The protein half-life of TBP is greatly enhanced by blocking its SUMOylation, suggesting that triggering proteolysis might be one of the most prominent functions of TBP SUMOylation. However, how does SUMO alter TBP protein stability remains unclear. SUMOylation of TBP might direct participate in regulating its stability. Alternatively, the longer half-life of TBP 6KR could be an indirect effect caused by other defects in the SUMO-deficient mutant. Both of these two possibilities will be discussed below.
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Figure 9-1. A proposed model for function of SUMOylation on TBP promoter dynamics. (A). A
structural model illustrating the ―open‖ and ―close‖ configurations of TBP, which are defined by the position of the N-terminal domain relative to the TBP main body. The C-terminal core domain is a blue saddle. The black stick is the N-terminal domain containing six SUMO sites labelled in red. The position of K47 is underneath the concave side of the saddle. The switch between two configurations is regulated by SUMOylation (green oval). (B). The open form of TBP can be stably bound to DNA, and the exposure of SUMO sites promotes SUMOylation. Once SUMOylated, the physical hindrance from K47 as well as the ―closing‖ of the N-terminal domain renders TBP unstable on DNA, which in turn releases TBP from DNA. Unbound TBP are able to rebind to DNA as soon as it is deSUMOylated and switched to an open form.
9.4.1. SUMOylation of TBP might actively promote proteolysis of TBP proteins
A recent report revealed that SUMOylation of Gcn4, which is a yeast transcription activator, facilitates its promoter clearance via Srb10-mediated
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degradation (Rosonina et al., 2012). In this study, Gcn4 is SUMOylated via two lysine residues within the activation domain, and, compared to its wild-type counterpart, SUMO-deficient Gcn4 displays higher promoter occupancy at Gcn4-targeted genes and leads to elevated expression levels of these targets when Srb10- and SUMO- independent degradation pathway is inhibited. In this case, SUMOylation directly facilitates degradation of a transcription factor.
As mentioned in the Introduction, a protein family named ―SUMO-targeted ubiquitin ligases‖ (STUbLs) is able to recognise SUMOylated substrates and facilitate SUMO-dependent ubiquitylation, thereby promoting protein degradation (Praefcke et al., 2012). In Saccharomyces cerevisiae, two classes of STUbL have been identified. These include Uls1 and the Slx5-Slx8 complex (Lescasse et al., 2013; Mullen and Brill, 2008; Uzunova et al., 2007). They have been shown to be involved in proteolytic control of SUMO conjugates. If SUMO directly facilitates ubiquitin-dependent degradation of TBP, STUbL could be the bridge between the modifications.
Many studies have pointed out the importance of quantitative and temporal control of transcription factors. The overexpression of a transcription factor or expressing it at improper timing could result in deleterious consequences. For instance, the cellular level of Myc, which activates genes involved in cell growth and proliferation, is tightly regulated by ubiquitin-mediated proteolysis, and its de-regulation is associated with oncogenic activity (King et al., 2013; Thomas and Tansey, 2011). Another example is p53, whose turnover is rapid under normal condition and whose stability is increased upon DNA damage to trigger cell cycle arrest (Blattner et al., 1999; Pellegata et al., 1996). Similar quantity controls are observed in TBP. In mouse F9 embryonal carcinoma cells as well as C2C12 myoblasts, TBP and its associated factor TAF4 are selectively depleted via
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proteasome-dependent degradation during cell differentiation, and the phenomenon is not observed when the differentiation is impaired or abolished (Perletti et al., 2001). This represents another line of evidence which highlights a critical role for cellular TBP levels. In chicken DT40 lymphoid cells, reducing TBP protein population by half contributes to slow growth, delayed mitosis and increased apoptosis. By contrast, overexpression of TBP can lead to oncogenic transformation, and TBP levels are indeed greatly increased in Ras oncogene transformed cells (Bush et al., 2008; Davidson, 2003). Overall, these findings imply that expression of TBP might be under tightly spatiotemporal regulation.
9.4.2. Higher protein stability of TBP 6KR might be caused by longer retention on
promoters
In this study, I observed an extended protein half-life for TBP after blocking its SUMOylation. Based on Gcn4 where SUMOylation directly triggers its degradation and facilities the clearance from promoters, it might be tempting to speculate that SUMO plays an active role in regulating proteolysis of TBP. However, TBP is different from Gcn4 in many aspects.
Firstly, in contrast to the high promoter occupancy of non-SUMOylatable Gcn4, TBP 6KR has much lower enrichment at all the promoters I tested. It was subsequently proved by CLK and FRAP that the low occupancy is caused by decreased promoter dynamics. Therefore, non-SUMOylatable TBP is less mobile upon binding to DNA, thereby remaining in a bound-state. Secondly, a recent report revealed a vital role for de-ubiquitylating enzyme Ubp3 (ubiquitin-specific protease 3) in TBP-mediated transcriptional activation (Chew et al., 2010). Deletion of ubp3 not only reduces TBP protein stability and causes transcriptional defects but also contributes to an accumulation of poly-ubiquitylated TBP when the proteasome-based
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proteolysis system is impaired. Furthermore, ChIP analysis revealed that Ubp3 is recruited to GAL1 and HIS3 promoters upon induction, suggesting that promoter-bound TBP is prevented from degradation by the de-ubiquitylation activity of Ubp3.
According to this evidence, promoter-bound TBP is less likely to be degraded due to protection by Ubp3, and promoter-bound TBP 6KR is less likely to be released from DNA due to its low mobility. These facts together imply that the higher protein stability of non-SUMOylatable TBP 6KR is an indirect consequence of its low dynamics on the promoters rather than a direct effect from SUMOylation.