SUMO protease FUG1, histone reader AL3 and chromodomain protein LHP1 are integral to repeat expansion-induced gene silencing in Arabidopsis thaliana.

Epigenetic gene silencing induced by expanded repeats can cause diverse phenotypes ranging from severe growth defects in plants to genetic diseases such as Friedreich's ataxia in humans. The molecular mechanisms underlying repeat expansion-induced epigenetic silencing remain largely unknown. Using a plant model with a temperature-sensitive phenotype, we have previously shown that expanded repeats can induce small RNAs, which in turn can lead to epigenetic silencing through the RNA-dependent DNA methylation pathway. Here, using a genetic suppressor screen and yeast two-hybrid assays, we identified novel components required for epigenetic silencing caused by expanded repeats. We show that FOURTH ULP GENE CLASS 1 (FUG1)-an uncharacterized SUMO protease with no known role in gene silencing-is required for epigenetic silencing caused by expanded repeats. In addition, we demonstrate that FUG1 physically interacts with ALFIN-LIKE 3 (AL3)-a histone reader that is known to bind to active histone mark H3K4me2/3. Loss of function of AL3 abolishes epigenetic silencing caused by expanded repeats. AL3 physically interacts with the chromodomain protein LIKE HETEROCHROMATIN 1 (LHP1)-known to be associated with the spread of the repressive histone mark H3K27me3 to cause repeat expansion-induced epigenetic silencing. Loss of any of these components suppresses repeat expansion-associated phenotypes coupled with an increase in IIL1 expression with the reversal of gene silencing and associated change in epigenetic marks. Our findings suggest that the FUG1-AL3-LHP1 module is essential to confer repeat expansion-associated epigenetic silencing and highlight the importance of post-translational modifiers and histone readers in epigenetic silencing.

1,10-phenanthroline inhibits sumoylation and reveals that yeast SUMO modifications are highly transient.

The steady-state levels of protein sumoylation depend on relative rates of conjugation and desumoylation. Whether SUMO modifications are generally long-lasting or short-lived is unknown. Here we show that treating budding yeast cultures with 1,10-phenanthroline abolishes most SUMO conjugations within one minute, without impacting ubiquitination, an analogous post-translational modification. 1,10-phenanthroline inhibits the formation of the E1~SUMO thioester intermediate, demonstrating that it targets the first step in the sumoylation pathway. SUMO conjugations are retained after treatment with 1,10-phenanthroline in yeast that express a defective form of the desumoylase Ulp1, indicating that Ulp1 is responsible for eliminating existing SUMO modifications almost instantly when de novo sumoylation is inhibited. This reveals that SUMO modifications are normally extremely transient because of continuous desumoylation by Ulp1. Supporting our findings, we demonstrate that sumoylation of two specific targets, Sko1 and Tfg1, virtually disappears within one minute of impairing de novo sumoylation. Altogether, we have identified an extremely rapid and potent inhibitor of sumoylation, and our work reveals that SUMO modifications are remarkably short-lived.

Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast.

Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.

Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II.

Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

A conserved role for transcription factor sumoylation in binding-site selection.

Large numbers of eukaryotic transcription factors (TFs) are modified by SUMO post-translational modifications. Whereas the effect of TF sumoylation on the expression of target genes is largely context-dependent, it is not known whether the modification has a common function in regulating TFs throughout eukaryotic species. Here, I highlight four studies that used genome-wide chromatin-immunoprecipitation analysis (ChIP-seq) to examine whether sumoylation affects the selection of sites on the genome that are bound by human and yeast TFs. The studies found that impairing sumoylation led to deregulated binding-site selection for all four of the examined TFs. Predominantly, compared to wild-type forms, the sumoylation-deficient forms of the TFs bound to numerous additional non-specific sites, pointing to a common role for the modification in restricting TF binding to appropriate sites. Evidence from these studies suggests that TF sumoylation influences binding-site selection by modulating protein-protein interactions with other DNA-binding TFs, or by promoting conformational changes in the TFs that alter their DNA-binding specificity or affinity. I propose a model in which, prior to their sumoylation, TFs initially bind to chromatin with reduced specificity, which leads to spurious binding but also ensures that all functional sites become bound. Once the TFs are bound to DNA, sumoylation then acts to increase specificity and promotes release of the TFs from non-specific sites. The similar observations from these four genome-wide studies across divergent species suggest that binding-site selection is a general and conserved function for TF sumoylation.

