Complexity of Translationally Controlled Transcription Factor Sp3 Isoform Expression

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.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-specifically expressed genes, viral genes, and cell cycle-regulated genes (1, 2). Sp3 contains a highly conserved DNA-binding domain close to the C terminus and two glutamine-rich activation domains in the N-terminal moiety. The expression pattern, the structure, and the DNAbinding properties of Sp3 are very similar to Sp1, which suggested originally that these two proteins exert similar functions. The physiological roles of Sp1 and Sp3, however, appear to be significantly different. Sp1 knock-out mouse embryos are severely retarded in growth, and die after day 10 of embryonic development (3). Sp3-deficient embryos develop until birth, but die invariably of respiratory failure immediately after birth (4). In addition, late tooth and bone developmental processes are impaired in Sp3Ϫ/Ϫ mice (4, 5).Functional analyses of the transcriptional properties of Sp1 and Sp3 also revealed significant differences between these two transcription factors (6). On many reporter constructs containing multiple Sp-binding sites Sp3 is, unlike Sp1, inactive or acts only as a weak activator (7). The molecular basis for the inactivity of Sp3 under these conditions has been mapped to an inhibitory domain located between the second glutamine-rich activation domain and the zinc finger region (8). More recently, 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).All previously published studies with Sp3 (more than 500 ...

Sp3‐Mediated VEGF regulation is dependent on phosphorylation by extra‐cellular signals regulated kinases (Erk)

“…On the contrary, for Sp3, we have observed striking differences of protein half life depending on the presence of growth factors. Our data further increase the complexity of the regulation of Sp3 in particular the fact that different Sp3 isoforms result for internal initiation of translation (Kennett et al, 1997) but also the recent advances showing that sumoylation of Sp3 represses its transcriptional activity (Sapetschnig et al, 2002(Sapetschnig et al, , 2004Spengler et al, 2005). It is interesting to note that the Erk phosphorylation site is situated in the long Sp3 isoform, which behaves as an activator of transcription (Kennett et al, 1997) or a repressor depending on the promoter (Lambiase et al, 2005).…”

“…Sp3 can act as a positive transcription factor as well as a repressor (Hagen et al, 1994;Dennig et al, 1996;Kennett et al, 1997). It is expressed as a full-length 110 kDa protein but some shorter Sp3 isoforms corresponding to internal initiation of translation were also described (Kennett et al, 1997;Sapetschnig et al, 2004). Whether Sp3 acts as an activator or a repressor seems to be regulated by sumoylation (Sapetschnig et al, 2002;Sapetschnig et al, 2004).…”

“…It is expressed as a full-length 110 kDa protein but some shorter Sp3 isoforms corresponding to internal initiation of translation were also described (Kennett et al, 1997;Sapetschnig et al, 2004). Whether Sp3 acts as an activator or a repressor seems to be regulated by sumoylation (Sapetschnig et al, 2002;Sapetschnig et al, 2004). As we have previously demonstrated the importance of phosphorylation by Erks on Sp1 function, we have investigated whether Sp3 was also a target of Erk and if such phosphorylation was required for or influence its activity.…”

Transcription of PRDM1, the master regulator for plasma cell differentiation, depends on an SP1/SP3/EGR‐1 GC‐box

“…Another change was observed for the slower mobility bands indicated as complex 6. These multiple bands related to SP3 protein could be due to the occurrence of the different isoforms for this protein [25]. All these results indicate that this GC-box is a binding site for at least TF SP1 and SP3.…”

Sp1/Sp3 compound heterozygous mice are not viable: Impaired erythropoiesis and severe placental defects

“…However, detailed biochemical and molecular studies have revealed significant differences. For instance, Sp1 but not Sp3 is a highly O-linked glycosylated protein (Jackson and Tjian, 1988;Sapetschnig et al, 2004), and four expressed isoforms of Sp3 (Sapetschnig et al, 2004) are targets for SUMO modification mediating transcriptional repression (Ross et al, 2002;Sapetschnig et al, 2002Sapetschnig et al, , 2004.…”