Final Report Summary - SUMO AND CHROMATIN (Regulating nuclear organisation in telomere maintenance and DNA repair: the role of SUMO modification)
The genetic information of all cells is contained within the sequence of DNA that makes up its genome. Importantly however, DNA is not naked within the nucleus but is complexed with histone proteins to form chromatin. It has been shown that chromatin structure can be modulated to regulate access to the underlying DNA. Thus, chromatin is a key means of regulating where and when DNA can be accessed and is therefore important for all aspects of DNA metabolism. Recently, growing evidence has suggested that not only is chromatin structure important but also chromatin localisation, i.e. where within the nucleus it is found.
Telomeres contain a specialised chromatin structure that consist of TG rich DNA sequence repeats and associated binding proteins that form the ends of all linear chromosomes. These serve to protect chromosome ends from being recognised as double strand breaks (DSBs) which would otherwise result in either cell cycle arrest or eventual genome instability. In budding yeast, telomeres show a particularly distinctive nuclear localisation; being reversibly bound to the nuclear periphery. The organisation of chromatin into spatially and functionally distinct compartments is thought to promote the efficient completion of fundamental nuclear processes such as transcription and DNA repair. This is perhaps especially important for telomeres, where it has been shown that altered nuclear localisation and dynamics can result in an inappropriate repair response.
Telomere anchoring in S. cerevisiae occurs via two partially redundant genetic pathways acting via either Sir4 or the Yku70/80 heterodimer. Interestingly, the relative influence of these pathways changes throughout the cell cycle and also from telomere to telomere, suggesting a dynamic regulation of anchoring by these factors. Given that several proteins implicated in telomere anchoring have been identified in large-scale screens for sumoylated proteins, we hypothesised that SUMO (small ubiquitin-like modifier) modification may impact upon telomere anchoring.
During our research, we were able to show that deletion of the PIAS-like SUMO E3 ligase SIZ2 but not the related SIZ1 enzyme delocalises telomeres from the nuclear periphery. This coincides with an increased association of telomerase with telomeres and a telomerase dependent increase in TG repeat length. Remarkably, the localisation and length phenotypes seen in siz2delta cells appear to be functionally linked as both are suppressed by additional deletion of the DNA helicase PIF1. Together, these data suggest that the anchoring of telomeres to the nuclear periphery may impair the ability of telomerase to lengthen telomeres even though telomerase itself can help anchor telomeres in S-phase cells. Furthermore, we find that critically short telomeres detach from the nuclear periphery in the first S-phase of zygote formation when they are likely being elongated.
Both telomere anchoring pathways (Sir4 and Yku70/80) are positively regulated by Siz2 and, consistently, both anchors are targets of Siz2 dependent sumoylation in vivo. Importantly, we see that the defect in the Yku80-4 (an allele of Yku80 that does not bind Sir4) mediated chromatin anchoring seen in siz2? cells can be overcome by artificially sumoylating Yku80-4. Our data suggests that SUMO modification impacts upon telomere maintenance by affecting telomere localisation, a role which may be conserved in higher eukaryotes. Indeed, in human cancer cell using the alternative lengthening of telomeres (ALT) pathway, the localisation of telomeres to PML bodies is driven by the sumoylation of telomere binding proteins. Therefore, using a simplified system like budding yeast to gain a deeper mechanistic understanding of these processes will be of benefit to the treatment of human disorders such as cancer that show significant telomere misregulation.
Telomeres contain a specialised chromatin structure that consist of TG rich DNA sequence repeats and associated binding proteins that form the ends of all linear chromosomes. These serve to protect chromosome ends from being recognised as double strand breaks (DSBs) which would otherwise result in either cell cycle arrest or eventual genome instability. In budding yeast, telomeres show a particularly distinctive nuclear localisation; being reversibly bound to the nuclear periphery. The organisation of chromatin into spatially and functionally distinct compartments is thought to promote the efficient completion of fundamental nuclear processes such as transcription and DNA repair. This is perhaps especially important for telomeres, where it has been shown that altered nuclear localisation and dynamics can result in an inappropriate repair response.
Telomere anchoring in S. cerevisiae occurs via two partially redundant genetic pathways acting via either Sir4 or the Yku70/80 heterodimer. Interestingly, the relative influence of these pathways changes throughout the cell cycle and also from telomere to telomere, suggesting a dynamic regulation of anchoring by these factors. Given that several proteins implicated in telomere anchoring have been identified in large-scale screens for sumoylated proteins, we hypothesised that SUMO (small ubiquitin-like modifier) modification may impact upon telomere anchoring.
During our research, we were able to show that deletion of the PIAS-like SUMO E3 ligase SIZ2 but not the related SIZ1 enzyme delocalises telomeres from the nuclear periphery. This coincides with an increased association of telomerase with telomeres and a telomerase dependent increase in TG repeat length. Remarkably, the localisation and length phenotypes seen in siz2delta cells appear to be functionally linked as both are suppressed by additional deletion of the DNA helicase PIF1. Together, these data suggest that the anchoring of telomeres to the nuclear periphery may impair the ability of telomerase to lengthen telomeres even though telomerase itself can help anchor telomeres in S-phase cells. Furthermore, we find that critically short telomeres detach from the nuclear periphery in the first S-phase of zygote formation when they are likely being elongated.
Both telomere anchoring pathways (Sir4 and Yku70/80) are positively regulated by Siz2 and, consistently, both anchors are targets of Siz2 dependent sumoylation in vivo. Importantly, we see that the defect in the Yku80-4 (an allele of Yku80 that does not bind Sir4) mediated chromatin anchoring seen in siz2? cells can be overcome by artificially sumoylating Yku80-4. Our data suggests that SUMO modification impacts upon telomere maintenance by affecting telomere localisation, a role which may be conserved in higher eukaryotes. Indeed, in human cancer cell using the alternative lengthening of telomeres (ALT) pathway, the localisation of telomeres to PML bodies is driven by the sumoylation of telomere binding proteins. Therefore, using a simplified system like budding yeast to gain a deeper mechanistic understanding of these processes will be of benefit to the treatment of human disorders such as cancer that show significant telomere misregulation.