engleski

MULTIPLE ROLES OF SAGA IN THE ACTIVATION OF THE PHO5 PROMOTER

Activation of the yeast PHO5 gene upon phosphate starvation is accompanied by a profound structural alteration of the promoter chromatin structure, making it a suitable model to study the process of chromatin remodeling and the role chromatin plays in transcriptional regulation. Remodeling of the promoter chromatin structure is triggered by the binding of Pho4, the principal activator of PHO5, to the promoter, which is then thought to recruit chromatin modifying multiprotein complexes, such as SAGA and Swi/Snf. The molecular mechanisms, however, by which these multiprotein coactivators function are not well understood. We have previously shown that even though the final level of activated PHO5 transcription is not affected by a GCN5 deletion, the rate of chromatin remodeling and consequently the rate of promoter activation is strongly decreased (1). Here we have addressed the possible role(s) of other SAGA components in transcriptional activation of the PHO5 promoter. First, we have found that deletion of SPT7 or SPT20 which results in SAGA disassembly, has a much stronger effect on promoter activation than a GCN5 deletion, dramatically reducing not only the rate but also the final level of transcriptional activation. This shows that SAGA contributes to promoter activation not only through Gcn5 activity. Deletion of the SAGA subunit ADA2 had practically the same effect as deletion of GCN5. Moreover, disruption of the Ada2 SANT domain, which is not essential for SAGA assembly but plays a critical role in Gcn5 catalytic activity (2), also results in a gcn5 phenotype. These results demonstrate an essential, physiologically relevant role of the Ada2 SANT domain in controlling Gcn5 activity at the PHO5 promoter. Finally, we have found that the absence of Spt3 results in a significantly decreased promoter activity, affecting, in contrast to Gcn5, not the rate but the extent of transcriptional activation. Moreover, deletion of both, SPT3 and GCN5 results in a much stronger defect than deletion of only one. The double disruption has almost the same effect as an SPT7 or SPT20 deletion. Importantly, the rate of chromatin remodeling is not affected by the absence of Spt3, suggesting that Spt3 is required at a step subsequent to chromatin opening and thus has a function that is distinct from the function of Gcn5. These results show that SAGA plays at least two independent roles in PHO5 activation.

SAGA; PHO5 promoter; chromatin remodeling

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