New molecular mechanisms of aging regulation
Sen, Ilke
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Chapter 4
DAF-16/FOXO
requires Protein
Phosphatase 4 to initiate the
transcription of stress resistance and
longevity promoting genes
Ilke Sen, Xin Zhou, Alexey Chernobrovkin, Andrea Stöhr, Nataly Puerta Cavanzo, Xin-Xuan Lin, Bora Baskaner, Simone Brandenburg, Roman Zubarev, Christian Riedel
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Abstract:
The conserved transcription factor DAF-16/FOXO is one of the most powerful aging regulators known to date. It responds to diverse aging-regulatory stimuli, most notably low insulin/IGF-like signaling (IIS), and relays them into the expression of stress resistance and longevity promoting genes. However, in this task DAF-16 alone is not sufficient. Instead, it relies on regulators and cofactors that orchestrate its activity. Maybe the most potent positive regulator of DAF-16 is SMK-1, which is essential for most of DAF-16’s functions under low IIS. But despite its discovery dating back more than a decade, the mechanism by which SMK-1 fullfills this crucial role has remained unknown. Here we show that SMK-SMK-1 functions as part of a specific protein phosphatase 4 complex (PP4SMK-1), which is essential for DAF-16 to extend lifespan and promote resistance to specific stresses under low IIS. Consistently, we found that loss of PP4SMK-1 suppresses many of the gene expression changes mediated by DAF-16 under low IIS. Investigating these PP4SMK-1-dependent DAF-16 target genes more closely, we observed that a) they are particularly dependent on transcriptional initiation as a rate-limiting step and that b) loss of PP4SMK-1 specifically impairs here the recruitment of RNA polymerase II (Pol II) and transcriptional initiation. We eventually sought the relevant substrate of PP4SMK-1 by phospho-proteomics and functionally evaluated emerging candidates by RNAi screening. This led to identification of the conserved transcriptional initation/elongation factor SPT-5, whose loss phenocopies the loss of PP4SMK-1 with regard to DAF-16’s functions. We eventually propose a model, whereby SPT-5 undergoes a phosphoregulatory cycle in which it needs to be dephosphorylated to efficiently bind promoters and drive Pol II recruitment and transcriptional initiation. In absence of PP4SMK-1, SPT-5 accumulates in a phosphorylated state that is unable to license new rounds of transcription, particularly affecting genes that are transcriptional-initiation-dependent, like many of DAF-16’s targets, and thus impairing DAF-16’s functions.
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107 Introduction:
Insulin/IGF-like signaling (IIS) is one of the most prominent aging-regulatory pathways, conserved from worms to mammals (Kimura et al., 1997; Tatar et al., 2001). It has crucial roles in the regulation of metabolism, reproduction, stress resistance, and longevity (Kenyon et al., 1993; Lee et al., 2003). DAF-16/FOXO is a conserved transcription factor downstream of IIS, mediating most of its influence on stress resistance and longevity. Under normal conditions, IIS is active and phosphorylates DAF-16 via AKT and SGK kinases, which leads to its sequestration by 14-3-3 proteins in the cytoplasm and thus away from its target genes. However, under dire conditions, in particular those leading to low IIS, this phosphorylation ceases, allowing DAF-16 to translocate into the nucleus, where it eventually binds to the promoters of many stress resistance and longevity promoting genes and drives their transcription (Calnan and Brunet, 2008).
Although nuclear translocation of DAF-16 is a prerequisite, it is not sufficient for DAF-16 to regulate its target genes (Lin et al., 2001). Additional regulators and cofactors are needed. One of the most powerful positive regulators of DAF-16 was described already a decade ago, the conserved protein SMK-1/SMEK (Wolff et al., 2006). It is essential for DAF-16 to exert most of its functions under low IIS, including the resistance to many stresses (with the exception of heat stress) and the extension of lifespan. However, little is known about the mechanism by which SMK-1 confers this crucial role.
In this study, we were able to elucidate this mechanism. We show that SMK-1 exerts its DAF-16 dependent stress resistance and lifespan promoting roles as part of a specific protein phosphatase 4 complex, PP4SMK-1. In turn, we found that this phosphatase complex binds and dephosphorylates the transcription initiation/elongation factor SPT-5, thereby promoting efficient RNA polymerase II (Pol II) recruitment, transcriptional initiation, and eventually Pol II release from transcriptional end sites (TESs) under low IIS. This mechanism is required for induction of a large subset of DAF-16 target genes – genes which need to be rapidly induced and are particularly dependent on transcriptional initiation as a rate-limiting step – and thus renders PP4SMK-1 so crucial for DAF-16’s functions.
Results:
SMK-1 is part of a nuclear Protein Phosphatase 4 complex (PP4SMK-1).
In order to find the mechanism by which SMK-1 is acting, we first looked for other proteins that SMK-1 might be binding to. For this, we used C. elegans expressing SMK-1::mCherry, grew them to large scale, lysed them, immunoprecipitated the bait using anti-mCherry antibody, and then analyzed the precipitated material by silver staining (Fig. 1A) and mass spectrometry (LC-MS/MS, Fig. 1B). 16 proteins were identified that co-purified with SMK-1::mCherry (Fig. 1B,C). The most abundant of these were subunits of protein phosphatase 4 (PP4), namely the catalytic subunits PPH-4.1 and PPH-4.2 as well as the regulatory subunit PPFR-2 (Fig. 1B,C). According to previous work in yeast and mammals, PP4 complexes exist in different defined subunit compositions: Each complex contains the catalytic PP4 subunit, the closest C. elegans orthologs to which would be PPH-4.1 and PPH-4.2, and then it contains a selection out of four regulatory subunits, which help to target PP4 to its substrates (Chen et al., 2008; Chowdhury et al., 2008; Gingras et al., 2005). Closest C. elegans orthologs
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to three of these regulatory subunits are PPFR-1, PPFR-2, and PPFR-4. Strikingly, closest ortholog to the fourth one is SMK-1. Together with our large scale IP data, this suggests that SMK-1 is actually a regulatory subunit of a specific type of PP4 complex, hereafter referred to as PP4SMK-1, and that this complex has a specific subunit composition, containing besides the catalytic subunits also the structural or regulatory subunit PPFR-2, but not PPFR-1 or PPFR-4 (see also Fig. 1C). Next, we sought to confirm this result by independent large-scale immunoprecipitation experiments (IPs), using a different epitope tag on SMK-1 and different affinity matrix (SMK-1::GFP and GFP-Trap, respectively). At the same time, given that SMK-1 is required for DAF-16 functions specifically when IIS activity is low, we also wanted to test, if the incorporation of SMK-1 into the PP4SMK-1 complex is IIS-dependent. Thus, we altered IIS activity in this experiment using daf-2(e1370) (a mutant of the insulin/IGF receptor, yielding low IIS activity) or daf-18(mg198) (a mutant of PTEN, yielding high IIS) animals. As can be seen in Tables S2 and S3, we were able to confirm the existence and composition of the PP4SMK-1 complex, while the formation of this complex was not affected by the level of IIS activity. One observation surprised us in all the above SMK-1 large-scale IPs: In addition to C. elegans orthologs of known PP4 specific subunits, we also found the phosphatase subunit PAA-1 to be an abundant component of PP4SMK-1 (Fig. 1B,C, Tables S2,S3). PAA-1 is a well-established scaffold subunit of protein phosphatase 2A (PP2A) complexes but had not been found in PP4 complexes before (Lange et al., 2013). However, our data suggests that at least in C. elegans, PAA-1 forms the scaffold of some PP4 complexes, too – at least of PP4SMK-1.
