Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation

The Srs2 DNA helicase of Saccharomyces cerevisiae affects recombination in multiple ways. Srs2 not only inhibits recombination at stalled replication forks but also promotes the synthesis-dependent strand annealing (SDSA) pathway of recombination. Both functions of Srs2 are regulated by sumoylation-sumoylated PCNA recruits Srs2 to the replication fork to disfavor recombination, and sumoylation of Srs2 can be inhibitory to SDSA in certain backgrounds. To understand Srs2 function, we characterize the mechanism of its sumoylation in vitro and in vivo. Our data show that Srs2 is sumoylated at three lysines, and its sumoylation is facilitated by the Siz SUMO ligases. We also show that Srs2 binds to SUMO via a C-terminal SUMO-interacting motif (SIM). The SIM region is required for Srs2 sumoylation, likely by binding to SUMO-charged Ubc9. Srs2's SIM also cooperates with an adjacent PCNA-specific interaction site in binding to sumoylated PCNA to ensure the specificity of the interaction. These two functions of Srs2's SIM exhibit a competitive relationship: sumoylation of Srs2 decreases the interaction between the SIM and SUMO-PCNA, and the SUMO-PCNA-SIM interaction disfavors Srs2 sumoylation. Our findings suggest a potential mechanism for the equilibrium of sumoylated and PCNA-bound pools of Srs2 in cells.     

Sumoylation of the budding yeast kinetochore protein Ndc10 is required for Ndc10 spindle localization and regulation of anaphase spindle elongation

Posttranslational modification by the ubiquitin-like protein SUMO (small ubiquitin-like modifier) is emerging as an important regulator in many cellular processes, including genome integrity. In this study, we show that the kinetochore proteins Ndc10, Bir1, Ndc80, and Cep3, which mediate the attachment of chromosomes to spindle microtubules, are sumoylated substrates in budding yeast. Furthermore, we show that Ndc10, Bir1, and Cep3 but not Ndc80 are desumoylated upon exposure to nocodazole, highlighting the possibility of distinct roles for sumoylation in modulating kinetochore protein function and of a potential link between the sumoylation of kinetochore proteins and mitotic checkpoint function. We find that lysine to arginine mutations that eliminate the sumoylation of Ndc10 cause chromosome instability, mislocalization of Ndc10 from the mitotic spindle, abnormal anaphase spindles, and a loss of Bir1 sumoylation. These data suggest that sumoylation of Ndc10 and other kinetochore proteins play a critical role during the mitotic process.     

Enhancer sequences influence the role of the amino-terminal domain of bicoid in transcription

Bicoid (Bcd) is a Drosophila melanogaster morphogenetic gradient that controls embryonic patterning by activating target gene expression in a concentration-dependent manner. In this study we describe experiments to determine how different enhancers respond to Bcd distinctively, focusing on two natural Bcd-responsive enhancer elements, hunchback (hb) and knirps (kni). Our results show that, on the hb enhancer element, the amino-terminal domain of Bcd (residues 1 to 91) plays primarily an inhibitory role, whereas on the kni enhancer element this same Bcd domain plays a positive role at low protein concentrations. We further demonstrate that while the amino-terminal domain is largely dispensable for cooperative binding to the hb enhancer element, it is preferentially required for cooperative binding to the kni enhancer element. Alteration of the arrangement of Bcd binding sites in the kni enhancer element reduces the role of the amino-terminal domain in cooperative DNA binding but increases the effectiveness of the self-inhibitory function. In addition, elimination of symmetric pairs of Bcd binding sites in the kni enhancer element reduces both DNA binding and activation by Bcd. We propose that the amino-terminal domain of Bcd is an enhancer-specific switch that contributes to the protein's ability to activate different target genes in distinct manners.     

Sumoylation of Drosophila SU(VAR)3-7 is required for its heterochromatic function

In Drosophila, SU(VAR)3-7 is an essential heterochromatin component. It is required for proper chromatin condensation, and changing its dose modifies position-effect variegation. Sumoylation is a post-translational modification shown to play a role in diverse biological processes. Here, we demonstrate that sumoylation is essential for proper heterochromatin function in Drosophila through modification of SU(VAR)3-7. Indeed, SU(VAR)3-7 is sumoylated at lysine K839; this modification is required for localization of SU(VAR)3-7 at pericentric heterochromatin, chromosome 4, and telomeres. In addition, sumoylation of SU(VAR)3-7 is a prerequisite for its ability to enhance position-effect variegation. Thus, these results show that the heterochromatic function of SU(VAR)3-7 depends on its own sumoylation, and unveil a role for sumoylation in Drosophila heterochromatin.     

Sumoylation of CCAAT/enhancer-binding protein alpha and its functional roles in hepatocyte differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.     

Ribosomal protein S3 is stabilized by sumoylation

Human ribosomal protein S3 (rpS3) acts as a DNA repair endonuclease. The multiple functions of this protein are regulated by post-translational modifications including phosphorylation and methylation. Using a yeast-two hybrid screen, we identified small ubiquitin-related modifier-1 (SUMO-1) as a new interacting partner of rpS3. rpS3 interacted with SUMO-1 via the N- and C-terminal regions. We also observed sumoylation of rpS3 in Escherichia coli and mammalian cell systems. Furthermore, we discovered that one of three lysine residues, Lys18, Lys214, or Lys230, was sumoylated in rpS3. Interestingly, sumoylated rpS3 was resistant to proteolytic activity, indicating that SUMO-1 increased the stability of the rpS3 protein. We concluded that rpS3 is covalently modified by SUMO-1 and this post-translational modification regulates rpS3 function by increasing rpS3 protein stability.