Browsing by Author "Jonathan M. Horowitz, Committee Chair"

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    Expression and developmental requirement for transcription factor Sp2
    (2009-08-10) Yin, Haifeng; Jonathan M. Horowitz, Committee Chair
    The Sp-family of DNA-binding proteins is comprised by nine members, and controls the expression of many mammalian genes. Most Sp-family members have been well studied with respect to their patterns of expression as well as requirement for mammalian development. However, little information regarding the expression and function of Sp2 was available. Our laboratory has reported that Sp2 is widely expressed in murine and human cell lines, Sp2 DNA-binding activity and trans-activation are negatively regulated in vitro, and that the vast majority of Sp2 localizes to sub-nuclear foci associated with the nuclear matrix. To extend these studies, expression of the mouse Sp2 locus was analyzed in detail and the requirement for Sp2 for mouse development was evaluated via the creation of a conditional "knock-out" mouse strain. To initiate the analysis of Sp2 I first identified transcriptional start sites using a PCR-assisted (5'RACE) strategy. Sequencing of 5’RACE clones showed that transcriptional start sites clustered within three regions of the Sp2 locus producing three types of transcripts: Exon 2A (type I), Exon 4 (type II) and Exon 5 (type III). Synthesis of type I transcripts was shown to be directed by a promoter that contains sequences encoding at least four discrete enhancer and inhibitory elements. Additional promoters were not identified within the Sp2 locus. Using RT-PCR and RNA in situ hybridization assays, Sp2 was shown to be widely expressed at embryonic and post-natal stages and it's expression was noted to be particularly concentrated in brain sub-regions. Sp2 conditional "knock-out" mice were generated via the insertion of loxP sites flanking the first two Sp2 coding exons. The developmental consequences of loss of Sp2 function were evaluated following matings with three Cre recombinase-carrying strains that express Cre in a widespread (CMV-Cre) or tissue-restricted fashion (Keratin 14-Cre and Keratin14-Cre/Estrogen receptor fusion). Sp2 hemizygous animals are indistinguishable from wild-type animals, whereas nullizygous animals perish early in gestation. These data indicate that Sp2 is an essential gene at the organismal level, yet we have also shown that Sp2 nullizygous cells are viable in adult, mosaic animals. Constitutive (Keratin 14-Cre) or induced (Keratin 14-Cre/Esr) expression of the Cre recombinase in basal keratinocytes resulted in a dramatic increase in stem cell proliferation and the loss of Keratin 5 and Keratin 15 expression in these cells. Taken together, we conclude that Sp2 is required for early embryogenesis and regulates the proliferation and differentiation of adult progenitor cells.
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    Regulation of Sp2 DNA-Binding Activity and trans-Activation
    (2005-08-02) Moorefield, Kristopher Scott; Jonathan M. Horowitz, Committee Chair; Robert C. Smart, Committee Member; Spencer V. Muse, Committee Member; Jim W. Mahaffey, Committee Member
    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.