Sumoylation of DNA-bound transcription factor Sko1 prevents its association with nontarget promoters.

Sequence-specific transcription factors (TFs) represent one of the largest groups of proteins that is targeted for SUMO post-translational modification, in both yeast and humans. SUMO modification can have diverse effects, but recent studies showed that sumoylation reduces the interaction of multiple TFs with DNA in living cells. Whether this relates to a general role for sumoylation in TF binding site selection, however, has not been fully explored because few genome-wide studies aimed at studying such a role have been reported. To address this, we used genome-wide analysis to examine how sumoylation regulates Sko1, a yeast bZIP TF with hundreds of known binding sites. We find that Sko1 is sumoylated at Lys 567 and, although many of its targets are osmoresponse genes, the level of Sko1 sumoylation is not stress-regulated and the modification does not depend or impinge on its phosphorylation by the osmostress kinase Hog1. We show that Sko1 mutants that cannot bind DNA are not sumoylated, but attaching a heterologous DNA binding domain restores the modification, implicating DNA binding as a major determinant for Sko1 sumoylation. Genome-wide chromatin immunoprecipitation (ChIP-seq) analysis shows that a sumoylation-deficient Sko1 mutant displays increased occupancy levels at its numerous binding sites, which inhibits the recruitment of the Hog1 kinase to some induced osmostress genes. This strongly supports a general role for sumoylation in reducing the association of TFs with chromatin. Extending this result, remarkably, sumoylation-deficient Sko1 binds numerous additional promoters that are not normally regulated by Sko1 but contain sequences that resemble the Sko1 binding motif. Our study points to an important role for sumoylation in modulating the interaction of a DNA-bound TF with chromatin to increase the specificity of TF-DNA interactions.

Regulation of transcription factors by sumoylation.

Transcription factors (TFs) are among the most frequently detected targets of sumoylation, and effects of the modification have been studied for about 200 individual TFs to date. TF sumoylation is most often associated with reduced target gene expression, which can be mediated by enhanced interactions with corepressors or by interference with protein modifications that promote transcription. However, recent studies show that sumoylation also regulates gene expression by controlling the levels of TFs that are associated with chromatin. SUMO can mediate this by modulating TF DNA-binding activity, promoting clearance of TFs from chromatin, or indirectly, by influencing TF abundance or localization.

Chromatin Association of Gcn4 Is Limited by Post-translational Modifications Triggered by its DNA-Binding in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae transcription factor Gcn4 is expressed during amino acid starvation, and its abundance is controlled by ubiquitin-mediated proteolysis. Cdk8, a kinase component of the RNA polymerase II Mediator complex, phosphorylates Gcn4, which triggers its ubiquitination/proteolysis, and is thought to link Gcn4 degradation with transcription of target genes. In addition to phosphorylation and ubiquitination, we previously showed that Gcn4 becomes sumoylated in a DNA-binding dependent manner, while a nonsumoylatable form of Gcn4 showed increased chromatin occupancy, but only if Cdk8 was present. To further investigate how the association of Gcn4 with chromatin is regulated, here we examine determinants for Gcn4 sumoylation, and how its post-translational modifications are coordinated. Remarkably, artificially targeting Gcn4 that lacks its DNA binding domain to a heterologous DNA site restores sumoylation at its natural modification sites, indicating that DNA binding is sufficient for the modification to occur in vivo Indeed, we find that neither transcription of target genes nor phosphorylation are required for Gcn4 sumoylation, but blocking its sumoylation alters its phosphorylation and ubiquitination patterns, placing Gcn4 sumoylation upstream of these Cdk8-mediated modifications. Strongly supporting a role for sumoylation in limiting its association with chromatin, a hyper-sumoylated form of Gcn4 shows dramatically reduced DNA occupancy and expression of target genes. Importantly, we find that Cdk8 is at least partly responsible for clearing hyper-sumoylated Gcn4 from DNA, further implicating sumoylation as a stimulus for Cdk8-mediated phosphorylation and degradation. These results support a novel function for SUMO in marking the DNA-bound form of a transcription factor, which triggers downstream processes that limit its association with chromatin, thus preventing uncontrolled expression of target genes.

Sumoylation controls the timing of Tup1-mediated transcriptional deactivation.