Next, we wondered about the localization of the PP4SMK-1 complex, to get an indication of the tissues and cellular compartments in which the complex may regulate DAF-16 function under low IIS. For this we studied wild-type as well as daf-2(e1370) animals expressing either SMK-1::GFP or PPH-4.1::GFP under their endogenous promoters. Animals expressing DAF-16::GFP served as an additional control. We found that SMK-1::GFP and PPH-4.1::GFP were both globally expressed, similar to DAF-16::GFP (Fig. 1D, see also (Henderson and Johnson, 2001; Wolff et al., 2006)). Within cells, SMK-1::GFP localized strictly to the nucleus, while PPH-4.1::GFP could be seen in both, the nucleus and the cytoplasm (Fig. 1D). Both, expression pattern and subcellular localization of SMK-1::GFP and PPH-4.1::GFP were not affected by changes in IIS activity. This data suggested that under low IIS the PP4SMK-1 complex can form globally in all tissues, where it is localized and thus should act in the nucleus.
Taking our IP and localization data together, they suggest that SMK-1 acts as part of a specific protein phosphatase 4 complex, PP4SMK-1, that acts in the nucleus to promote diverse functions of DAF-16 under low IIS.
PP4SMK-1 is required for DAF-16 to promote longevity.
Given that SMK-1 is part of a PP4 complex, we wondered if SMK-1 fulfills the regulation of DAF-16 functions under low IIS as part of this complex, in particular via its catalytic activity. To test this, we first explored the role of PP4SMK-1 in the regulation of lifespan: We began by knocking down all the different PP4SMK-1 complex subunits by RNAi in daf-2 mutant animals, which are long-lived due to constitutive DAF-16 activity, and in 2; daf-16 mutant animals, in which this DAF-daf-16-driven effect is absent. Consistent with SMK-1 acting as part of PP4SMK-1, smk-1 but also pph-4.1, pph-4.2, or ppfr-2 were all required for the full longevity of daf-2 mutant animals (Fig. 2A). Importantly, these lifespan effects were
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fully dependent on the presence of daf-16 (Fig. 2B). Next, we wanted to determine the specificity of the PP4SMK-1 complex over other PP4 complexes in regulating DAF-16 functions. Thus we extended the previous experiment also to knockdowns of 1 and ppfr-4, two PP4 subunits that we found not to be part of PP4SMK-1 (Fig. 1B,C). Consistent with a specific role for PP4SMK-1 in regulating DAF-16 functions, loss of ppfr-1 had no effect on lifespan (Fig. S1A,B), and loss of ppfr-4 shortened lifespan of daf-2 animals – but in a manner independent of daf-16 (Fig. S1A,B).
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Figure 1. SMK-1 is part of a nuclear Protein Phosphatase 4 complex (PP4SMK-1) and
PP4SMK-1 co-localizes with DAF-16 in the nucleus in IIS mutants
A) SDS-PAGE and silver stain analysis of large-scale anti-mCherry immunoprecipitation from whole-worm lysates of animals expressing SMK-1::mCherry in WT background. Untagged whole-worms were used for the control purification in order to subtract non-specific proteins as background. Immunoprecipitated material was analyzed by mass spectrometry (LC-MS/MS). B) List of binding partners of SMK-1 according to the MS results, containing the spectral counts and MW of the proteins. C) A model of the formation of a specific PP4 complex including SMK-1 based on MS data. D) Localization studies with DAF-16::GFP, SMK-1::GFP and PPH-4.1::GFP transgenic worms in WT and
daf-2(e1370) mutant background. Worms were grown at 15°C, and were then shifted to 25°C for 16
hours before the pictures were taken. Arrows are pointing the nuclear localization of PPH-4.1 in both WT and daf-2(e1370) mutant background.
In Figure 2A, RNAi of pph-4.1 or pph-4.2 had a lesser effect on DAF-16 driven longevity than RNAi of smk-1. We attributed this to the possible redundancy of these two PP4 catalytic subunit paralogs. To test this, we conducted double-RNAi experiments targeting pph-4.1 and pph-4.2 together. Indeed, combined knockdown of both catalytic subunits led to additive suppression of the longevity in daf-2 animals, in extent now much more similar to smk-1 RNAi. And again, these lifespan phenotypes were dependent on daf-16, as illustrated by conducting the same knockdowns in wild-type (Fig. S1C) or daf-2; daf-16 mutant animals (Fig. S1D).
Next, we validated the phenotypes obtained from knockdown of the PP4SMK-1 catalytic subunits by use of a null mutant allele of pph-4.1. Consistent with our RNAi data, also this pph-4.1(tm1445) allele specifically suppressed the longevity of daf-2 mutant animals, while it had little effect in wild-type and no effect in daf-2; daf-16 mutant animals (Fig. 2D,E). Finally we wanted to see, if it is truly the catalytic activity and not a structural property of these subunits that is needed for DAF-16-mediated longevity. Thus, we made transgenic lines overexpressing either wildtype pph-4.1 or an allele encoding an R262L mutant form of PPH-4.1, which is known to be catalytically inactive, as shown in studies of PP4 in C. elegans and mammals (Sato-Carlton et al., 2014; Zhou et al., 2002). Consistent with the catalytic activity of PP4SMK-1 being required, overexpression of the catalytic dead for of PPH-4.1 led to lifespan shortening in daf-2 mutant animals – a phenotype which was lost upon knockdown of daf-16 (Fig. 2F).
We conclude that SMK-1 is acting as part of a specific PP4 complex and employs its catalytic activity to promote DAF-16-driven longevity under low IIS.
PP4SMK-1 is required for DAF-16 to promote resistance to oxidative stress and to UV
irradiation but not to heat stress.
Much of the DAF-16-driven longevity in daf-2 mutant animals can be attributed to improved stress responses. Consistently, a previous study had shown that SMK-1 was required for the daf-16-dependent improved resistance of daf-2 animals to oxidative stress and UV irradiation (Wolff et al., 2006). Notably though, SMK-1 showed surprising specificity in mediating stress resistance, by not affecting the daf-16-dependent increased resistance to heat stress in these animals (Wolff et al., 2006). We now tested, if SMK-1 confers stress resistance as part
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of PP4SMK-1, too. For this we knocked down the complex’s catalytic subunits, pph-4.1 and pph-4.2, by combined RNAi in either daf-2 or daf-2; daf-16 mutant animals and exposed them at day 2 of adulthood to either 1500 J/m2 UV light or shifted them to 6 mM t-BOOH (oxidative stress) or to 32°C (heat stress). Survival under these stresses was determined using a lifespan scoring machine (Stroustrup et al., 2013) to obtain data of particularly high resolution. We found that just like RNAi against smk-1, also RNAi against pph-4.1/pph-4.2 led to a reduction of the resistance to t-BOOH and to UV irradiation but not to heat (Fig. 3A,C,E); and consistent with these being daf-16-dependent effects, all these phenotypes were suppressed when daf-16 activity was reduced in wild-type animals (Fig. S2) and entirely missing in daf-16 null mutant animals (Fig. 3B,D,F).
