Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single base-pair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bahΔ, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3 overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

Centrosomal Enrichment and Proteasomal Degradation of SYS-1/-β-catenin Requires the Microtubule Motor Dynein

The C. elegans Wnt/β-catenin Asymmetry (WβA) pathway utilizes asymmetric regulation of SYS-1/β-catenin and POP-1/TCF coactivators. This differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased towards their appropriate cell fate at birth. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Due to the mitosis-specific mobility of centrosomal SYS-1 and 'traffic jam' like enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNAi-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe that depletion of microtubules by Nocodazole treatment or RNAi of putative dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose retrograde microtubule-mediated trafficking enables SYS-1 and negative regulators to enrich at centrosomes, enhancing their interaction and perhaps implicating the centrosome as a mitotic sink for proteins targeted for degradation.

Myosin XI drives polarized growth by vesicle focusing and local enrichment of F-actin in Physcomitrium patens

In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell’s tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin’s local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

The RNA helicase Ddx52 functions as a growth switch in juvenile zebrafish

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-Dependent RNA Helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.

Decreasing Mitochondrial RNA Polymerase Activity Reverses Biased Inheritance of Hypersuppressive mtDNA

Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that regions of rho+ mtDNA persisted after hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced hypersuppressive biased inheritance. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance relative to hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.

Assessing adaptive requirements and breeding potential of spelt under Mediterranean environment

The rising demand for spelt wheat (Triticum aestivum ssp. spelta) as a high-value grain crop has raised interest in its introduction into non-traditional spelt growing areas. This study aimed to assess adaptive constrains of spelt under short Mediterranean season. At first screening of a wide spelt collection for phenology and allelic distribution at the photoperiod (PPD) and vernalization (VRN) loci was done. In addition an in-depth phenotypic evaluation of a selected panel (n = 20) was performed, including agronomically important traits and concentration of grain mineral (GMC) and grain protein (GPC) content. Results from both wide screening and in-depth in panel (group of 18 spelt lines and two bread wheat lines) evaluation shows that the major adaptive constraint for spelt under Mediterranean conditions is late heading, caused by day length sensitivity, as evident from phenology and allelic profile (PPD and VRN). All lines carrying the photoperiod-sensitive allele (PPD-D1b) were late flowering (> 120DH). Based on the panel field evaluations those consequently suffer from low grain yield and poor agronomic performances. As for minerals, GMC for all but Zn, significantly correlated with GPC. In general, GMC negatively correlated with yield which complicated the assessment of GMC per-se and challenge the claim for higher mineral content in spelt grains. The exceptions were, Fe and Zn, which did not correlate with yield. Spelt lines showing high Fe and Zn concentration in a high-yield background illustrate their potential for spelt wheat breeding. Improving spelt adaptation to Mediterranean environments could be mediated by introducing the insensitive-PPD-D1a allele to spelt wheat background. Following this breeding path spelt could better compete with bread wheat under short season with limited and fluctuating rain fall.

Comprehensive Synthetic Genetic Array Analysis of Alleles That Interact with Mutation of the Saccharomyces cerevisiae RecQ Helicases Hrq1 and Sgs1

Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgs1-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiae eco1-1 allele

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

Comprehensive synthetic genetic array analysis of alleles that interact with mutation of the Saccharomyces cerevisiae RecQ helicases Hrq1 and Sgs1

ABSTRACTMost eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgsl-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1

Using a temperature-sensitive allele, we find that the phosphatidylinositol 4-phosphatase Sac1 is required for ER homeostasis, endosomal degradation, and basal patterning in the developing Drosophila retina.