THE DEC1 TRANSCRIPTION FACTOR: ONCOGENIC INVOLVEMENT AND MOLECULAR MECHANISMS ON TRANSCRIPTION REGULATION

The basic helix-loop-helix (bHLH) proteins are int imately associated with developmental events such as cell differentiation and lineage commitment. The HLH domain in the bHLH motif is responsible for dimerization whereas the basic region mediates sequence specific DNA binding. Human DEC, mouse STRA and rat SHARP proteins represent a new c lass of bHLH proteins. In each species, two members are identified wi th a sequence identi ty of>90% in the bHLH region and 40% in the total protein, respectively. Based on sequence alignment and domain analysis, DEC/STRA/SHARP proteins show high s imilarities (40%) to Drosoph ila Hairy and E(spl) as well as the mammalian homologues (e.g., HES) in the bHLH domain. However, they lack the C-terminal WRPW motif which is present at all other Ha iry/E(spl)/Hes proteins and mediates transcription repression. These structural d ifferences distinguish DEC/STRA/SHARPs from other bHLH proteins and indicate that they have dist inct biological functions. The purpose of this dissertation is to elucidate the oncogenic roles of DEC I and determine the molecular actions of DEC I on transcription regulation. Expression measurments demonstrate that DEC I is abundantly expressed in cancer tissues but not in adjacent nonmal tissues. In stable transfectants, DEC I inhibits ce ll proliferation, antagonizes serum deprivation-induced apoptosis, and selectively decreases the activities of several major caspases. Western blott ing analyses identify that antiapoptot ic protein survivin is a fu nctional mediator responsible for DEC I-directed antiapoptotic activity. DEC I and survivin exhibit a paralleled expression pattern in paired tumor-normal tissues. In co-transfection experiments, DEC I stimulates the survivin promoter, and this mechanism relies on the physical interactions with Sp I sites in the proximal promoter. In contrast, DEC I and its structurally related protein DEC2 show an inverted expression pattern in paired tumor-normal tissues. Forced expression of DEC I causes proportional decreases in the expression of DEC2 in stable transfectants. Co-transfection with DEC I represses the activity of a DEC2 promoter reporter by as much as 90%. The DEC I mediated transcription repression is achieved by direct binding to the E-box element in the proximal promoter of DEC2. The established activation of the survivin promoter provides a molecular explanation for its oncogenic involvement, and the differential activities toward DEC2 and survivin promoters establish that DEC I can act as a repressor or an activator depending on the genomic context of a target gene. ACKNOWLEDGEMENT This dissertation is a culmination of several years of work, during which time, I have greatly benefited from interactions wi th a great many friends, teachers, and family members who have taught me, encouraged me and supported me. I would first like to express my s incere gratitude to my mentor Dr. Bingfang Yan for his leadership, discipline and knowledge that guided me throughout this endeavor. I consider mysel f extremely fortunate to have worked with him. I would also like to express my sincere appreciation to the members of my dissertation commettee, Ors Nasser H. Zawia, Gongqin Sun, Kayvous Parang, and Fatem Akahlaghi, for their time, their consideration and their valuable suggestions. I would also thank all of the past the present members of Dr. Yan 's research group for their friendship, camaraderie and readiness to help. Special thanks to Dr. He Zhang for his friendship and help, to Dr. Mingxing Xie for conducting the western blots described in manuscript Ill , to Dr. Xiulong Song for cloning the mutant reporters described in manuscript IJI and to Dr. Ryaz Mohammad fo r his help with the gel shift assay. Finally, I wou ld like to thank my wife Ying Wang. She supported me throughout my whole study period. I would also like to thank to my parents and my parents-in-law, for their love, support and encouragement. TABLE OF CONTENTS ABSTRACT . ii ACKNOWLEDGEMENT. iv TABLE OF CONTENTS . v LIST OF TABLES . LIST OF FIGURES . •. •.