Abstract
Sp3 is a ubiquitous transcription factor closely related to Sp1. Both proteins contain a highly conserved DNA-binding domain close to the C terminus and two glutamine-rich domains in the N-terminal moiety. Immunoblot analyses of Sp3 reveal a striking complex protein pattern of up to eight distinct species. This pattern is not observed in Sp3-deficient cell lines showing that all signals reflect Sp3 antigen. In this study, we have unraveled the complexity of Sp3 expression. We show that four isoforms of Sp3 that retain different parts of the N terminus are expressed in vivo. The four isoforms derive from alternative translational start sites at positions 1, 37, 856, and 907. An upstream open reading frame located at position -47 to -18 regulates expression of the two long isoforms. Unlike Sp1, none of the Sp3 isoforms is glycosylated. However, all four isoforms become SUMO-modified in vivo and in vitro specifically and exclusively at lysine residue 551. The transcriptional activity of the two long isoforms strongly depends on the promoter settings, whereas the small isoforms appear to be inactive. The transcriptional activity of all the Sp3 isoforms is regulated by SUMO modification. Our results demonstrate that Sp3 has many unique features and is not simply a functional equivalent of Sp1.
Highlights
The transcription factor Sp3 is a ubiquitously expressed member of the Sp family of transcription factor that is involved in the expression and regulation of many genes, including housekeeping genes, tissue- expressed genes, viral genes, and cell cycle-regulated genes [1, 2]
It was shown that Sp3 is post-translationally modified by the small ubiquitin-like modifier (SUMO)1 within its inhibitory domain and that SUMO modification leads to inactivation [9, 10]
In vitro translation products of wild-type Sp3 as well as of the Sp3⌬1.AUG mutant lacking the first isoform of Sp3 but not of the Sp3K551R mutant are targets for SUMOylation in vitro (Fig. 4C). These results show that lysine 9 within the VKQE motif, which is only present in the longest Sp3 isoform, is not a target for SUMO modification, neither in vivo nor in vitro
Summary
The transcription factor Sp3 is a ubiquitously expressed member of the Sp family of transcription factor that is involved in the expression and regulation of many genes, including housekeeping genes, tissue- expressed genes, viral genes, and cell cycle-regulated genes [1, 2]. Expression of the four Sp3 isoforms observed in immunoblots can be reconstituted in a coupled in vitro transcription/translation assay using the full-length Sp3 cDNA clone (Fig. 1B, lane 2). The additional Sp3 species observed exclusively in SDS-lysed cell extracts can be reconstituted when in vitro translated Sp3 proteins were subjected to an in vitro SUMO modification reaction [10] (Fig. 1B, lane 1).
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