Figure 2. SMK-1 regulates DAF-16-mediated lifespan as part of a PP4 complex
Lifespan phenotypes caused by loss of daf-16 or specific PP4 subunits in various genetic backgrounds. All strains used in Figure A-C have eri-1(mg366) mutation in their backgrounds in order to yield better knockdown efficiencies. Animals of indicated genotypes were grown from the L1 stage at 15°C and then shifted to 25°C at the L4 stage and their lifespan was monitored. Specific PP4 complex components including SMK-1 are required for the longevity of IIS mutants, in a DAF-16-dependent manner. Lifespan curves showing the single knockdown phenotypes of daf-16, PP4 catalytic subunits; pph-4.1
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and pph-4.2 and regulatory subunits ppfr-2 and smk-1 and their comparison with control RNAi condition in daf-2(e1370) mutants (A) daf-16(mgDf47); daf-2(e1370) mutants (B). Catalytic subunits of PP4 are redundant and required both for the longevity of IIS mutants. (C) Lifespan curves showing the single and double knockdown phenotypes of the catalytic subunits in daf-2(e1370) mutants. Although, there is almost no lifespan effect in WT, loss of function mutant of pph-4.1(tm1445) is sufficient to suppress DAF-16-mediated longevity. D) Lifespan comparisons of N2 and
pph-4.1(tm1445) mutant worms on control and daf-2 RNAi. Promotion of longevity by PPH-4.1 depends
on daf-16. E) Lifespan curves of daf-2(e1370) and daf-2(e1370); pph-4.1(tm1445) animals on control and daf-16 RNAi. SMK-1 promotes DAF-16-mediated longevity via the catalytic activity of PP4. F) Lifespan curves of daf-2(e1370) mutant animals expressing catalytic active PPH-4.1 and catalytic dead
PPH-4.1 (R262L) transgenes. The detailed results with statistics are shown in Table S5.
This data showed that also for conferring daf-16-dependent resistance to stress under low IIS, SMK-1 functions as part of the PP4SMK-1 complex, relying on its catalytic subunits. And we were able to confirm that PP4SMK-1 is selective in promoting DAF-16 to confer oxidative stress and UV resistance, while it is not required for DAF-16’s benefits to the survival of heat stress.
PP4SMK-1 is required for a significant part of the DAF-16-mediated gene expression
changes in low IIS mutants
Given the requirement of PP4SMK-1 for many of DAF-16’s functions under low IIS, and given that DAF-16 is a transcription factor, we wondered if PP4SMK-1 acts by influencing the expression of DAF-16-regulated genes. To address this question, we carried out mRNA-seq experiments: We used either wild-type or daf-2 mutant C. elegans, treated them from the L1 stage with either control, daf-16, or smk-1 RNAi, and then extracted and sequenced their RNAi at young adulthood. Using the resulting data, we first determined the genes differentially expressed by the reduced IIS in daf-2 mutant animals, identifying 1120 upregulated and 2073 downregulated genes (Fig. 4A). Next we asked, how these differentially expressed genes are affected by RNAi against daf-16 or smk-1 in daf-2 animals. Most of the gene expression changes that occur in daf-2 mutant animals are known to be mediated by DAF-16 (Murphy et al., 2003; Riedel et al., 2013). Thus, RNAi of daf-16 suppressed 62 % of gene activation and 87 % of the gene repression events in daf-2 mutant animals. RNAi of smk-1 suppressed many of these gene expression changes, too, namely 41.4 % of the gene activation and 18.7 % of the gene repression events that occur in daf-2 animals. These findings are consistent with PP4SMK-1 indeed influencing the lifespan-extending and largely DAF-16-induced gene expression changes under low IIS. Next, we determined the actual genes that were regulated by DAF-16 and SMK-1 under low IIS as well as their overlap (Fig. 4B,C). As expected already from the results in Figure 4A, we found significant overlaps in the genes activated or repressed by DAF-16 and SMK-1. And consistent with both, DAF-16 and SMK-1, being required for the longevity under low IIS, we found these co-regulated genes to be enriched for aging-related genes (based on GO-term enrichment analyses, Fig. 4D,E).
Taken together, PP4SMK-1 is required and thus likely confers its phenotypes by licensing the transcriptional regulation of a subset of aging-related DAF-16 target genes.
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Figure 3. PP4SMK-1 is required for DAF-16-driven oxidative stress and UV stress
resistance but not heat stress resistance in daf-2(e1370) mutants
Stress survival phenotypes caused by loss of daf-16 and PP4SMK-1. Animals of indicated genotypes were
grown at 15°C from L1 to L4 stage. Then, the temperature was shifted to 25°C and the next day, day 1 adults were either transferred to 6mM tBOOH containing RNAi plates (oxidative stress) or exposed to
1500J/m2 UV light (UV stress) or transferred to 32°C (heat stress). All strains used in Figure 3A-F have
eri-1(mg366) mutation in their backgrounds. PP4SMK-1 is involved in survival of the worms that are
exposed to oxidative stress (A) or UV stress in daf-2(e1370) mutants (C). B, D) There is no additional
effect of PP4SMK-1 loss on survival of the daf-16(mgDf47); daf-2(e1370) mutants. However, PP4SMK-1
is not essential for heat stress response neither in 2(e1370) mutants (E) nor in 16(mgDf47);
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Figure 4. PP4SMK-1 is required for a significant part of the DAF-16-driven gene
expression changes in low IIS mutants
C. elegans with the genotypes, daf-2(e1370), daf-2(e1370); daf-16 (RNAi), and daf-2(e1370); smk-1
(RNAi) were grown from L1 to L4 stage at 15°C, then shifted for 16 h to 25°C, harvested, and their transcriptomes determined by mRNA-seq. All the strains have eri-1(mg366) mutation in their backgrounds. Similar to DAF-16, SMK-1 at least partially suppresses the gene expression changes of long-lived daf-2 animals. The scatter plots on the left show the genes significantly upregulated and the scatter plots on the right show the genes significantly downregulated in daf-2(e1370) compared to WT animals and the effect of loss of daf-16 and smk-1 on these gene sets. B, C) Venn diagrams illustrate the number of genes significantly regulated by the DAF-16 or SMK-1 in daf-2(e1370) mutants as well as their overlap. D, E) GO-term enrichment analyses, conducted on the co-activated and co-repressed
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genes shown in Figures 4B, C. Only GO-terms of significant enrichment are shown (DAVID score ≥ 1.3). Arrows highlight aging-related GO-terms.PP4SMK-1 only mildly affects nuclear entry of DAF-16 and its binding to chromatin Having by now established that PP4SMK-1 is important for DAF-16 functions under low IIS, most importantly the regulation of aging-related target genes, we wondered how this function arises. Between the initial DAF-16-activating stimulus of low IIS activity and the successful regulation of target genes resides a long series of events that all need to occur. At which level of this cascade is PP4SMK-1 acting? To determine this, we first studied the kinetics of nuclear entry and exit of DAF-16 upon inactivation and reactivation of the temperature-sensitive daf-2(e1370) allele, respectively (Fig. S3A). Previous work, only comparing translocation endpoints, had found no impact of SMK-1 on DAF-16’s nuclear translocation (Wolff et al., 2006). Consistently, we observed no difference in the endpoints of our nuclear entry and nuclear exit time courses (Fig. S3B,C). Only when we followed the kinetics of nuclear translocation, we observed that knockdown of smk-1 led to a mild delay, in particular in the entry of DAF-16 caused by daf-2 inactivation (Fig. S3B,C). However, we deemed this phenotype as very moderate and thus unlikely to explain the strong impact of PP4SMK-1 on DAF-16’s functions.
