Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes.
Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation. Our findings demonstrate that residues 115-125 within the yeast Top1p N-terminal domain are required for the complementation of the top1∆ rDNA phenotypes. In chromatin immunoprecipitation and co-immunoprecipitation experiments, we further demonstrate the physical interaction between Top1p and Sir2p. Our genetic and biochemical studies support a model whereby Top1p recruits Sir2p to the rDNA and clarifies a structural role of DNA topoisomerase I in the epigenetic regulation of rDNA, independent of its known catalytic activity.
Dipartimento di Biologia e Biotecnologie, Università di Roma Sapienza, Roma 00185, Italy. Electronic address: dalfonsoanna@yahoo.it.
Anna
A
D'Alfonso
Dipartimento di Biologia e Biotecnologie, Università di Roma Sapienza, Roma 00185, Italy. Electronic address: francesca.difelice@uniroma1.it.
Francesca
F
Di Felice
Dipartimento di Biologia e Biotecnologie, Università di Roma Sapienza, Roma 00185, Italy. Electronic address: vale611@hotmail.it.
Valentina
V
Carlini
Department of Pharmacology and Toxicology, University of Birmingham at Alabama, Birmingham, AL 35294, USA. Electronic address: cwright7@uab.edu.
Christine M
CM
Wright
Department of Pharmacology and Toxicology, University of Birmingham at Alabama, Birmingham, AL 35294, USA. Electronic address: marla.hertz@gmail.com.
Marla I
MI
Hertz
Department of Pharmacology and Toxicology, University of Birmingham at Alabama, Birmingham, AL 35294, USA. Electronic address: bjornsti@uab.edu.
Mary-Ann
MA
Bjornsti
Dipartimento di Biologia e Biotecnologie, Università di Roma Sapienza, Roma 00185, Italy; Istituto Pasteur, Fondazione Cenci Bolognetti, Università di Roma Sapienza, Roma 00185, Italy. Electronic address: giorgio.camilloni@umiroma1.it.
Giorgio
G
Camilloni
D047369:Chromatin Immunoprecipitation; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000378:metabolism*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D011485:Protein Binding; D012335:RNA, Ribosomal / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000378:metabolism*; D056565:Sirtuin 2 / Q000378:metabolism*; D014158:Transcription, Genetic
D004275:DNA, Ribosomal; D012335:RNA, Ribosomal; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2; D004264:DNA Topoisomerases, Type I
DNA topoisomerase I; Sir2p; histone acetylation; rDNA; transcriptional silencing