Elaboration, Diversification and Regulation of the Sir1 Family of Silencing Proteins in Saccharomyces

This study quantifies the effects of naturally occurring X-linked variation on immune response in Drosophila melanogaster to assess associations between immunity genotypes and innate immune response. We constructed a set of 168 X-chromosomal extraction lines, incorporating X chromosomes from a natural population into co-isogenic autosomal backgrounds, and genotyped the lines at 88 SNPs in 20 X-linked immune genes. We find that genetic variation in many of the genes is associated with immune response phenotypes, including bacterial load and immune gene expression. Many of the associations act in a sex-specific or sexually antagonistic manner, supporting the theory that with the selective pressures facing genes on the X chromosome, sexually antagonistic variation may be more easily maintained.THE deep evolutionary conservation of many specific genes in innate immunity underscores the potent forces of natural selection maintaining this vital function. While it is widely accepted as the ancestral form of immune response, its role in the activation of adaptive immune response further motivates investigation into variation in its function (Medzhitov and Janeway 1997). Drosophila has been used as a valuable model organism to identify and characterize functions of the components of innate immune pathways as well as the evolutionary patterns present among the genes comprising these pathways (reviewed in Brennan and Anderson 2004; Irving et al. 2004; Ferrandon et al. 2007). The humoral response, resulting in the production of antimicrobial peptides in response to bacterial or fungal infection, relies mainly on Toll and imd signal transduction pathways, both of which are highly homologous to pathways in mammalian immunity (reviewed in Kimbrell and Beutler 2001). The cellular component, on the other hand, incorporates phagocytic engulfment as well as melanization and encapsulation of infecting particles. While less well defined in the Drosophila model, portions of other systems also appear to affect the effectiveness of immune response, including JAK/STAT and JNK signaling pathways, hematopoesis, and iron metabolism.Population genetic analysis can be used to determine whether sequence polymorphism and divergence patterns among Drosophila genes in innate immune pathways are consistent with signatures of selection acting within and between species of flies. If, for example, the innate immune pathways are involved in an evolutionary “arms race” with pathogenic organisms, genes in these pathways would be expected to show signs of positive selection driven by evolutionary pressure to counter virulence mechanisms of invading microbes. When signs of selection (as inferred from sequence comparisons within Drosophila simulans populations and between D. simulans and D. melanogaster) in immune genes and nonimmune genes were evaluated, immune genes as a group were found to have higher K_A/K_S ratios than nonimmune genes, providing evidence for elevated adaptive evolution (Schlenke and Begun 2003). Since receptor, effector, and signaling proteins function in different portions of the immune response pathways, these may be exposed to differing levels of contact with invading microbes and may display nonuniform levels of functional redundancy or pleiotropy. Thus, genes from different functional groups may be exposed to distinct selective pressures. Antimicrobial peptides, which might be expected to encounter unique selective pressures due to their direct interactions with invading microbes, have shown little sign of positive selection, bearing low levels of amino acid divergence (Clark and Wang 1997; Date et al. 1998; Ramos-Onsins and Aguadé 1998; Lazzaro and Clark 2003). Furthermore, sequence analyses of immune-related receptors have shown evidence for purifying selection in peptidoglycan recognition proteins (PGRPs), while others, including some scavenger receptors (SRs), appear to be rapidly evolving under pressures consistent with positive selection (Jiggins and Hurst 2003; Lazzaro 2005). On a deeper evolutionary timescale, sequence comparisons between immune genes in multiple Drosophila species (based on full-genome sequence data) have shown striking differences among functional groups of immune genes, with recognition molecules showing much more positive selection than either signaling or effector genes (Sackton et al. 2007).Beyond using sequence data and the analysis of polymorphism and divergence to infer levels and modes of selection that have previously acted on immune genes (either individually or in functional groups), other studies have investigated correlations between autosomal variation in genotype and immune response phenotype in natural populations of Drosophila (Lazzaro et al. 2004, 2006). These experiments tested associations between naturally occurring genetic variation in immune-related genes and postinfection bacterial load. In these studies, genetic variation in many of the immune genes was found to associate significantly with one or more of the bacterial load phenotypes. Specifically, polymorphisms in autosomal genes encoding recognition and signaling proteins (but not antimicrobial peptides) associate consistently with bacterial load phenotypes, suggesting that not all functional classes of immune-related genes harbor equally influential genetic variation.The focus of this study is X-linked immune genes, which may be under unique regulatory and selective pressures simply because they are hemizygous in males, are dosage compensated, and face elevated influence of random genetic drift due to their smaller effective population size. As a consequence, the X chromosome should favor the more rapid fixation of beneficial recessive alleles and more rapid loss of harmful recessive alleles compared to the autosomes (Charlesworth et al. 1987; Singh et al. 2008). Thus, with different selective pressures compared to autosomal genes, X-linked immunity genes are expected to bear different standing levels of variation, and segregating polymorphisms in these genes may have different impacts on phenotype.Different exposures of X-linked genes to selection in males and females can also contribute to sexual dimorphism. Rice (1984) suggested that X-linked sexually antagonistic alleles may more freely influence sexually dimorphic traits than can those on autosomes. In fact, the X chromosome appears to favor the maintenance of sexually antagonistic variation (Gibson et al. 2002); if a given allele is slightly deleterious in one sex, it may be maintained in the population by being beneficial to the other sex. Immune-related genes may be particularly prone to bearing sexual dimorphism in Drosophila, since males and females have been shown to have different evolutionary optima for energetic expenditure on immune response, and thus their respective immune responses may differ on the basis of conditions such as food or reproductive resource availability (McKean and Nunney 2001, 2005). If sexually antagonistic traits are responsible for some of the observed sexual dimorphism, variation in X-linked genes could contribute to phenotypic differences, and so X-linked variation in immune genes could face unique selective pressures.In this report we investigate the standing levels of variation in X-linked immune genes in natural populations of D. melanogaster and quantify the impacts of that variation on immune response phenotypes. We genotyped 168 lines at single-nucleotide polymorphisms (SNPs) across 20 X-linked immunity loci and quantified postinfection bacterial load and immune gene expression phenotypes. We found significant variation across the lines for bacterial load after infection, and we were able to identify polymorphisms in immune-related genes that associate with immune response phenotypes individually and in interacting pairs of SNPs. Additionally, some of the genetic variation was found to associate with a sex difference in immune competence, with alleles acting in either a sex-specific or a sexually antagonistic manner. This provides evidence for X-linked genetic variation in immune-related loci associating with both phenotypic variation among lines and sex differences in these phenotypes.     