The small ubiquitin-like modifier (SUMO) is implicated in various cellular activities, including transcriptional regulation. We previously showed that the yeast activator Gcn4 becomes sumoylated during activation, facilitating its eventual promoter eviction and transcriptional shut off. Here we show that the corepressor Tup1 is sumoylated, at two specific lysines, under various stress conditions. Mutation of these sites has no effect on Tup1 recruitment or RNAP II promoter occupancy immediately following induction. However, Tup1 levels subsequently decrease, while RNAP II and transcription increase in Tup1 mutant cells. Consistent with this, a Tup1 mutant displaying increased sumoylation led to reduced transcription. We also show that coordinated sumoylation of Gcn4 and Tup1 enhances Gcn4 promoter eviction and that multiple Tup1-interacting proteins become sumoylated after stress. Together, our studies provide evidence that coordinated sumoylation of Gcn4, Tup1 and likely other factors dampens activated transcription by stabilizing Tup1 binding and stimulating Gcn4 and RNAP II removal.

Threonine-4 of the budding yeast RNAP II CTD couples transcription with Htz1-mediated chromatin remodeling.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of repeated YSPTSPS heptapeptides and connects transcription with cotranscriptional events. Threonine-4 (Thr4) of the CTD repeats has been shown to function in histone mRNA 3'-end processing in chicken cells and in transcriptional elongation in human cells. Here, we demonstrate that, in budding yeast, Thr4, although dispensable for growth in rich media, is essential in phosphate-depleted or galactose-containing media. Thr4 is required to maintain repression of phosphate-regulated (PHO) genes under normal growth conditions and for full induction of PHO5 and the galactose-induced GAL1 and GAL7 genes. We identify genetic links between Thr4 and the histone variant Htz1 and show that Thr4, as well as the Ino80 chromatin remodeler, is required for activation-associated eviction of Htz1 specifically from promoters of the Thr4-dependent genes. Our study uncovers a connection between transcription and chromatin remodeling linked by Thr4 of the CTD.

Sumoylation of transcription factor Gcn4 facilitates its Srb10-mediated clearance from promoters in yeast.

The small ubiquitin-related modifier (SUMO) is a conserved factor that post-translationally regulates proteins involved in many cellular processes, including gene transcription. We previously demonstrated that promoter-bound factors become sumoylated during activation of inducible genes in yeast, but the identity of these factors, and the role of sumoylation in their function, was unknown. Here we show that the transcriptional activator Gcn4 is sumoylated on two specific lysine residues and in a manner that depends on its ability to bind DNA, indicating that sumoylation occurs after Gcn4 binding to target promoters. Importantly, this functions to facilitate the subsequent removal of the activator from these promoters after recruitment of RNA polymerase II, which can prevent inappropriate transcription of target genes. Furthermore, we show that clearance of sumoylated Gcn4 requires the protein kinase and Mediator complex subunit Srb10, linking activator removal with target gene transcription. Our study demonstrates an unexpected role for protein sumoylation in the process of transcriptional activation.

Sub1 globally regulates RNA polymerase II C-terminal domain phosphorylation.

The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3'-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

SUMO functions in constitutive transcription and during activation of inducible genes in yeast.

Transcription factors represent one of the largest groups of proteins regulated by SUMO (small ubiquitin-like modifier) modification, and their sumoylation is usually associated with transcriptional repression. To investigate whether sumoylation plays a general role in regulating transcription in yeast, we determined the occupancy of sumoylated proteins at a variety of genes by chromatin immunoprecipitation (ChIP) using an antibody that recognizes the yeast SUMO peptide. Surprisingly, we detected sumoylated proteins at all constitutively transcribed genes tested but not at repressed genes. Ubc9, the SUMO conjugation enzyme, was not present on these genes, but its inactivation reduced SUMO at the constitutive promoters and modestly decreased RNA polymerase II levels. In contrast, activation of the inducible GAL1, STL1, and ARG1 genes caused not only a striking accumulation of SUMO at all three promoter regions, but also recruitment of Ubc9, indicating that gene activation involves sumoylation of promoter-bound factors. However, Ubc9 inactivation, while reducing sumoylation at the induced promoters, paradoxically resulted in increased transcription. Providing an explanation for this, the reduced sumoylation impaired the cell's ability to appropriately shut off transcription of the induced ARG1 gene, indicating that SUMO can facilitate transcriptional silencing. Our findings thus establish unexpected roles for sumoylation in both constitutive and activated transcription, and provide a novel mechanism for regulating gene expression.

Alternative polyadenylation blooms.