Upon DAF-16’s nuclear entry, the next crucial step towards regulation of its target genes should be its binding to target promoters (Fig. S3A). To see if this step was in any way influenced or regulated by PP4SMK-1, we determined the genome-wide binding of DAF-16 to its target promoters in the presence or absence of SMK-1. We used daf-2 mutant animals expressing DAF-16::GFP, treated them with either control or smk-1 RNAi, and then subjected them to ChIP-seq analysis using anti-GFP antibody. Looking across 2824 DAF-16 binding sites described in previous work (Lin et al., 2018), we found no significant change in the binding of DAF-16 to its target regions (Fig. S3D).
In summary, absence of SMK-1 led to only a slight delay in DAF-16 nuclear entry and no significant change in DAF-16’s binding to target promoters. Thus we expected that PP4 SMK-1 would influence DAF-16 functions yet further downstream.
PP4SMK-1 is required for efficient promoter recruitment and transcriptional initiation of
RNA Pol II at the genes co-activated by DAF-16 and SMK-1
16 is predominantly a transcriptional activator (Riedel et al., 2013). Thus, once DAF-16 has bound to its target promoters, the next key downstream steps should lead to target gene activation and thus be: 1) the recruitment of RNA Polymerase II (Pol II) to the promoter region, 2) transcriptional initiation, during which Pol II moves from the promoter region into the gene body, where it will pause around the +50 position (Maxwell et al., 2014), and 3) transcriptional elongation, during which Pol II gets released from pausing and eventually transcribes the entire gene (Fig. 5A). To evaluate these steps, we used daf-2 mutant animals, subjected them to either control or smk-1 RNAi, and eventually determined the genome-wide distribution of Pol II on chromatin by ChIP-seq using an antibody against the large subunit of Pol II, AMA-1. Strikingly, while Pol II recruitment was unaffected by smk-1 RNAi when looking cumulatively at all promoter regions genome-wide, we saw a significant reduction
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in Pol II recruitment to promoters whose downstream genes would be activated by DAF-16 and at the same time depend also on SMK-1 for their activation (genes “co-activated” by DAF-16 and SMK-1 according to Figure 4B) (Fig. 5B). Additionally, we observed an unexpected distribution of Pol II at these promoters. When we looked cumulatively at the regions around transcriptional start sites (TSSs) genome-wide, most of Pol II has already undergone transcriptional initiation and localized to the pause site in the gene body (Fig. 5C). The same distribution of Pol II can be found when looking cumulatively at the genes activated by DAF-16 (Fig. 5C). However, specifically at the genes that depend on both, DAF-16 and SMK-1, for their activation, the distribution differed: A substantial amount of Pol II was found localized still to the promoter regions and thus has not yet undergone transcriptional initiation (Fig. 5C). This argued that transcriptional initiation at these genes occurred slower and thus may be particularly rate-limiting for the transcription of these genes (Reppas et al., 2006; Wade and Struhl, 2008). Strikingly, upon smk-1 RNAi, while the distribution of Pol II did not change across TSS regions genome-wide, specifically at the genes co-activated by DAF-16 and SMK-1 Pol II shifted yet further into the promoter regions. In fact, the summit of Pol II distribution now repositioned from the pause site in the gene body to the promoter region, arguing that SMK-1 is not only required for efficient Pol II recruitment but also for transcriptional initiation at these promoters. To further support this notion, we repeated our ChIP-seq experiments using an antibody that detects only the initiating form of Pol II, phosphorylated at the Ser5 position of the C-terminal repeats (CTR) within its C-terminal domain (CTD) (Chen et al., 2013; Garrido-Lecca and Blumenthal, 2010). Consistent with a defect in transcriptional initiation, we found significantly less initiating Pol II in the TSS regions of DAF-16/SMK-1-coactivated genes (Figure 5D). Thus, we conclude that PP4SMK-1 is required for efficient Pol II recruitment and transcriptional initiation at a subset of DAF-16-activated target genes; and we propose that these functions are the major reason why
PP4SMK-1 is required for DAF-16-driven induction of this target gene set and eventually the
DAF-16-driven increased stress resistance and longevity under low IIS.
PP4SMK-1 regulates RNA Pol II recruitment and transcriptional initiation by
dephosphorylating the transcriptional initiation/elongation factor SPT-5
By now we had identified the steps in the cascade from DAF-16 activation to target gene expression which were dependent on PP4SMK-1. However, PP4SMK-1 is a phosphatase and we still did not know the relevant substrate by whose dephosphorylation PP4SMK-1 would promote Pol II recruitment and transcriptional initiation at DAF-16 target genes. First, we took a candidate approach and tested whether DAF-16 itself could be the target. However, neither by large-scale IPs (Fig. 1A,B, Tables S2,S3, and (Riedel et al., 2013)) nor by low-stringency co-IPs (data not shown) we could find evidence for a physical interaction between
PP4SMK-1 and DAF-16 – a result that was also consistent with previous work (Wolff et al.,
2006). Additionally, we investigated the smk-1-dependent phosphorylation status of DAF-16 by phos-tag gel-electrophoresis and by mass spectrometry: For the former, we grew DAF-16::GFP expressing C. elegans containing either daf-2(e1370) or daf-18(mg198) and exposed them to either control or smk-1 by RNAi. As expected from high IIS activity leading to phosphorylation of DAF-16, we observed an upshift of DAF-16::GFP in 18 versus daf-2 mutant protein extracts, when analyzed by a phos-tag gel and western blotting, but RNAi of smk-1 had no effect on the migratory behavior of DAF-16::GFP in this assay (Fig. S4). For the mass spectrometric evaluation of DAF-16, we grew daf-2 mutant animals expressing
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DAF-16::GFP to large scale, exposed them to either control or smk-1 by RNAi, lysed the animals, immunoprecipitated the DAF-16::GFP, and determined its phosphorylation status by LC-MS/MS. Again, no effect of smk-1 RNAi could be found (data not shown). Thus, we concluded that the relevant substrate of PP4SMK-1 should be distinct from DAF-16.Figure 5. PP4SMK-1 is required for efficient promoter recruitment and transcriptional
initiation of RNA Pol II at the genes co-activated by DAF-16 and SMK-1
A) Model showing the general rate limiting steps of transcriptional regulation. ChIP-seq experiments
were carried out by pulling down Pol II to understand the role of PP4SMK-1 in these processes.
daf-2(e1370) animals were grown synchronously on control and smk-1 RNAi containing plates at 15°C,
then shifted for 16 hrs to 25°C, until the animals were harvested. B) Loss of smk-1 did not affect the binding of Pol II at the TSS of all genes in the genome (n=20389) but there is significantly less recruited
Pol II at TSS of the genes that are co-activated by DAF-16 and PP4SMK-1 (p=0-012) with a summit shift
towards the upstream of TSS. C) ChIP-seq data showing the binding profile of RNA Pol II at the TSS
of all genes, DAF-16-activated genes and DAF-16 and PP4SMK-1 co-activated genes. The red arrow
points to the binding region of RNA Pol II at co-activated genes (n=150), where RNA Pol II accumulates more upstream of the TSS compared to its binding at the TSS of all genes and
DAF-16-4
activated genes, which centers at the pause site. D) A (Ser5) phospho-modified antibody was used to
detect specifically the initiating form of Pol II in the presence or absence of PP4SMK-1 in daf-2(e1370)
mutants. Binding of initiating RNA Pol II is significantly reduced (p=0.014) at the TSS of DAF-16 and
PP4SMK-1 co-activated genes (n=150). PP4SMK-1 is required for efficient transcriptional initiation at the
genes co-activated by DAF-16 and PP4SMK-1.
