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Functional complementation of sir2? yeast mutation by the human orthologous gene SIRT1.

ABSTRACT: Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2? mutant strain. The structure of chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2? shows a series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2? strains. By strong SIRT1 overexpression in sir2? cells, we found that specific molecular phenotypes of the mutant revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that: i) SIRT1 gene is able to complement different molecular phenotypes of the sir2? mutant at rDNA ii) the in vivo screening of Sirt1 activity is possible in yeast.

SUBMITTER: Gaglio D

PROVIDER: S-EPMC3859646 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Project description:BackgroundMultiprotein-bridging factor 1 (MBF1) is a transcriptional co-activator that bridges a sequence-specific activator (basic-leucine zipper (bZIP) like proteins (e.g. Gcn4 in yeast) or steroid/nuclear-hormone receptor family (e.g. FTZ-F1 in insect)) and the TATA-box binding protein (TBP) in Eukaryotes. MBF1 is absent in Bacteria, but is well- conserved in Eukaryotes and Archaea and harbors a C-terminal Cro-like Helix Turn Helix (HTH) domain, which is the only highly conserved, classical HTH domain that is vertically inherited in all Eukaryotes and Archaea. The main structural difference between archaeal MBF1 (aMBF1) and eukaryotic MBF1 is the presence of a Zn ribbon motif in aMBF1. In addition MBF1 interacting activators are absent in the archaeal domain. To study the function and therefore the evolutionary conservation of MBF1 and its single domains complementation studies in yeast (mbf1Δ) as well as domain swap experiments between aMBF1 and yMbf1 were performed.ResultsIn contrast to previous reports for eukaryotic MBF1 (i.e. Arabidopsis thaliana, insect and human) the two archaeal MBF1 orthologs, TMBF1 from the hyperthermophile Thermoproteus tenax and MMBF1 from the mesophile Methanosarcina mazei were not functional for complementation of an Saccharomyces cerevisiae mutant lacking Mbf1 (mbf1Δ). Of twelve chimeric proteins representing different combinations of the N-terminal, core domain, and the C-terminal extension from yeast and aMBF1, only the chimeric MBF1 comprising the yeast N-terminal and core domain fused to the archaeal C-terminal part was able to restore full wild-type activity of MBF1.However, as reported previously for Bombyx mori, the C-terminal part of yeast Mbf1 was shown to be not essential for function. In addition phylogenetic analyses revealed a common distribution of MBF1 in all Archaea with available genome sequence, except of two of the three Thaumarchaeota; Cenarchaeum symbiosum A and Nitrosopumilus maritimus SCM1.ConclusionsThe absence of MBF1-interacting activators in the archaeal domain, the presence of a Zn ribbon motif in the divergent N-terminal domain of aMBF1 and the complementation experiments using archaeal- yeast chimeric proteins presented here suggests that archaeal MBF1 is not able to functionally interact with the transcription machinery and/or Gcn4 of S. cerevisiae. Based on modeling and structural prediction it is tempting to speculate that aMBF1 might act as a single regulator or non-essential transcription factor, which directly interacts with DNA via the positive charged linker or the basal transcription machinery via its Zn ribbon motif and the HTH domain. However, also alternative functions in ribosome biosynthesis and/or functionality have been discussed and therefore further experiments are required to unravel the function of MBF1 in Archaea.

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Functional complementation of sir2? yeast mutation by the human orthologous gene SIRT1.

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