Introduction
Sfp1 is found to be a relatively stable protein (Figure 35). Cycloheximide treatment experiments shown here reveal that even after 1 hour of translation inhibition Sfp1 protein is still at half of its initial level as judged by WB. Nevertheless, several different extracellular conditions were shown to influence Sfp1 by controlling its subcellular localization (Marion et al., 2004). Therefore is not surprising that Sfp1 function can be controlled at the post-translational level, for example by phosphorylation. Indeed it has been shown that Sfp1 is a direct target of TORC1 and that TORC1-dependent phosphorylation is important for proper Sfp1 nuclear localization under optimal growth conditions (Lempiainen et al., 2009). However, analysis of the Sfp1 primary sequence (Figure 36) suggests that other kinases, such as PKA or Sch9, could phosphorylate Sfp1. Indeed, constitutively high PKA activity or hyperactive Ras signaling leads to a ~2-fold higher ChIP signal for Sfp1 on some selected RPG promoters (Jorgensen et al., 2004; Marion et al., 2004). These observations prompted us to further analyze Sfp1 phosphorylation under different growth conditions and using mutant strains for PKA and Sch9 kinases. In this chapter, Sfp1-MYC phosphorylation is assessed using different growth conditions and upon inactivation of the SCH9 analog-sensitive allele (sch9as), whose kinase activity can be rapidly inhibited by addition of the ATP analog 1NM-PP1 to the medium.
Figure 35. Sfp1 protein stability
Logarithmic growing cells expressing Sfp1-HA tagged protein were treated with cycloheximide for the indicated time points and samples were collected for TCA protein extraction and SDS-PAGE analysis. Quantification of WB signal intensity is reported in the bottom graph as relative value compared to the WB intensity for the untreated sample.
Figure 36. Sfp1 protein sequence contains PKA and Sch9 possible target sites
Sfp1 protein sequence is represented with RxxS (green) and RRxS (blue) motifs highlighted MDFTTMTMASNMATSTTTTATSAHASINSSSNFNIDIDSNQNTPSILINNNSDSSNGKNTDFNGVNNI HQKNIMNNTNNVHLYSPNIMDQTLLTPQDIAKLRRESIAHSQGMGGVSWGSISVGSWLRDEIISRRN SIVPASANGAASAAASATTTATNTLQIQQPTKRPSVSNPPYHRGYSISPQIAYTAYLPNLEKQYCKDY SCCGLSLPGLHDLLRHYEEAHISTSPNTTNMSQIPMNSAGNTSSSVRMTNNTSSANYNLQNNMAAN TKNAGHKTNTMQAHSSNATNNTSINNMHANLQSNMDSNSTIRQSQHPHHQQNIIQQQLQSNSVNHT SGAVPTPSVMGSATASSTTANPNVISITGAPNSGLSMANHSQQLHLNGNLVDAVSTNDVFLRTSNSP SRHVPHNKQINSNNNSGININNNTSHNSNINMGSKNAMVNRPHTFNNYSLNKTSRNPIQHQSRKIDP HQTDLSPLVLVQDIDLSFMDDDILGPSNHNSMNSVVNPTTGSHNYNTFHSSVHAKSSQNMVEDQDI DDIDDDDDVDDDDDDDDDDDTENGSSSNGKSVHNNNYKMPQQAYIDDPARRLYVMDHEEQKPFK CPVIGCEKTYKNQNGLKYHRLHGHQNQKLHENPDGTFSVIDPDSTDSFGDGMGSAKDKPYRCEVC GKRYKNLNGLKYHRGHSTH*
Sfp1 phosphorylation level signals extracellular changes
In order to define the physiological relevance of phosphorylation as a post-translational control mechanism for Sfp1 activity, we first asked whether growing cells in the presence of different carbon sources could affect Sfp1 phosphorylation. Sfp1-MYC protein mobility was examined by SDS-PAGE in the presence of the reagent Phos-tag that works as a phosphate-binding reagent and retards the mobility of phosphorylated proteins. Cells were grown either in glucose- or glycerol-containing medium and subsequently shifted to glycerol or glucose respectively. After 10, 30 and 60 minutes following the carbon source shift, sample were collected for protein extraction. This experimental set-up allows one to analyze the effect of glucose starvation or, conversely, glucose addition to Sfp1 phosphorylation.
To begin with, we found that Phos-tag containing SDS-PAGE analysis of Sfp1-MYC protein extracted from glucose-grown cells reveals several different mobility-species, suggesting that Sfp1 is a highly phosphorylated protein (Figure 37). However, a phosphatase treatment experiment needs to be done to demonstrate that the lower-mobility species observed here are indeed due to phosphorylation of Sfp1. In the absence of this experiment we will assume for the time being that changes in Sfp1 mobility in the Phos-Tag gels accurately reflect changes in Sfp1 phosphorylation (Figure 37).