To eventually find the relevant substrate of PP4SMK-1, we turned to an unbiased phosphoproteomics approach: We grew daf-2 mutant animals, treated them with either control or smk-1 by RNAi, lysed them, and eventually determined their phosphoproteome by TiO2-based phosphopeptide enrichment and LC-MS/MS (see also Fig. 6A). This identified 208 phosphopeptides whose abundance had increased upon smk-1 RNAi and thus should be direct or indirect targets of PP4SMK-1 (Fig. 6A, Table S4). These 208 phosphopeptides derived from 150 proteins (Table S4), of which we selected 41 for further evaluation, based on their annotation and the confidence in their smk-1-dependent differential phosphorylation. To determine the functional relevance of these candidate proteins, we used a transcriptional reporter for the canonical DAF-16 target gene sod-3. This reporter is induced by DAF-16 under low IIS – an induction that requires SMK-1 (Wolff et al., 2006). Thus, we knocked down each of the 41 candidates by RNAi in wild-type or daf-2 mutant animals expressing sod-3p::GFP and evaluated their GFP expression on day 3 of adulthood. Knockdown of several candidates led to substantial changes in the expression of this reporter (Fig. 6B). Additionally, we searched our SMK-1 large-scale IP data, to see whether any of the candidates co-purified with PP4SMK-1 – a behavior that often can be observed for phosphatase substrates (Sacco et al., 2014). Indeed, we found that several of our candidate substrates co-purified with SMK-1 (Fig. 1B,6B, Table S2,S3). Taken together, these analyses highlighted one candidate in particular: The conserved transcriptional initiation/elongation factor SPT-5 (Fig. 6B). Under low IIS, SPT-5 harbors four phosphorylation sites that are dependent on
PP4SMK-1 (Fig. 6B), SPT-5 physically associates with PP4SMK-1 (Fig. 1B,6B, Table S2,S3),
and SPT-5 influences the expression of sod-3 (Fig. 6B). The significance of SPT-5 differential phosphorylation has the best p-value of all sod-3-influencing candidates. Furthermore, two transcriptional initiation/elongation factors from the same SPT family which closely synergize with SPT-5 (Kaplan et al., 2000) have emerged from our study: 1) EMB-5 (Kato et al., 2013), which just like SPT-5 is a candidate substrate of PP4SMK-1 (Fig. 6B), and SPT-4, which just like SPT-5 is bound to PP4SMK-1 (Tables S2,S3) and which together with SPT-5 forms the so-called DSIF (DRB sensitivity inducing factor) complex, important for transcriptional initiation and elongation (Ding et al., 2010). We then knocked down the different SPT family members by RNAi and determined their impact on DAF-16-driven longevity and increased resistance to oxidative and heat stress under low IIS. RNAi of emb-5 or spt-4 did not result in any daf-16-dependent lifespan or stress-resistance phenotypes (data not shown). However strikingly, loss of spt-5 led to phenotypes perfectly reminiscent of the loss of smk-1, impairing the longevity and oxidative stress resistance but not the heat stress resistance of daf-2 mutant animals, while spt-5 RNAi had no effect in wild type animals or in daf-16 null mutants (Fig. 6C-E). Overall, this data is consistent with SPT-5 being the relevant substrate, by which PP4SMK-1 promotes Pol II recruitment and transcriptional initiation at many DAF-16 target genes and thus fulfills its lifespan and stress resistance regulatory roles under low IIS.
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Figure 6. PP4SMK-1 regulates RNA Pol II recruitment and transcriptional initiation by
dephosphorylating the transcriptional initiation/elongation factor SPT-5
Identification of PP4SMK-1 targets by unbiased phosphoproteomics and functional validation of the
candidates. A) Workflow of the unbiased phosphoproteomics experiment and the RNAi screen for
sod-3p::GFP expression changes. B) Results for the screen showing the list of substrate candidates that
induced (in WT) and/or reduced (in daf-2(e1370) mutants) the expression of GFP driven by the sod-3 promoter. The most prominent candidates SPT-5 and EMB-5 highlighted with red color, belong to the same transcription initiation/elongation factor family (SPTs). All strains used in Figure 6C-E present
eri-1(mg366) mutation in their backgrounds. Animals at L4 stage were fed with different RNAi
bacteria. C) Lifespan results of WT, daf-2(e1370) and daf-16(mgDf47); daf-2(e1370) mutant animals on control and spt-5 RNAi. D-E) Stress survival results of 2(e1370) and 16(mgDf47);
daf-2(e1370) worms on spt-5, daf-16 or control RNAi upon exposure to oxidative stress (D) or heat stress
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PP4SMK-1 is required for efficient release of RNA Pol II and SPT-5 at the end of
transcription and SPT-5’s recycling to promoter regions for a new round of transcription.
SPT-5 forms together with SPT-4 the DSIF complex, which is conserved across metazoans (Hartzog and Fu, 2013). It has been shown that the DSIF complex binds to Pol II already prior to transcriptional initiation and that it remains associated with Pol II throughout transcription. Consistent with our observations, the DSIF component SPT-5 has been found required for efficient recruitment of Pol II to promoter regions (Fitz et al., 2018). Further consistent with our data, presence of the DSIF complex in promoter regions is required for the recruitment of several factors involved in transcriptional initiation, thus promoting this step – especially at genes that require rapid induction (Diamant et al., 2016; Grohmann et al., 2011). During the various stages of transcription, SPT-5 undergoes a crucial phosphorylation cycle, in particular in its C-terminal repeat (CTR) domain (Parua et al., 2017): When SPT-5 arrives at the promoter, bound to Pol II, it is unphosphorylated and remains in this state, until it has traveled with Pol II to the pause site in the gene body. Presumably SPT-5 has to be unphosphorylated to fulfill these tasks. At least for the task of recruiting the mRNA capping enzyme during transcriptional initiation, this has been proven experimentally (Wen and Shatkin, 1999). Once SPT-5 arrives at the pause site, phosphorylation of the CTR domain has to occur, to allow for the release from the pause site and thus for the transition from transcriptional initiation to elongation (Lu et al., 2016; Yamada et al., 2006). The CTR domain of SPT-5 remains then phosphorylated while moving with Pol II through the gene body, until it finally gets dephosphorylated again near the transcriptional end site (TES), presumably to facilitate the release of the DSIF complex and Pol II from DNA and to allow their recycling for another round of transcription (see also Fig. 7A for the model).
In our unbiased phosphoproteomics approach (Fig. 6A,B), we found 4 phospho-sites on SPT-5 that were targeted by PP4SMK-1, two of which were located in the CTR domain of SPT-5. Thus, we proposed that PP4SMK-1 has a central role in the removal of CTR phosphorylations, and that in the absence of PP4SMK-1, SPT-5 no longer is efficiently dephosphorylated at the end of transcription, leading to possibly a delayed release from DNA and the eventual lack of unphosphorylated SPT-5 which would be needed to license new rounds of Pol II recruitment and transcriptional initiation at promoter regions (see also Fig. 7B for the model). While our model was consistent with all the data presented so far in this study, we eventually concluded our study by testing two additional implications from this model. First, we tested if in the absence of PP4SMK-1, there would indeed be less SPT-5 available to associate with TSS regions (Fig. 7B, steps 1 and 2) and thus promote Pol II recruitment and transcriptional initiation. Indeed, by ChIP-seq analyses using SPT-5::GFP expressing daf-2 mutant C. elegans grown on either control or smk-1 RNAi, we found a mild but significant reduction of SPT-5 binding to TSS regions throughout the genome (Fig. 7C). Second, we tested if a lack of PP4SMK-1 would affect the release of SPT-5 and Pol II from TESs. Indeed, by ChIP-seq analyses using SPT-5::GFP expressing daf-2 mutant C. elegans grown on either control or smk-1 RNAi, we saw a moderate shift of the SPT-5 and Pol II summits at the TESs into the downstream direction at genes co-activated by DAF-16 and SMK-1 (Fig. 7C,D). This observation was also supported by a recent study showing that artificially increased phosphorylation of SPT-5 by removal of protein phosphatase 1 led to delayed Pol II release beyond the normal TESs (Parua et al., 2017).
