| Popis: |
The massive transition of chromatin structure at the PHO5 promoter from a repressed, closed, to an active, open state was clearly demonstrated to be a prerequisite for promoter activation. We have previously shown that chromatin structure remodelling at the PHO5 promoter involved the SWI/SNF and Ino80 remodelling complexes, the Gcn5 HAT activity and the histone chaperone Asf1, but no essential chromatin cofactor has been identified yet. The RSC complex is one of the most abundant chromatin-remodelling complexes in yeast. It contains the ATPase subunit Sth1, which is paralogue to Snf2, and has been shown to disassemble nucleosomes in vitro. Here we show that the RSC complex has a prominent role in chromatin remodelling and consequently in activation of the PHO5 promoter in vivo. Since inactivation of the RSC subunit Sth1 is lethal, we employed a conditional, temperature sensitive sth1td mutant. Already at conditions of depletion rather than complete elimination of Sth1, remodelling at the PHO5 promoter under physiological induction conditions in phosphate-free medium is significantly affected. This effect is even more pronounced under weaker induction conditions that bring about PHO5 induction in otherwise repressive phosphate-rich medium. Under such semi-inducing conditions (inactivation of Pho85 or Pho80) a higher stringency of cofactor requirement is generally required, and indeed, no significant opening of PHO5 promoter chromatin was detected upon ablation of the RSC complex. Simultaneous inactivation of the SWI/SNF and RSC complexes completely prevents remodelling at the PHO5 promoter even under full induction conditions, demonstrating a functional interplay of the two complexes. Chromatin remodelling and consequent activation of a PHO5 promoter variant, which is activated by Gal4 instead of the native Pho4 activator, was similarly affected by the absence of RSC activity. Finally, assessing the effects of RSC inactivation on the regulation of chromatin structure at the PHO8 and PHO84 promoters, which are coactivated with PHO5, showed a differential involvement of the RSC complex at these three PHO promoters. |
