@article{moorefield_yin_nichols_cathcart_simmons_horowitz_2006, title={Sp2 localizes to subnuclear foci associated with the nuclear matrix}, volume={17}, ISSN={["1939-4586"]}, DOI={10.1091/mbc.E05-11-1063}, abstractNote={We have reported that extracts prepared from many human and mouse cell lines show little or no Sp2 DNA-binding activity and that Sp2 has little or no capacity to stimulate transcription of promoters that are activated by Sp1, Sp3, and Sp4. Using an array of chimeric Sp1/Sp2 proteins we showed further that Sp2 DNA-binding activity and trans-activation are each negatively regulated in mammalian cells. As part of an ongoing effort to study Sp2 function and regulation we characterized its subcellular localization in comparison with other Sp-family members in fixed and live cells. We report that 1) Sp2 localizes largely within subnuclear foci associated with the nuclear matrix, and 2) these foci are distinct from promyelocytic oncogenic domains and appear to be stable during an 18-h time course of observation. Deletion analyses identified a 37 amino acid sequence spanning the first zinc-“finger” that is sufficient to direct nuclear matrix association, and this region also encodes a bipartite nuclear localization sequence. A second nuclear matrix targeting sequence is encoded within the Sp2 trans-activation domain. We conclude that Sp2 preferentially associates with the nuclear matrix and speculate that this subcellular localization plays an important role in the regulation of Sp2 function.}, number={4}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Moorefield, KS and Yin, HF and Nichols, TD and Cathcart, C and Simmons, SO and Horowitz, JM}, year={2006}, month={Apr}, pages={1711–1722} }
 @article{spengler_kennett_moorefield_simmons_brattain_horowitz_2005, title={Sumoylation of internally initiated Sp3 isoforms regulates transcriptional repression via a Trichostatin A-insensitive mechanism}, volume={17}, ISSN={["1873-3913"]}, DOI={10.1016/j.cellsig.2004.06.007}, abstractNote={Sp3 is a ubiquitously expressed member of the Sp family of transcription factors that encodes three proteins, Sp3, M1 and M2, with differing capacities to stimulate or repress transcription. As part of ongoing efforts to study the functions of Sp3 isoforms, we employed a yeast “two-hybrid” screen to identify Sp3-binding proteins. This screen resulted in the identification of Ubc9, a SUMO-1 conjugating enzyme, as an M2-binding protein, and consistent with these results sequence analyses identified consensus sumoylation motifs within several Sp family members. Western blots probed with anti-Sp3 detected a high molecular weight Sp3 isoform that is stabilized by a SUMO-1 hydrolase inhibitor, and this protein is also bound by anti-SUMO-1 antiserum. Transient transfection assays with epitope-tagged-SUMO-1 and GFP-SUMO-1 fusion proteins confirmed that Sp3, M1 and M2 proteins are sumoylated in vivo. Substitution of arginine for lysine at one putative site of sumoylation, lysine551, blocked sumoylation of all Sp3 isoforms in vivo and led to a marginal increase in Sp3-mediated trans-activation in insect and mammalian cells. In contrast, introduction of this amino acid substitution within M1 converted it into a potent transcriptional trans-activator. We conclude that Sp3 isoforms are sumoylated in vivo and this post-translational modification plays an important role in the regulation of Sp3-mediated transcription.}, number={2}, journal={CELLULAR SIGNALLING}, author={Spengler, ML and Kennett, SB and Moorefield, KS and Simmons, SO and Brattain, MG and Horowitz, JM}, year={2005}, month={Feb}, pages={153–166} }
 @article{moorefield_fry_horowitz_2004, title={Sp2 DNA binding activity and trans-activation are negatively regulated in mammalian cells}, volume={279}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M313589200}, abstractNote={Previous studies have indicated that Sp2 binds poorly to GC-rich sequences bound by Sp1 and Sp3, and further functional analyses of Sp2 have been limited. To study Sp2-mediated transcription, we employed a PCR-based protocol to determine the Sp2 consensus DNA-binding sequence (5′-GGGCGGGAC-3′) and performed kinetic experiments to show that Sp2 binds this consensus sequence with high affinity (225 pm) in vitro. To determine the functional consequence of Sp2 interaction with this sequence in vivo, we transformed well characterized Sp-binding sites within the dihydrofolate reductase (DHFR) promoter to consensus Sp2-binding sites. Incorporation of Sp2-binding sites within the DHFR promoter increased Sp2-mediated trans-activation in transient co-transfection experiments but also revealed Sp2 to be a relatively weak trans-activator with little or no capacity for additive or synergistic trans-activation. Using chimeric molecules prepared with portions of Sp1 and Sp2 and the human prostate-specific antigen promoter, we show that Sp2 DNA binding activity and trans-activation are negatively regulated in mammalian cells. Taken together, our data indicate that Sp2 is functionally distinct relative to other Sp family members and suggest that Sp2 may play a unique role in cell physiology.}, number={14}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Moorefield, KS and Fry, SJ and Horowitz, JM}, year={2004}, month={Apr}, pages={13911–13924} }
 @article{kennett_moorefield_horowitz_2002, title={Sp3 represses gene expression via the titration of promoter-specific transcription factors}, volume={277}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.M108661200}, abstractNote={We have determined previously that Sp3 encodes three distinct gene products as follows: a full-length protein (Sp3) that is an activator of transcription and two isoforms (M1 and M2) derived via internal translational initiation that function as transcriptional repressors. To identify amino acids and functions required for transcriptional repression, we employed PCR-directed mutagenesis to create a panel of mutated M2 proteins. Biochemical and functional analyses of these mutated proteins indicate that functions encoded by the M2 carboxyl terminus, such as DNA binding activity and the capacity to form multimeric complexes, are not required or sufficient for transcriptional repression. Instead, a 93-amino acid portion of the trans-activation domain was shown to be the minimal portion of M2 required to block Sp-dependent gene expression. Transcriptional analysis of three Sp-dependent promoters showed that mutations sustained by many M2 proteins result in promoter-specific effects. Regions of M2 required for physical interactions with five TATA box-associated factors (TAFIIs) were mapped, and mutations that disrupt the interaction of M2 with two of these proteins, TAFII70 and TAFII40, were identified. We conclude that Sp3- mediated transcriptional repression is due, at least in part, to competition for promoter-specific transcription factors.}, number={12}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Kennett, SB and Moorefield, KS and Horowitz, JM}, year={2002}, month={Mar}, pages={9780–9789} }