Isolation of YY1 genomic clones. 20 i Plasmids. 20 Sequencing of the human YY1 promoter. 22 Primer extension. 22 Cell line, transfection, luciferase assays, and CAT assays. 23 Recombinant proteins. 23 In vitro acetylation reactions. 24 Immunoprecipitation and western blot analysis. 24 In vitro protein-protein interaction assays. 25 Histone deacetylation assays. 25 Immunofluorescence analysis. 26 Electrophoretic mobility shift assays. 26 Gel filtration analysis of YY1. 27 Purification of a YY1 complex by anion exchange and immobilized- metal affinity chromatography. 27 Coomassie staining and silver staining. 27 Purification of a YY1 complex by anion exchange and antibody- affinity chromatography. 28 Purification of the F-YY1 complex. 28 Radioimmunoprecipitation analysis (RIPA) of F-YY1. 28 Accession number. 29 RESULTS 30 Determination of the transcriptional start site of the human YY1 gene. 30 ii Transcriptional analysis of the human YY1 promoter. 31 Sequence analysis of the human YY1 promoter. 33 E1A-mediated repression of the human YY1 promoter. 35 YY1 does not autoregulate its own promoter. 37 YY1 is acetylated by p300 and PCAF. 38 Lysine-to-arginine mutations within YY1 residues 170-200 significantly reduce the transcriptional repression activity of YY1. 45 Acetylation of YY1 residues 170-200 increases YY1 binding to HDACs. 46 HDAC1 and HDAC2 deacetylate YY1 170-200 but not the C- terminal region of YY1. 46 HDACs bind YY1 at multiple regions. 52 YY1 contains associated histone deacetylase activity, which localizes 52 to C-terminal residues 261-333 of YY1. Acetylation of YY1 at the C-terminal zinc finger domain decreases 54 the DNA-binding activity of YY1. YY1 exists in a high molecular weight protein complex. 57 Purification of native YY1 complexes. 57 Purification of a Flag-tagged YY1 complex. 65 DISCUSSION 70 REFERENCES 80 ABOUT THE AUTHOR End Page iii LIST OF FIGURES FIG. 1. Step-wise assembly of the transcriptional initiation complex. 5 FIG. 2. Regulation of transcription: a model. 7 FIG. 3. Determination of the 5’ end of the human YY1 transcript. 30 FIG. 4. Expression of luciferase enzymatic activity driven by the human YY1 promoter in transiently transfected cells. 32 FIG. 5A. DNA sequences upstream of the translational start codon of the human YY1 gene. 33 FIG. 5B. Comparison of the YY1 promoter DNA sequences between mouse and human. 34 FIG. 6. Adenovirus E1A proteins repress the YY1 promoter. 36 FIG. 7. The YY1 protein does not autoregulate the YY1 promoter. 37 FIG. 8. Multiple functional domains of transcription factor YY1. 38 FIG. 9A. Acetylation of YY1 by PCAF. 39 FIG. 9B. Acetylation of YY1 by p300. 40 FIG. 9C. Acetylation of YY1 is dependent on p300 or PCAF. 41 FIG. 9D. YY1 is acetylated in vivo. 42 FIG. 9E. Identification of regions of YY1 acetylated in vivo. 43 FIG. 9F, G. Determination of the number of acetylated lysines by mass spectrometry. 44 iv FIG. 10. The effect of acetylation of YY1 residues 170-200 on the transcriptional repressor activity of YY1. 45 FIG. 11. Increased HDAC-binding to YY1 residues 170-200 by acetylation. 47 FIG. 12A. YY1 peptide deacetylation by HDACs. 48 FIG. 12B. YY1 protein deacetylation by HDACs. 49 FIG. 12C. Deacetylation of YY1 serial deletion proteins by HDAC1. 50 FIG. 13. Mapping of the HDAC-interaction domains of YY1. 51 FIG. 14A. Histone deacetylase activity of endogenous YY1 in HeLa cells. 52 FIG. 14B, C. Identification of amino acid 261-333 as the histone deacetylase activity domain of YY1. 53 FIG. 14D. Expression of F-YY1 deletion mutants. 54 FIG. 14E. Sub-cellular localization of F-YY1 deletion constructs. 55 FIG. 15A, B. The effect of acetylation on YY1’s sequence-specific DNA- binding activity. 56 FIG. 16. Gel filtration analysis of YY1 in HeLa cells. 57 FIG. 17A, B. Purification of a native YY1 complex from HeLa cells using anion exchange chromatography and IMAC. (A) A Coomassie-stained SDS-polyacrylamide gel of the Q Sepharose fractions. (B) Western blot analysis of the Q Sepharose fractions. 58 FIG. 17C, D. Purification of a native YY1 complex from HeLa cells using v anion exchange chromatography and IMAC. (C). EMSA of the Q Sepharose fractions using the AAV P5+1 YY1 binding sequence as a probe. (D). Histone deacetylation assay of the Q Sepharose fractions. 59 FIG. 17E, F. Purification of a native YY1 complex from HeLa cells using anion exchange chromatography and IMAC. (E) Western blot analysis of the Ni2+ column fractions. (F) EMSA of the 300 mM imidazole-eluted fractions using the AAV P5+1 YY1 binding sequence as a probe. 60 FIG. 17G. Purification of a native YY1 complex from HeLa cells using anion exchange chromatography and IMAC. (G) Silver- staining analysis of the second fraction from the 300 mM imidazole-eluted fractions. 61 FIG. 18A, B. Purification of a native YY1 complex from HeLa cells using anion exchange chromatography and antibody-affinity chromatography. (A) A Coomassie-stained SDS- polyacrylamide gel of the Q Sepharose fractions. (B) Western blot analysis of the Q Sepharose fractions. 62 FIG. 18C, D. Purification of a native YY1 complex from HeLa cells using anion exchange chromatography and antibody-affinity chromatography. (C) EMSA of the Q Sepharose fractions using the AAV P5+1 YY1 binding sequence as a probe. (D) Histone deacetylation assay of the Q Sepharose fractions. 63 vi FIG. 18E. Purification of a native YY1 complex from HeLa cells using anion exchange chromatography and antibody-affinity chromatography: (E) Silver-staining analysis of fractions from the anti-YY1 H-10 column (lane 2) and the protein G control column (lane 1). 64 FIG. 18F. Purification of a native YY1 complex from HeLa cells using anion exchange chromatography and antibody-affinity chromatography. (F) Histone deacetylation assay from the protein G control column (protein G) and the anti-YY1 H-10 column (H-10) against the H4 peptide. 65 FIG. 19A. Silver-staining analysis of the second fractions from the Flag-alone (lane 1) column and the F-YY1 column (lane 2). 66 FIG. 19B. Histone deacetylation analysis of the second fractions from the Flag alone column and the F-YY1 column against the H4 peptide. 67 FIG. 20A. A representative autoradiograph showing proteins bound to Flag alone or F-YY1. 68 FIG. 20B. A representative autoradiograph showing proteins bound to different F-YY1. 69 FIG. 21 Summary of regulation of YY1 by acetylation and deacetylation. 72 vii REGULATION OF YY1, A MULTIFUNCTIONAL TRANSCRIPTIONAL FACTOR by YA-LI YAO An Abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medical Microbiology and Immunology College of Medicine University of South Florida May 2001 Major Professor: Edward Seto, Ph.D.