When cells are starved for glucose and transferred to glycerol containing medium, the migration pattern observed 10 minutes after the shift strongly resemble the one observed upon rapamycin treatment. Interestingly, after 30 minutes the intensity of the fast migrating species decreases and returns to initial level after 1 hour. On the other hand, when the opposite experiment is performed and starved cells are shifted to glucose, the overall migration pattern is strongly retarded after 10 minutes. As previously observed, after 30 and 60 minutes, the initial condition is restored. Moreover, even though the resolution of the different migration-species is not optimal and the technique requires further improvement, is still possible to distinguish bands that migrate differently according to the condition. As indicated by the arrow in Figure 37, the migrating species just below the 100 KDa reference band is visible in extracts
of unperturbed cultures (lane 0’ “more exposed” blot) and becomes more intense upon rapamycin treatment. Interestingly, this band is completely undetectable upon glucose starvation suggesting that this phosphorylation state of Sfp1 is dependent on glucose but is not affected by TORC1 inactivation. Taken together, these results suggest that shifting cells from glucose to a less favorable carbon source such as glycerol leads to transient de-phosphorylation Sfp1, whereas glucose addition causes a massive increase in Sfp1 phosphorylation. Therefore, Sfp1 phosphorylation is rapidly modified in response to specific extracellular signals but returns to the initial steady-state level shortly thereafter, despite a change in cell growth rate.
Next we wanted to identify other kinases that in addition to TORC1 could phosphorylate Sfp1.
The nutrient sensitive kinases Sch9 and PKA are good candidates since their specific targets sites are found in the Sfp1 primary sequence (Figure 36) and previously data showed (i) increased Sfp1 binding on RPG promoters in the presence of hyperactive PKA activity (Jorgensen et al., 2004; Marion et al., 2004) and (ii) that Sch9 is important for proper activation of RiBi genes, whose transcription is strongly affected by Sfp1 (Huber et al., 2011; Jorgensen et al., 2002). We initially analyzed Sfp1-MYC phosphorylation using the previously described analog-sensitive allele of SCH9 (sch9as; YST460 Table 1; (Jorgensen et al., 2004)).
Interestingly, inhibition of sch9as causes Sfp1 dephosphorylation after 15 minutes from the treatment (Figure 38). Afterwards, as already observed for the carbon shift experiment, initial levels of Sfp1 phosphorylation are rapidly restored (Figure 38). Therefore, this experiment suggests that Sfp1 phosphorylation could be affected by Sch9. Expression profiles for sch9as were determined 30, 60 and 90 minutes after addition of 1NM-PP1 (Jorgensen et al., 2004):
interestingly, RPG expression was constitutively reduced by Sch9 inactivation whereas RiBi genes were only transiently affected. We can speculate that the transient Sfp1 dephosphorylation observed upon sch9as inactivation could (at least in part) be responsible for the decreased RiBi gene expression and, therefore, when Sfp1 initial phosphorylation is
Figure 37. Glucose availability signals through Sfp1 by dynamically modifying its phosphorylation level
SDS-PAGE of TCA protein extracts obtained after shift of logarithmically growing cells to a different carbon source. The different time points indicate time from the treatment. Analysis of Sfp1 phosphorylation was obtained by addition of the Phos-tag reagent into the SDS-PAGE gel. Phos-tag interacts with phosphates and decreases the mobility of a phosphorylated protein consequently. The rectangle indicates the hyper-phosphorylation obtained when glucose is added to previously starved cells. The arrow indicates a possible Sfp1 migrating specie whose phosphorylation level could be dependent on the carbon source.
Figure 38. Sch9 inhibition transiently dephosphorylates Sfp1
A. SDS-PAGE of TCA protein extracts obtained after sch9as inhibition by 1NM-PP1 treatment. Cells were collected after 5, 15, 30 and 60 minutes from the inhibition and rapamycin treated cells were used as control for Sfp1 dephosphorylation. Analysis of Sfp1 phosphorylation was obtained by addition of the Phos-tag reagent into the SDS-PAGE gel. Phos-tag interacts with phosphates and decreases the mobility of a phosphorylated protein consequently. B. Figure from P. Jorgensen et al 2004 showing microarray mRNA analysis of RPG and RiBi gene expression in different mutants sch9as included. It is interesting to compare the transient RiBi gene down-regulation obtained in this experiment after Sch9 inactivation to the transient decrease in Sfp1 phosphorylation obtained using the same conditions.
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Discussion
The work presented in this thesis provides new insights into the mechanism of action of Sfp1, which has until now remained a relatively poorly characterized transcriptional regulator in budding yeast. Results described here allow us to propose that Sfp1 acts as a key factor coordinating growth and cell cycle progression by directly regulating transcription of genes require for ribosomes production, translation and the G1-S transition. Interestingly, the genome-wide analysis of Sfp1 distribution and Sfp1-dependent transcriptional effects described in this thesis suggest that Sfp1 could be involved in TBP redistribution and therefore in the appropriate adaptation of the overall gene expression program in response to growth and stress signals.
