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MASTERMIND-LIKE 1-Dependent Notch Target Gene Activation Requires a Sequence-Paired Site and a TATA Box

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MASTERMIND-LIKE 1-Dependent Notch Target Gene Activation Requires a Sequence-Paired Site and a TATA Box San Jose State University SJSU ScholarWorks Master's Theses Master's Theses and Graduate Research Summer 2013 MASTERMIND-LIKE 1-Dependent Notch Target Gene Activation Requires a Sequence-Paired Site and a TATA Box Cassandra Agbayani Ramos San Jose State University Follow this and additional works at: https://scholarworks.sjsu.edu/etd_theses Recommended Citation Ramos, Cassandra Agbayani, "MASTERMIND-LIKE 1-Dependent Notch Target Gene Activation Requires a Sequence-Paired Site and a TATA Box" (2013). Master's Theses. 4360. DOI: https://doi.org/10.31979/etd.rh9z-z2ej https://scholarworks.sjsu.edu/etd_theses/4360 This Thesis is brought to you for free and open access by the Master's Theses and Graduate Research at SJSU ScholarWorks. It has been accepted for inclusion in Master's Theses by an authorized administrator of SJSU ScholarWorks. For more information, please contact [email protected]. MASTERMIND-LIKE 1-DEPENDENT NOTCH TARGET GENE ACTIVATION REQUIRES A SEQUENCE-PAIRED SITE AND A TATA BOX A Thesis Presented to The Faculty of the Department of Biological Sciences San José State University In Partial Fulfillment of the Requirements for the Degree Master of Science by Cassandra Agbayani Ramos August 2013 © 2013 Cassandra Agbayani Ramos ALL RIGHTS RESERVED The Designated Thesis Committee Approves the Thesis Titled MASTERMIND-LIKE 1-DEPENDENT NOTCH TARGET GENE ACTIVATION REQUIRES A SEQUENCE-PAIRED SITE AND A TATA BOX by Cassandra Agbayani Ramos APPROVED FOR THE DEPARTMENT OF BIOLOGICAL SCIENCES SAN JOSÉ STATE UNIVERSITY August 2013 Dr. Brandon White Department of Biological Sciences Dr. Elizabeth Skovran Department of Biological Sciences Dr. Brooke Lustig Department of Chemistry ABSTRACT MASTERMIND-LIKE 1-DEPENDENT NOTCH TARGET GENE ACTIVATION REQUIRES A SEQUENCE-PAIRED SITE AND A TATA BOX by Cassandra Agbayani Ramos Notch signaling plays an important role in mammalian cellular proliferation, apoptosis, and differentiation. To activate target genes, the Notch intracellular domain (NICD) forms a complex with CBF1 and Mastermind-like protein (MAM). The Notch activation complex binds to a response element containing the consensus sequence RTGRGAR (R= purine) on target gene promoters. The promoter of one well characterized target gene, Hes1, contains four Notch response elements (NREs). Site- directed mutagenesis and reporter gene assays were used to examine the activation of Hes1 by mutating individual and combinations of NREs. Results indicated that NREs 2 and 4 are important for Hes1 activity, confirming previous results that this sequence- paired site (SPS) is an important feature of Notch target genes. Orientation and spacing between elements on the SPS were mutated in these studies. Other mutations included the Hes1 TATA box and the spacing between the SPS and TATA box. Additionally, a charged residue on NICD was mutated; this residue is believed to be responsible for Notch complex dimers. These results showed that SPS spacing and orientation as well as dimerization of Notch complexes are important for the promoter activity of Hes1. The TATA box was found to be necessary for promoter activation, and this activity is independent of spacing between the SPS and TATA box up to 173 base pairs. Taken together, these findings suggest that NICD- and MAM-mediated activation of target genes requires orientation- and spacing-dependent SPS and TATA box elements. ACKNOWLEDGMENTS I extend my deepest gratitude to my principal investigator and thesis advisor, Dr. Brandon White. I will always be grateful for the research opportunities he has given me. Working with Dr. White is a constant pleasure and I am proud to say that I have had an ideal M.S. program experience in his research laboratory. He has been an outstanding mentor, who has always pushed me to become a better scientist. I am grateful for all his helpful discussions, practice presentations, career advice, and generous support. Not only is Dr. White a wonderful advisor, but he is a deeply caring individual who has always been dedicated to my personal and professional growth. I would also like to thank my thesis committee members, Drs. Elizabeth Skovran and Brooke Lustig. Their helpful comments, suggestions, and encouragement have been invaluable for my thesis progress. They have been a treasured support system for me as I complete my M.S. program. I am also grateful for the wonderful collaboration and support from my fellow labmates: Dr. Elaina Chambers, Zackery Bevens, Anthony Bortolazzo, Christine Ha, Danny Ha, Asmara Hoo, Tetiana Lialiutska, Vy Ly, Nikki Nguyen, Anh Pham, Anh Truong, and Ivan Zein. I also extend my thanks to Thomas Burke, a former student in