Genetic Analysis of Rap1p/Sir3p Interactions in Telomeric and Hml Silencing in Saccharomyces Cerevisiae

We have identified three SIR3 suppressors of the telomeric silencing defects conferred by missense mutations within the Rap1p C-terminal tail domain (aa 800-827). Each SIR3 suppressor was also capable of suppressing a rap1 allele (rap1-21), which deletes the 28 aa C-terminal tail domain, but none of the suppressors restored telomeric silencing to a 165 amino acid truncation allele. These data suggest a Rap1p site for Sir3p association between the two truncation points (aa 664-799). In SIR3 suppressor strains lacking the Rap1p C-terminal tail domain, the presence of a second intragenic mutation within the rap1s domain (aa 727-747), enhanced silencing 30-300-fold. These data suggest a competition between Sir3p and factors that interfere with silencing for association in the rap1(s) domain. rap1-21 strains containing both wild-type Sir3p and either of the Sir3 suppressor proteins displayed a 400-4000-fold increase in telomeric silencing over rap1-21 strains carrying either Sir3p suppressor in the absence of wild-type Sir3p. We propose that this telomere-specific synergism is mediated in part through stabilization of Rap1p/Sir3p telomeric complexes by Sir3p-Sir3p interactions.     

Loss of Pol32 in Drosophila melanogaster Causes Chromosome Instability and Suppresses Variegation

Pol32 is an accessory subunit of the replicative DNA Polymerase δ and of the translesion Polymerase ζ. Pol32 is involved in DNA replication, recombination and repair. Pol32’s participation in high- and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32^rd1 and pol32^rds in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutantssuppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.     

The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.     

Enhancement of Telomere-Plasmid Segregation by the X-Telomere Associated Sequence in Saccharomyces Cerevisiae Involves Sir2, Sir3, Sir4 and Abf1

We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG(1-3)) tracts segregate efficiently relative to analogous plasmids lacking the TG(1-3) tract and this efficient segregation is dependent upon RAP1. While a long TG(1-3) tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abflp-binding site within the X-TAS. Mutation of the Abflp-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abflp-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG(1-3) tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.     

Evolution and Functional Trajectory of Sir1 in Gene Silencing

We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles of SIR and RNA interference (RNAi) genes in T. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography of chromatin at the HML and HMR loci than was observed in S. cerevisiae. S. cerevisiae Sir1, enriched at the silencers of HMLα and HMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast to S. cerevisiaeSIR1''s partially dispensable role in silencing, the T. delbrueckiiSIR1 paralog KOS3 was essential for silencing. KOS3 was also found at telomeres with T. delbrueckii Sir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing, T. delbrueckii RNAi genes AGO1 and DCR1 did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.     

Hec1p, an Evolutionarily Conserved Coiled-Coil Protein, Modulates Chromosome Segregation through Interaction with SMC Proteins

hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in M-phase progression in mammalian cells. A Saccharomyces cerevisiae protein, identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in structure and biological function to hsHec1p. Budding yeast cells deleted in the scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by hsHEC1 under control of the endogenous scHEC1 promoter. At the nonpermissive temperature, significant mitotic delay, chromosomal missegregation, and decreased viability were observed in yeast cells with temperature-sensitive (ts) alleles of hsHEC1. In the hshec1-113 ts mutant, we found a single-point mutation changing Trp395 to a stop codon, which resulted in the expression of a C-terminally truncated 45-kDa protein. The binding of this mutated protein, hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while its binding to human SMC1 protein and yeast Smc1p was ts. Hec1p also interacts with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts. Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the interactions between Hec1p and Smc1p and -2p are biologically significant. These results suggest that Hec1 proteins play a critical role in modulating chromosomal segregation, in part, through their interactions with SMC proteins.     

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 ^-/- ;Ybx3 ^-/- double mutants using a previously reported Ybx2 ^-/- model and a newly generated global Ybx3 ^-/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.