We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2-related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa2CysXaa20CysXaa2Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation.

Sporothrix schenckii (S. schenckii) is a dimorphic fungus that produces lymphocutaneous lesions. The signature characteristic of S. schenckii is a temperature-induced phase transition. Silent information regulator (Sir) has been proven to be involved in phenotypic switching in Saccharomyces cerevisiae (S. cerevisiae) and Candida albicans (C. albicans) by organizing chromatin structure. In this study, we isolated and characterized a Sir homologue gene, designated as SsSir2, from the yeast form of S. schenckii. The full-length SsSir2 cDNA sequence is 1753 bp in size and contains an open reading frame of 1329 bp encoding 442 amino acids. The predicted molecular mass of SsSir2 is 48.1 kDa with an estimated theoretical isoelectric point of 4.6. The SsSir2 kinase domain shows a 78% identity with that of Hst2, a Sir2 Ib gene from S. cerevisiae. Three exons and two introns were identified within the 1472‑bp SsSir2 genomic DNA sequence of S. schenckii. A three-dimensional model of SsSir2 was constructed using a homology modeling method, and its reliability was evaluated. The active site of SsSir2 was identified by docking simulation, which indicated that several important residues, such as Asn127 and Asp129, play an important role in the histone deacetylase activity of Sir2 family proteins. The differential expression of the SsSir2 in two stages was demonstrated by real-time RT-PCR. The expression of SsSir2 was higher in the yeast stage compared with that in the mycelial one, which indicated that SsSir2 may be involved in the phenotypic switching and morphogenesis of the yeast phase in S. schenckii.

Heterochromatin formation in the yeast Saccharomyces cerevisiae is characterized by the assembly of the Silent Information Regulator (SIR) complex, which consists of the histone deacetylase Sir2 and the structural components Sir3 and Sir4, and binds to unmodified nucleosomes to provide gene silencing. Sir3 contains an AAA+ ATPase-like domain, and mutations in an exposed loop on the surface of this domain abrogate Sir3 silencing function in vivo, as well in vitro binding to the Sir2/Sir4 subcomplex. Here, we found that the removal of a single methyl group in the C-terminal coiled-coil domain (mutation T1314S) of Sir4 was sufficient to restore silencing at the silent mating-type loci HMR and HML to a Sir3 version with a mutation in this loop. Restoration of telomeric silencing required further mutations of Sir4 (E1310V and K1325R). Significantly, these mutations in Sir4 restored in vitro complex formation between Sir3 and the Sir4 coiled-coil, indicating that the improved affinity between Sir3 and Sir4 is responsible for the restoration of silencing. Altogether, these observations highlight remarkable properties of selected amino-acid changes at the Sir3-Sir4 interface that modulate the affinity of the two proteins.

Positioning of telomeres at the nuclear periphery can have dramatic effects on gene expression by establishment of heritable, transcriptionally repressive subdomains. However, little is known about the integral membrane proteins that mediate telomere tethering at the nuclear envelope. Here, we find a previously unrecognized function for the Saccharomyces cerevisiae Sad1-UNC-84 domain protein Mps3 in regulating telomere positioning in mitotic cells. Our data demonstrate that the nucleoplasmic N-terminal acidic domain of Mps3 is not essential for viability. However, this acidic domain is necessary and sufficient for telomere tethering during S phase and the silencing of reporter constructs integrated at telomeres. We show that this is caused by the role of the Mps3 acidic domain in binding and localization of the silent information regulator protein Sir4 to the nuclear periphery. Thus, Mps3 functions as an integral membrane anchor for telomeres and is a novel nuclear receptor for the Sir4 pathway of telomere tethering and gene inactivation.

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC "affinity" genome-wide by performing an ORC ChIP-on-chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1-resistant ORC-interacting chromosomal sites (ORF-ORC sites) that did not function as replication origins or silencers. Instead, ORF-ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC-silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF-ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism.

Telomere chromatin structure is pivotal for maintaining genome stability by regulating the binding of telomere-associated proteins and inhibiting the DNA damage response. In Saccharomyces cerevisiae, silent information regulator (Sir) proteins bind to terminal repeats and to subtelomeric X-elements, resulting in transcriptional silencing. Herein, we show that sir2 mutant strains display a specific loss of a nucleosome residing in the X-elements and that this deficiency is remarkably consistent between different telomeres. The X-elements contain several binding sites for the transcription factor Reb1 and we found that Sir2 and Reb1 compete for stabilizing/destabilizing this nucleosome, i.e. inactivation of Reb1 in a sir2 background reinstated the lost nucleosome. The telomeric-repeat-containing RNAs (TERRAs) originate from subtelomeric regions and extend into the terminal repeats. Both Sir2 and Reb1 repress TERRAs and in a sir2 reb1 double mutant, TERRA levels increased synergistically, showing that Sir2 and Reb1 act in different pathways for repressing TERRAs. We present evidence that Reb1 restricts TERRAs by terminating transcription. Mapping the 5'-ends of TERRAs from several telomeres revealed that the Sir2-stabilized nucleosome is the first nucleosome downstream from the transcriptional start site for TERRAs. Finally, moving an X-element to a euchromatic locus changed nucleosome occupancy and positioning, demonstrating that X-element nucleosome structure is dependent on the local telomere environment.

Apoptotic-like programmed cell death (PCD) is one of the main strategies for fungi to resist environmental stresses and maintain homeostasis. The apoptosis-inducing factor (AIF) has been shown in different fungi to trigger PCD through upregulating reactive oxygen species (ROS). This study identified a mitochondrial localized AIF homolog, CcAIF1, from Coprinopsis cinerea monokaryon Okayama 7. Heterologous overexpression of CcAIF1 in Saccharomyces cerevisiae caused apoptotic-like PCD of the yeast cells. Ccaif1 was increased in transcription when C. cinerea interacted with Gongronella sp. w5, accompanied by typical apoptotic-like PCD in C. cinerea, including phosphatidylserine externalization and DNA fragmentation. Decreased mycelial ROS levels were observed in Ccaif1 silenced C. cinerea transformants during cocultivation, as well as reduction of the apoptotic levels, mycelial growth, and asexual sporulation. By comparison, Ccaif1 overexpression led to the opposite phenotypes. Moreover, the transcription and expression levels of laccase Lcc9 decreased by Ccaif1 silencing but increased firmly in Ccaif1 overexpression C. cinerea transformants in coculture. Thus, in conjunction with our previous report that intracellular ROS act as signal molecules to stimulate defense responses, we conclude that CcAIF1 is a regulator of ROS to promote apoptotic-like PCD and laccase expression in fungal-fungal interactions. In an axenic culture of C. cinerea, CcAIF1 overexpression and H2O2 stimulation together increased laccase secretion with multiplied production yield. The expression of two other normally silent isozymes, Lcc8 and Lcc13, was unexpectedly triggered along with Lcc9. KEY POINTS: • Mitochondrial CcAIF1 induces PCD during fungal-fungal interactions • CcAIF1 is a regulator of ROS to trigger the expression of Lcc9 for defense • CcAIF1 overexpression and H2O2 stimulation dramatically increase laccase production.