学位论文详细信息
Regulation of Sp2 DNA-Binding Activity and trans-Activation
DNA-binding;regulation;Sp2
Moorefield, Kristopher Scott ; Jonathan M. Horowitz, Committee Chair,Robert C. Smart, Committee Member,SpencerV.Muse, Committee Member,Jim W. Mahaffey, Committee Member,Moorefield, Kristopher Scott ; Jonathan M. Horowitz ; Committee Chair ; Robert C. Smart ; Committee Member ; SpencerV.Muse ; Committee Member ; Jim W. Mahaffey ; Committee Member
University:North Carolina State University
关键词: DNA-binding;    regulation;    Sp2;   
Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/3745/etd.pdf?sequence=1&isAllowed=y
美国|英语
来源: null
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【 摘 要 】

Regulated transcription requires the collaboration of a variety of transcription factors, including sequence-specific DNA-binding proteins.The Sp-family of DNA-binding proteins governs the expression of a wide variety of mammalian genes, including housekeeping, cell cycle-regulated, and developmentally regulated genes.A wealth of information has been obtained about the functional and biochemical properties of two Sp-family members, Sp1 and Sp3, whereas studies of other Sp proteins have been limited.The purpose of this research has been to characterize the functional and biochemical properties of Sp2, and to provide insights into mechanisms regulating Sp2 DNA-binding activity and trans-activation.To initiate my analysis of Sp2 I first identified its consensus DNA-binding sequence (5'-GGGCGGGAC-3') using a PCR-based protocol, and determined that Sp2 binds this sequence with high affinity in vitro (225 pM).Despite the incorporation of this consensus sequence within the promoter of a well-characterized Sp-dependent gene, I found that Sp2 was a relatively weak activator of transcription compared with Sp1 and Sp3.To begin to define mechanisms limiting Sp2 function, chimeric proteins carrying portions of Sp2 and Sp1 were created and analyzed.These studies demonstrated that Sp2 DNA-binding activity and trans-activation are each negatively regulated in mammalian cells.In mixing experiments I detected an activity in mammalian extracts that abrogates Sp2 DNA-binding activity.I showed further that an 84 kDa mammalian protein bound to the trans-activation domain of Sp2, but not that of Sp1 or Sp3.Phosphatase treatment revealed that Sp2 DNA-binding activity requires phosphorylation, and phosphoamino acid analysis confirmed that Sp2 is phosphorylated in vivo.Size-exclusion chromatography indicated that Sp2 is specifically phosphorylated in vitro by an activity in fractions carrying high-molecular weight proteins or protein complexes.Indirect immunofluorescence studies indicated that, unlike other Sp proteins, the vast majority of Sp2 localizes to sub-nuclear foci associated with the nuclear matrix.The data reported herein indicates that Sp2 is functionally distinct from other Sp proteins, and provides insight into mechanisms that negatively regulate Sp2-mediated transcription.Taken together, my results suggest that Sp2 may perform a highly specialized role in the regulation of gene expression.

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