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Figure 7. PP4SMK-1 is required for efficient release of RNA Pol II and SPT-5 from TES
and SPT-5’s recycling to promoter regions for a new round of transcription
A) Proposed mechanism. SPT-5 undergoes a phosphoregulatory cycle in which it needs to be dephosphorylated to efficiently bind promoters and drive RNA Pol II recruitment and transcriptional
initiation. B) In absence of PP4SMK-1, SPT-5 accumulates in a phosphorylated state that is unable to
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targets, which in turn affects DAF-16’s downstream lifespan regulatory functions. ChIP-seq experiments by SPT-5 and Pol II pulldowns in transgenic daf-2(e1370) mutants confirms our model via
revealing the consequence of the PP4SMK-1 loss on the binding profile of SPT-5 and RNA Pol II at TSS
and TES. C) There is a significant reduction in SPT-5 binding to the chromatin at TSS of all genes
(p=0.046) together with a delay in its release from TES of PP4SMK-1 and DAF-16 co-activated genes
due to the loss of PP4SMK-1. D) Delay in SPT-5 release also causes defects in efficient release of RNA
Pol II from TES to facilitate the transcription re-initiation of these specific genes.
Discussion:
DAF-16/FOXO is undoubtably one of the most central and powerful aging regulators across metazoans. However, it has become clear that DAF-16 is not self-sufficient in this role but depends on a variety of proteins that need to assist it. More than a decade ago, SMK-1 was discovered as the first protein of such kind (Wolff et al., 2006). SMK-1 is essential for DAF-16 to promote longevity and resistance to many stresses under low IIS, making it a very important aging regulator, even in itself. However, despite this prominence and the thorough genetic exploration of SMK-1’s functions (Wolff et al., 2006), it remained entirely unknown how SMK-1 was acting mechanistically. In this study we were finally able to reveal this mechanism. First, we showed that SMK-1 functions as part of a specific protein phosphatase 4 complex. This complex contains only some of the possible PP4 subunits, namely PPH-4.1/PPH-4.2, providing the catalytic activity, and PPFR-2 as well as SMK-1, two regulatory subunits. Notably, we found also PAA-1 in this complex, normally a structural subunit of PP2A complexes. It will be interesting to explore in the future, why this subunit is present in PP4SMK-1.
An established role of regulatory subunits in phosphatase complexes is the physical interaction with and thus recruitment of the complex to substrates. Indeed, we observed the DSIF complex component SPT-5 amongst proteins co-purifying with SMK-1, and we were able to show that PP4SMK-1 dephosphorylates this protein. Our data combined with published
knowledge on the DSIF complex suggest that this dephosphorylation promotes the release of RNA Pol II from DNA at the end of transcription and maintains a pool of unphosphorylated SPT-5, which in turn is required for efficient RNA Pol II recruitment to promoter regions and the subsequent transcriptional initiation.
Considering this mechanism by which PP4SMK-1 influences transcription, namely through a
general component of the transcriptional regulatory machinery, SPT-5, it is remarkable that PP4SMK-1 preferentially affects transcription of a subset of DAF-16 target genes and not
transcription in general. But as we could show in Figure 5C, these genes seem to be unique, in that already under normal conditions transcriptional initiation occurs here less efficiently and thus is more rate-limiting, compared to the bulk of genes in the genome. Thus, it only is consistent that loss of PP4SMK-1 and resulting impairment of Pol II recruitment and
transcriptional initiation would selectively impair the expression of this gene set. About the reason for these genes to be so transcriptional initiation dependent we can only speculate. But this may be a feature of genes that need the ability to get robustly induced in response to sudden stimuli, like here a drop in IIS.
Another interesting aspect of PP4SMK-1 is that, although it is required for most of the functions
of DAF-16 under low IIS, for some of its functions it is irrelevant – most notably for promoting resistance to heat stress. Consistently, we observed that loss of SPT-5 impaired
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longevity and oxidative stress resistance and but not heat stress resistance under low IIS (Fig. 6C-E); and a previous study had shown that knockdown of the DSIF complex by RNAi did not affect expression of the heat shock reporter HSP-16.2.::GFP (Shim et al., 2002). This indicates that some DAF-16 target genes are regulated in a fundamentally different manner that does not involve the PP4SMK-1 –SPT-5 axis, e.g. due to either different promoter
characteristics or DAF-16 fulfilling such functions in synergy with other transcription factors that impose different mechanisms of transcriptional control at these loci. In particular for heat shock response genes, it is already known that DAF-16 is regulating these genes combinatorially, together with HSF-1 (Hsu et al., 2003). Importantly, it has been shown that HSF-1 target genes are not controlled on the level of Pol II recruitment or transcriptional initiation but rather by promoting the pause-site release of Pol II and thus transcriptional elongation (Mahat et al., 2016).
In the end, the data presented here, in conjunction with published knowledge, paints the picture of PP4SMK-1 being essential for the induction of a specific subset of DAF-16 activated
genes under low IIS – genes for which transcriptional initiation is a rate limiting step and where PP4SMK-1, by supplying unphosphorylated SPT-5 that catalyzes Pol II recruitment and
initiation, helps to overcome this limitation.
It is very possible that PP4SMK-1 positively influences gene expression, not only of many
DAF-16 targets but also of targets that are regulated by other transcription factors. This may even occur under other long-lived conditions beyond low IIS, such as dietary restriction, since a previous study investigating eat-2 mutants was able to show that their longevity, although not dependent on DAF-16 but rather the C. elegans FOXA ortholog PHA-4, depended on SMK-1, too (Panowski et al., 2007). Similarly, it was also shown that SMK-1 is essential for the increased lifespan of mitochondrial electron transport chain (ETC) mutants (Wolff et al., 2006). This points to an involvement of PP4SMK-1 also with other
aging-regulatory signaling pathways and their downstream transcription factors, which will be interesting to explore in the future.
In summary, we were able to elucidate here a sophisticated mechanism by which SMK-1 fulfills its important aging-regulatory roles, in particular under low IIS. Given that all the components involved are well conserved across metazoans, it will be important to investigate the conservation also of this mechanism in the future – most importantly in humans. And given that a sizeable pool of non-phosphorylated SPT-5 appears beneficial for increased stress resistance and the prevention of aging, and that this pool can be manipulated in various ways, be it by controlling the expression of SPT-5, or by interfering with the enzymes placing or removing phosphorylations on SPT-5, the here described mechanism could actually be an interesting therapeutic target for interventions against aging and age-related diseases in humans.
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Methods:
Animals
All C. elegans strains were grown on streptomycin-resistant Escherichia coli OP50-1 using standard methods (Stiernagle, 2006).
Strains and alleles
For a complete list of strains used in this study, please see Table S1.
