Fission Yeast Iec1-Ino80-Mediated Nucleosome Eviction Regulates Nucleotide and Phosphate Metabolism

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.The structure of chromatin is modulated by chromatin-remodeling factors, such as histone-modifying enzymes and ATP-dependent chromatin-remodeling complexes (7, 35, 45, 54). The latter are usually multisubunit protein complexes containing an ATPase from the SWI2/SNF2 superfamily, which uses the energy derived from ATP hydrolysis to disrupt histone-DNA interactions (6, 13, 26).Ino80 is a member of this SWI2/SNF superfamily and is the catalytic subunit of the Ino80 chromatin-remodeling complex (65). Initially characterized in budding yeast (Saccharomyces cerevisiae), the Ino80 complex plays a central role in DNA-mediated processes, such as DNA double-strand break repair, homologous recombination, and the regulation of the DNA damage cell cycle checkpoint response, chromosomal-DNA replication, and transcription (5, 10, 18, 23, 24, 38, 39, 52, 56, 57, 65, 69, 74, 76, 80, 82; reviewed in reference 4). Ino80 complexes purified from budding yeast and mammalian cells contain core subunits, which are conserved across species, as well as species-specific proteins (36, 65). In budding yeast, the core Ino80 complex is composed of INO80, ARP5, ARP8, ARP4, RVB1, RVB2, IES2, and IES6 (63, 65). The actin-like proteins ARP5 and ARP8 are unique to the Ino80 complex and are required for an active complex in budding yeast (38, 63). ARP4 plays a role in the response to DNA damage in budding yeast and is also a member of the chromatin-remodeling complex SWR1 and the histone acetyltransferase complex NuA4 (16, 42, 52, 76). The Rvb1 and Rvb2 proteins (also called Pontin/Tip49 and Reptin/Tip48, respectively, in mammalian cells) are AAA⁺ ATPases related to the Holliday junction-resolving RUVB proteins of bacteria (65). They are integral members of the Ino80- and SWR1-remodeling complexes (36, 42, 43, 50, 65), as well as the histone acetylase Tip60 complex (34) and the transcription activator c-Myc complex (reviewed in references 25 and 81). Ies2 and Ies6 are conserved from yeast to humans and have direct roles in transcriptional regulation in mammalian cells (10, 63).A YY1-containing Ino80 complex was recently identified in mammalian and Drosophila cells (10, 41, 82). Yin yang-1 (YY1) is a zinc finger-containing Polycomb group (PcG) transcription factor that regulates genes essential for growth and development (1, 9, 15, 68). The mammalian YY1-Ino80 complex has been found to play roles in transcription and homologous-recombination-based repair (10, 82).The precise mechanisms by which Ino80 regulates cellular processes are unclear. Unlike budding yeast, which has very little higher-order chromatin, the fission yeast (Schizosaccharomyces pombe) offers a more complex chromatin structure that is more similar to that of higher eukaryotes in many respects (for a review, see reference 28). Therefore, the characterization of the Ino80 complex in this model organism may give new insights into the functions and mechanisms of this versatile complex.Here, we characterize the fission yeast Ino80 complex. The purified complex mediated ATP-dependent nucleosome remodeling in vitro. We found that the complex is highly conserved through evolution and contains a novel factor, Iec1 (67). Iec1 bears sequence similarity to YY1 and other members of the GLI-Krüppel family of zinc finger proteins. Iec1 and the Ino80 complex subunits arp8, ies6, and ies2 are important for efficient DNA damage repair and the response to replication stress. The double deletion of iec1 and ies2 suppresses defects of the single deletions, suggesting that the different Ino80-interacting proteins may program the complex in different, possibly opposing ways. We show that Iec1 is important for the regulation of several genes involved in nucleotide and phosphate metabolism. Iec1 is required for the binding of Ino80 to the promoter and downstream regions of these genes, leading to reduced nucleosome density over the locus. We suggest that the Iec1-Ino80 complex regulates the transcription of genes involved in nucleotide metabolism by mediating nucleosome eviction.