Dr. White’s lab, for creating the original Hes1 individual NRE site mutant plasmids used in my research. I would like to acknowledge the SJSU Department of Biological Sciences Scholarship Committee for awarding me the Glenn and Martha Vargas Scholarship and v Albert and Dorothy Ellis Fellowship. These funds have been greatly beneficial to my research progress. I would also like to acknowledge the CSU Program for Education and Research in Biotechnology and the American Society for Biochemistry and Molecular Biology for awarding me travel grants. These awards allowed me to present my thesis work and communicate with scientists from around the world. This experience provided much professional development and many memorable experiences. I owe many thanks to the wonderful professors who have helped me grow as a student and scientist through their classes: Drs. Luis Bonachea, Daniel Holley, Peter Nemes, Leslee Parr, Michael Sinensky, and Miri VanHoven. Finally, I would like to thank my mom, Daniel, my family, and my friends for lending helping hands through difficult times and sharing laughs and smiles in times of celebration and triumph. Completing this thesis would not have been possible without their loving support. vi DEDICATIONS This thesis is dedicated to my mom, Lorraine Agbayani. She is my personal hero and lifelong teacher. Her immense courage, strength, and love are always my inspiration. She reminds me that biomedical research is about helping the people that we hold dearest in our hearts. This thesis is also dedicated to Daniel F. Coronado, my loving and supportive partner who makes every day feel like a dream come true. vii TABLE OF CONTENTS INTRODUCTION .............................................................................................................. 1 LITERATURE REVIEW ................................................................................................... 7 Overview of Notch Signaling ........................................................................................ 7 Overview of Transcription in Class II Promoters .................................................... 13 Hes1 Promoter and Sequence-Paired Sites ............................................................... 16 EXPERIMENTAL PROCEDURES ................................................................................. 21 Materials and Equipment ........................................................................................... 21 Generation of Mutant Plasmids by Site-Directed Mutagenesis .............................. 22 Transfection into HeLa Cells ..................................................................................... 24 Reporter Gene Assays ................................................................................................. 28 Statistical Analysis ....................................................................................................... 28 RESULTS ......................................................................................................................... 30 Activation of Hes1 with Notch 1 and Mastermind-like 1 ........................................ 30 Notch 1- and Mastermind-like 1-Mediated Hes1 Activation with Mutated Notch Response Elements ........................................................................................... 31 Hes1 Promoter Activity with a Notch Dimerization Mutant .................................. 32 Notch- and Mastermind-like-Mediated Activation of Hes1 with a Sequence- Paired Site Separated by Additional Base Pairs ...................................................... 33 viii Notch- and Mastermind-like-Mediated Activation of Hes1 with Different Orientations of the Sequence-Paired Site .................................................................. 34 Notch- and Mastermind-like-Mediated Activation of Target Genes with a Mutated TATA Box .................................................................................................... 36 Notch- and Mastermind-like-Mediated Activation of Hes1 with Altered Base Pair Spacing between the Sequence-Paired Site and TATA Box ........................... 38 DISCUSSION ................................................................................................................... 40 REFERENCES ................................................................................................................. 46 ix LIST OF FIGURES FIGURE 1: Overview of Notch signaling ......................................................................... 2 FIGURE 2: Model of transcription initiation by Notch complex .................................. 2 FIGURE 3: Hes1 gene fragment used for promoter analysis studies ............................ 3 FIGURE 4: Crystal structure of Notch complex dimers assembled on the Hes1 promoter. ..........................................................................................................................
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