Making transgenics
To make PPH-4.1::GFP expressing transgenic lines, promoter of pph-4.1 was cloned from genomic DNA and pph-4.1 spliced mRNA sequence was cloned from cDNA of N2 worms. Later, these fragments were fused together with a PCR reaction. Then, the final product of fused promoter and coding sequence of pph-4.1 was cloned into the GFP coding sequence containing vector pPD95_75 from Fire Lab collection to make [pph-4.1P::pph-4.1::gfp] transgene. Complex arrays were prepared for injections by mixing the plasmid containing pph-4.1 transgene, ScaI digested DNA, and EcoRI digested pRF4 (rol-6) plasmid as a co-injection marker. Worms were injected by using InjectMan 4 connected to FemtoJet 4i (Eppendorf). Rollers in the F1 progeny were selected as transgenics carrying the extrachromosomal arrays of Ex[pph-4.1P::pph-4.1::gfp; rol-6(su1006)] and used for further crosses. To create a construct for the catalytically dead PPH-4.1, specific mutation was introduced in the catalytic core of PPH-4.1 by changing Arginine (R) to Leucine (L) at the amino acid position 262.
RNAi feeding
For the RNAi knockdowns, C. elegans were grown on the RNase III-deficient E. coli strain HT115 that contains dsRNA-expressing plasmids specific to the gene of interest. For the first lifespan experiment, in addition to the commercially available Vidal (Reboul et al., 2003) and Ahringer (Kamath et al., 2003) library clones, several other RNAi constructs were prepared by cloning approximately 1 kb region from both cDNA and gDNA sequences of each PP4 subunit. PCR products for each gene of interest were cloned into T-vector after A-tailing. HT115 strain containing the empty plasmid L4440 was used as a control.
Microscopy of the worms
For imaging, worms at L4 stage were used to eliminate background fluorescence observed mostly in the adult animals. Worms were paralyzed using 2,3-butanedione monoxime (BDM) (Sigma), mounted on 2% agarose pads and images were taken after 5 minutes of drug exposure on the slides by using a Zeiss Axio Observer.Z1 microscope and/or DeltaVision Microscope at 20x and 40x magnifications.
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Nuclear translocation assays
For nuclear translocation scoring, DAF-16::GFP expressing daf-2(e1370) mutant animals were treated with smk-1 RNAi and compared with the control RNAi conditions. Day 2 adults were analyzed after shifting the temperature to 25°C from 15°C in order to inactivate IIS and drive DAF-16 to the nucleus for scoring nuclear entry, and from 15°C to 25°C for scoring nuclear exit of DAF-16. Plates were scored blindly after each time point; 30 min, 1 hour, 2 hours, 4 hours and 6 hours using a Zeiss Axio Zoom.V16 microscope.
Lifespan assays
Worms were synchronized by bleaching and grown at 15°C on OP50-1 or, when specified, on RNAi bacteria-containing plates until the late L4 stage, unless any specific RNAi feeding led to larval arrest. In that case, worms were grown on HT115 bacteria-containing plates until L4 stage and transferred to the RNAi bacteria-containing plates after couple of washes with M9 buffer containing antibiotics mixture. FUDR was then added in both cases at late L4 stage to prevent progeny production and the plates were shifted to 25°C. Survival of the animals in all lifespan assays was measured every 2-3 days, as described previously (Hamilton et al., 2005).
Stress assays
Animals were synchronized by bleaching and grown at 15°C until the L4 stage, unless any specific RNAi feeding led to larval arrest. In that case, worms were grown on HT115 bacteria-containing plates until L4 stage. Then, they were transferred to RNAi bacteria- containing plates after couple of washes with M9 buffer containing antibiotics mixtures. Plates were then shifted to 25°C and FUDR was added to prevent progeny formation. At day 2 of adulthood, animals were transferred to plates containing 6 mM tBOOH (for oxidative stress assays) or the temperature was shifted to 32°C (for heat stress assays) or worms were exposed to 1500J/m2 UV light (for UV stress assays). Their survival was recorded and analyzed by a fully automated “lifespan machine” as previously described (Stroustrup et al., 2013).
Phos-tag gel run and western blot
SuperSep Phos-tag (50µM) 10% precast gels (Wako) were used for SDS-PAGE gel run followed by a western blot. Monoclonal mouse anti-GFP antibody (Roche) was used to detect the degree of DAF-16::GFP phosphorylation in daf-2 (e1370) and daf-18 (mg198) mutant backgrounds after treating worms with control and smk-1 RNAi.
Phospho-mapping of DAF-16
Transgenic worms overexpressing DAF-16::GFP protein in daf-2(e1370) background were grown on RNAi plates seeded with smk-1 and control RNAi bacteria. DAF-16 was purified
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in both conditions as described previously (Riedel et al., 2013) and phospho-modifications on DAF-16-related phosphopeptides were determined by LC-MS/MS.
Unbiased Phosphoproteomics: Sample preparation, lysis and digestion
daf-2(e1370) mutant worms were synchronized by bleaching. Then, L1s were seeded on the plates containing smk-1 and control RNAi and grown until the L4 stage. Plates were then shifted to 25°C and FUDR was added to prevent progeny formation. Day 2 adults were harvested and worm beads were prepared by drop-freezing in liquid nitrogen. Worm powders were prepared by using a cryomill (Retsch). Then 200mg/200ul of worm powder was taken for each sample and diluted with 300ul of lysis buffer (8M Urea, 1% SDS, 50mM Tris pH 8.5, Roche protease and phosphatase inhibitor). Samples were then vortexed and sonicated on ice using probe sonicator to shear DNA. After sonication, lysates were centrifuged at 4ºC, 13,000 rpm for 10 minutes. Clear supernatant was transferred to new tubes. Protein concentration in the samples was measured using BCA assay (Pierce). 600µg of proteins were reduced by adding DTT to final concentration of 5mM and incubated for 1 hour at 25°C. Samples were then alkylated by adding IAA to a final concentration of 15mM and incubated for 30 minutes at room temperature in the dark. Excess of iodoacetamide was quenched by adding an additional 10 mM DTT. Proteins were precipitated using methanol-chloroform protocol. Air dried protein pellet was re-dissolved in 100µl 8M Urea in 50mM Tris pH 8.5, and then diluted with equal volume of 50mM Tris pH 8.5. Proteins were digested by adding 10µg of LysC (Promega) and by incubating them in a thermo shaker at 300 rpm and 24ºC for 6 hours. Then, 3 volumes of 50mM Tris pH 8.5 was added, so the final Urea concentration was 1M. 10µg of LC grade trypsin (Promega) was added and samples were incubated at room temperature overnight. After digestion, TFA was added (to 0.5%) and samples were centrifuged at 4ºC, 13,000 rpm for 10 minutes. Pellet was discarded and supernatant was kept. Peptides were then desalted using C18 SepPak (Waters). Phosphopeptides were enriched by TiO2 Phosphopeptide Enrichment and Clean-up Kit (Pierce) using standard manufacturer protocol.
Unbiased Phosphoproteomics: LC-MS/MS
Chromatographic separation of peptides was achieved using EASY-Spray™ LC Columns, 50 cm (Thermo Scientific) connected to the EASY-LC 1000 chromatography system (Thermo Scientific). Peptides were eluted at 300 nL/min flow rate for 120 minutes at a linear gradient from 2% to 26% ACN in 0.1% formic acid. Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) analyzed the eluted peptides that were ionized with electrospray ionization. The survey MS spectrum was acquired at the resolution of 120,000 in the range of m/z 200-2000. MS/MS data for 20 most intense precursors were obtained with a higher-energy collisional dissociation (HCD) for ions with charge z>1 at a resolution of 15,000.
