The SNF2-family member Fun30 promotes gene silencing in heterochromatic loci.
Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.
Neves-Costa
Ana
A
Chromatin and Gene Expression, Babraham Institute, Cambridge, United Kingdom.
Will
W Ryan
WR
Vetter
Anna T
AT
Miller
J Ross
JR
Varga-Weisz
Patrick
P
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D004275:DNA, Ribosomal / Q000235:genetics; D017353:Gene Deletion; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter; D056426:Genetic Loci / Q000235:genetics*; D006570:Heterochromatin / Q000187:drug effects / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D016615:Telomere / Q000235:genetics; D059004:Topoisomerase I Inhibitors; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D004275:DNA, Ribosomal; C074000:FUN30 protein, S cerevisiae; D006570:Heterochromatin; D029701:Saccharomyces cerevisiae Proteins; D059004:Topoisomerase I Inhibitors; D014157:Transcription Factors; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C499333:SNF2 protein, S cerevisiae; D002166:Camptothecin