Sfp1 is an unusual transcription factor recruited to both promoters and gene bodies
Sfp1 is a Zn-finger protein that belongs from the yeast Cys2His2 protein family (Blumberg and Silver, 1991; Fingerman et al., 2003; Wolfe et al., 2000). It is a potent regulator of cell size (Jorgensen et al., 2002) that apparently exerts this function by controlling the expression of genes required for growth, such as ribosomal protein genes and genes required for ribosome biogenesis and translation (Blumberg and Silver, 1991; Cipollina et al., 2005; Cipollina et al., 2008; Fingerman et al., 2003; Jorgensen et al., 2002; Jorgensen et al., 2004; Lempiainen et al., 2009; Marion et al., 2004; Xu and Norris, 1998). DNA microarray analysis revealed that the overexpression of SFP1 induces transcription of these genes (Jorgensen et al., 2002):
shortly after SFP1 induction (15 minutes of galactose treatment) the steady state mRNA level of many genes implicated in ribosome biogenesis (RiBi) is strongly increased compared to the WT. On the other hand, the vast majority of the ribosomal protein encoding genes (RP) are induced only at later time points after SFP1 overexpression. Early ChIP experiments aimed to measure Sfp1 recruitment on these identified targets, reveals that Sfp1 was undetectable at
most RiBi gene promoters whereas it was found on the vast majority of the RPG (Jorgensen et al., 2004; Lempiainen et al., 2009; Marion et al., 2004). Therefore, a puzzling discord between ChIP targets (RPG) and the most rapid mRNA effect was underscored. Furthermore, studies performed using chemostat cultures (widely recognized as a valuable tool for physiological and metabolic analysis (Brauer et al., 2005; Hoskisson and Hobbs, 2005; Porro et al., 2003; Wu et al., 2006)) to compare the physiology of sfp1Δ cells with the reference WT strain, revealed decreased mRNA levels of the RiBi gene cluster, but not of the RP genes, in the absence of SFP1. Therefore, these results further support the notion that the RiBi genes are the primary Sfp1 transcriptional targets.
We have described in this thesis the genome-wide distribution of Sfp1 obtained by ChIP-seq experiments performed under normal growth conditions and as a consequence of elevated SFP1 expression driven by the GAL1 promoter in presence of galactose. We found that Sfp1 chromatin binding does not occur exclusively in the promoter region but is instead also observed to be significant on some gene ORFs (the effect being more evident upon overexpression). Therefore, by establishing an arbitrary cut-off, we characterized three different categories according to Sfp1 distribution between promoter and ORF: genes where Sfp1 was exclusively found on the promoter were classified as Category I. Genes where Sfp1 was found both on promoters and ORF were defined as Category II: the vast majority of the RPG were assigned to this class. Finally, Category III genes were defined as genes that have Sfp1 exclusively on their ORFs. As already stated, under normal growth conditions Sfp1 is mostly undetectable on RiBi gene promoters and ORFs. Therefore, we did not include RiBi genes in our categories except few example such as NSR1 that was positioned within category III. As reported on Tables 3, 4 and 5 gene ontology enrichments obtained for Cat I, II and III genes do not significantly reveal gene functional classes that are either occupied by Sfp1 in the promoter and/or through the ORF. The most significant identified clusters were RPGs (as previously noticed), genes involved in response to oxidative stress and in translational control.
genes besides RPGs and RiBi and may therefore perform its function in association with the gene specific transcriptional regulators.
The question of whether Sfp1 directly contacts the DNA through the recognition of specific DNA motifs is still unresolved. This interaction is at the moment, only inferred given the Zn-finger domains found on the Sfp1 protein sequence and ChIP-chip results where a Sfp1-binding motif was proposed (Fingerman et al., 2003; MacIsaac et al., 2006). However, distinguish direct vs indirect binding of a transcription factor to the DNA it is challenging.
Interestingly, when Protein Binding Microarray technology (PBM) was performed in parallel to CHIP-ChIP experiments to analyze Sfp1-DNA interaction, the previously inferred Sfp1 motif was found to coincides to the Rap1-binding site and therefore suggested to occur indirectly through the interaction with Rap1 (Zhu et al., 2009). Examples have been reported in yeast of transcription factors that do not directly contact the DNA to promote their function: the non-DNA-binding Met4 activator was shown to interact with multiple regulators that finally stabilize its DNA recruitment (Lee et al., 2010; Thomas D., 1992). Results presented in this thesis, further support the hypothesis of an indirect Sfp1-DNA interaction perhaps mediated by gene specific transcription factors (see next paragraph).
We went on by analyzing in more detail this peculiar Sfp1 distribution on different gene
We went on by analyzing in more detail this peculiar Sfp1 distribution on different gene