Data Analysis for Mass Spectroscopy
The mass spectrometric raw data was analyzed with the MaxQuant software (version 1.5.3.30). A false discovery rate (FDR) of 0.01 for proteins and peptides and a minimum peptide length of 6 amino acids were required. The Andromeda search engine was used to
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search the MS/MS spectra against the Uniprot C.elegans database (containing 27771 entries) combined with 262 common contaminants and concatenated with the reversed versions of all sequences. Enzyme specificity was set to trypsin specificity, allowing cleavage N-terminal to proline. Further modifications were cysteine carbamidomethylation (fixed) as well as protein N-terminal acetylation, asparagine and glutamine deamidation, methionine oxidation and phosphorylation of STY (variable).Analysis of the data provided by MaxQuant was performed in the R scripting and statistical environment. Protein relative intensities were normalized to the total protein signal and resulting values were log2 transformed. Differences in relative protein abundances between treated and control samples were assessed by moderated t-test using limma package (Ritchie et al., 2015). Benjamini-Hochberg correction for multiple comparison was used.
Genetic Screen by using sod-3p::GFP expressing transgenics
sod-3p::GFP-expressing WT and daf-2(e1370) mutant transgenic worms were used to detect the changes in the expression level of this canonical downstream target of DAF-16 upon RNAi treatment to knockdown selected PP4SMK-1 substrate candidates. All possible RNAi clones existed in both Ahringer (Kamath et al., 2003) and Vidal (Reboul et al., 2003) RNAi libraries were used for each gene of interest. First screen was conducted by L1 feeding on RNAi plates. Then, the second screen was conducted by L4 feeding with the RNAi clones that led larval arrest phenotype in the first screen. GFP expression levels were determined both in control and smk-1 knockdown conditions between the days 2-4 of animal adulthood by using Zeiss Axio Zoom V16 microscope with the same imaging conditions for each day of the analysis.
mRNA isolation and construction of libraries for high-throughput sequencing
Approximately 100 worms were synchronized by bleaching. daf-2(e1370) animals were treated with the control, smk-1 or daf-16 RNAi and grown at 15°C until the L4 stage, then FUDR was added and the plates were shifted to 25°C for 16 hours. Animals were then collected, washed with 1 x M9 buffer, and snap frozen with liquid nitrogen. Total RNA was extracted using Trizol (Sigma). mRNA-seq libraries were constructed using a TruSeq RNA SamplePrep V2 kit (Illumina) according to manufacturer’s instruction.
Chromatin immunoprecipitation (ChIP) and construction of libraries for high-throughput sequencing
For ChIP-seq experiments, DAF-16::GFP-expressing transgenic daf-2(e1370) animals were used for DAF-16 pulldown SPT-5::GFP-expressing transgenic worms in daf-2(e1370) mutant background were used for SPT-5 and Pol II pulldowns. For Pol II pulldowns, daf-2 (e1370) mutants were used. Animals were grown on OP-50_1 seeded plates at 15°C and synchronized by bleaching. L1s were seeded on plates containing control or smk-1 RNAi bacteria. When they reached late L4 stage, FUDR was added and the plates were shifted to 25°C for 16-18 hours before harvesting. ChIP experiment was performed as previously described with some modifications (Curran et al., 2009). Sequencing libraries of inputs and immunoprecipitated samples were constructed according to (Bowman et al., 2013).
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High-throughput sequencing
For both multiplexed mRNA-seq and ChIP-seq libraries, single-end sequencing was conducted for 50 cycles on an Illumina HiSeq 2000/3000 sequencer, according to the manufacturer’s instructions. Image analysis, base calling, and quality scoring were carried out in real time with the standard Illumina analysis pipeline using a phiX control.
Processing of mRNA-seq reads, differential expression analysis, and evaluation of gene list overlaps
Reads were mapped to the C. elegans genome (WS220) with the TopHat (v2.0.8b) software package (Trapnell et al., 2009) by using gene model annotations with the following parameters: --library-type fr-unstranded --b2-very-sensitive coverage-intron 10 --min-segment-intron 10 --microexon-search --no-novel-juncs. Transcript abundance (FPKM, fragments per kilo base of transcript per million fragments) and differential expression was calculated with Cuffdiff (v2.1.1) from the Cufflinks software package (Trapnell et al., 2010) using the following parameters: -u --FDR 0.05 --upper-quartile-norm --compatible-hits-norm --library-type fr-unstranded. All conditions were analyzed at least by using two biological replicates and the analyses were only limited to protein-coding genes. Statistically significant differentially expressed genes (DEGs) were identified by 5% FDR. Differential gene expression values were achieved by the calculation of the ratio of FPKM values between two conditons. To test the significance of the overlap between gene lists, hypergeometric test was applied by using phyper R function (R Development Core Team, 2011).
Processing of ChIP-seq reads, peak calling, and metagene analysis
Alignments of the reads to the C. elegans genome (WS220) was achieved by using Bowtie (v2.1) with the following parameter: -q. Mapped reads that include no more than one mismatch were used for peak calling and read density calculations. Identification of the enriched peaks for each condition was carried out by using MACS (v2) with the following parameters: --mfold 5,30 --bw 200 --keep-dup auto -q 0.05. Only statistically significant peaks (q < 1×10−6) were kept. Hotspots (genomic regions that are commonly identified in ChIP-seq experiments and that are considered potential artefacts) were removed from the peak data set as previously described (Niu et al., 2011). For the calculation of the genome-wide read densities around transcriptional start sites (TSS), the R package ngs.plot was used with the following parameter: -FL 200 (Shen et al., 2014).
p-value calculations determining whether RNA Pol II, RNA Pol II(Ser5Phos), or SPT-5 binding to TSS regions had significantly changed between the compared conditions were conducted in identical manner, only that regions from -600 to +600 bp around TSSs were investigated.
Gene functional enrichment analysis and annotation
Gene functional enrichments were determined by using the DAVID Bioinformatics Resources version 6.7 (Huang et al., 2009). Annotation clusters determined by DAVID
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(groupings of related annotation terms) having an enrichment score of ≥ 1.3 were considered significant and a representative naming for the cluster was derived from the contained Gene Ontology (GO) terms.
Statistical analysis
Lifespan and stress resistance assays were evaluated by Kaplan–Maier and log-rank tests. P values of the Venn diagram overlaps were determined by hypergeometric test. Statistical analyses for other experiments are stated in the respective methods sections, tables or figure legends. The detailed results, also of the replicate experiments are shown in Table S5.
Analyses were performed using either SPSS (IBM), OASIS (Yang et al., 2011), Origin (Originlab) or R (R Development Core Team, 2011).
Author contributions:
I.S., and C.G.R. conceived the project, I.S. performed most of the experiments and analyzed the results. X.Z. and A.S. performed part of the mammalian conservation experiments, A.C. performed MS-based phosphoproteomics analysis, N.P.C. was involved in transgenic making and related transgenic lifespan experiments, and X.X.L., was involved in the experimental part of the stress assays, B.B. and S.B. provided technical support, I.S. and C.R. wrote the manuscript.
Acknowledgements:
We thank the Proteomics Karolinska (PK/KI) for proteomic analysis. We thank Jianping Liu and Xidan Li from the Sequencing Facility at Integrated Cardio Metabolic Centre (ICMC), Patrick Muller and Fredrik Fagerström Billai from the Bioinformatics and Expression Analysis Core Facility (BEA) at Karolinska Institute and the Lansdorp lab at ERIBA for technical support. We thank the Caenorhabditis Genetic Center (CGC) for providing us variety of strains and all Riedel lab members for their support.
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