Alternative polyadenylation generates mRNAs with 3' untranscribed regions of different lengths, often affecting transcript stability. Hornyik et al., in this issue of Developmental Cell, and Liu et al. now demonstrate a role for alternative polyadenylation in gene silencing and the regulation of flowering time in Arabidopsis thaliana.

Telomere maintenance and survival in saccharomyces cerevisiae in the absence of telomerase and RAD52.

Telomeres are essential features of linear genomes that are crucial for chromosome stability. Telomeric DNA is usually replenished by telomerase. Deletion of genes encoding telomerase components leads to telomere attrition with each cycle of DNA replication, eventually causing cell senescence or death. In the Saccharomyces cerevisiae strain W303, telomerase-null populations bypass senescence and, unless EXO1 is also deleted, this survival is RAD52 dependent. Unexpectedly, we found that the S. cerevisiae strain S288C could survive the removal of RAD52 and telomerase at a low frequency without additional gene deletions. These RAD52-independent survivors were propagated stably and exhibited a telomere organization typical of recombination between telomeric DNA tracts, and in diploids behaved as a multigenic trait. The polymerase-delta subunit Pol32 was dispensable for the maintenance of RAD52-independent survivors. The incidence of this rare escape was not affected by deletion of other genes necessary for RAD52-dependent survival, but correlated with initial telomere length. If W303 strains lacking telomerase and RAD52 first underwent telomere elongation, rare colonies could then bypass senescence. We suggest that longer telomeres provide a more proficient substrate for a novel telomere maintenance mechanism that does not rely on telomerase, RAD52, or POL32.

Sub1 functions in osmoregulation and in transcription by both RNA polymerases II and III.

Sub1 is implicated in transcriptional activation, elongation, and mRNA 3'-end formation in budding yeast. To gain more insight into its function, we performed a synthetic genetic array screen with SUB1 that uncovered genetic interactions with genes involved in the high-osmolarity glycerol (HOG) osmoresponse pathway. We find that Sub1 and the HOG pathway are redundant for survival in moderate osmolarity. Chromatin immunoprecipitation analysis shows that Sub1 is recruited to osmoresponse gene promoters during osmotic shock and is required for full recruitment of TBP, TFIIB, and RNA polymerase II (RNAP II) at a subset of these genes. Furthermore, we detect Sub1 at the promoter of every constitutively transcribed RNAP II and, unexpectedly, at every RNAP III gene tested, but not at the RNAP I-transcribed ribosomal DNA promoter. Significantly, deletion of SUB1 reduced levels of promoter-associated RNAP II or III at these genes, but not TBP levels. Together these data suggest that, in addition to a general role in polymerase recruitment at constitutive RNAP II and RNAP III genes, during osmotic shock, Sub1 facilitates osmoresponse gene transcription by enhancing preinitiation complex formation.

From transcription to mRNA: PAF provides a new path.

Two new studies reveal a novel connection between transcription and pre-mRNA processing: the Paf1 elongation complex is involved in the 3' end formation of polyadenylated and nonpolyadenylated RNA polymerase II transcripts.

Role for PSF in mediating transcriptional activator-dependent stimulation of pre-mRNA processing in vivo.

In a recent study, we provided evidence that strong promoter-bound transcriptional activators result in higher levels of splicing and 3'-end cleavage of nascent pre-mRNA than do weak promoter-bound activators and that this effect of strong activators requires the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II). In the present study, we have investigated the mechanism of activator- and CTD-mediated stimulation of pre-mRNA processing. Affinity chromatography experiments reveal that two factors previously implicated in the coupling of transcription and pre-mRNA processing, PSF and p54(nrb)/NonO, preferentially bind a strong rather than a weak activation domain. Elevated expression in human 293 cells of PSF bypasses the requirement for a strong activator to promote efficient splicing and 3'-end cleavage. Truncation of the pol II CTD, which consists of 52 repeats of the consensus heptapeptide sequence YSPTSPS, to 15 heptapeptide repeats prevents PSF-dependent stimulation of splicing and 3'-end cleavage. Moreover, PSF and p54(nrb)/NonO bind in vitro to the wild-type CTD but not to the truncated 15-repeat CTD, and domains in PSF that are required for binding to activators and to the CTD are also important for the stimulation of pre-mRNA processing. Interestingly, activator- and CTD-dependent stimulation of splicing mediated by PSF appears to primarily affect the removal of first introns. Collectively, these results suggest that the recruitment of PSF to activated promoters and the pol II CTD provides a mechanism by which transcription and pre-mRNA processing are coordinated within the cell.