A novel allele ofSIR2reveals a heritable intermediate state of gene silencing

Genetic information acquires additional meaning through epigenetic regulation, the process by which genetically identical cells can exhibit heritable differences in gene expression and phenotype. Inheritance of epigenetic information is a critical step in maintaining cellular identity and organismal health. In Saccharomyces cerevisiae, one form of epigenetic regulation is the transcriptional silencing of two mating-type loci, HML and HMR, by the SIR-protein complex. To focus on the epigenetic dimension of this gene regulation, we conducted a forward mutagenesis screen to identify mutants exhibiting an epigenetic or metastable silencing defect. We utilized fluorescent reporters at HML and HMR, and screened yeast colonies for epigenetic silencing defects. We uncovered numerous independent sir1 alleles, a gene known to be required for stable epigenetic inheritance. More interestingly, we recovered a missense mutation within SIR2, which encodes a highly conserved histone deacetylase. In contrast to sir1Δ, which exhibits states that are either fully silenced or fully expressed, this sir2 allele exhibited heritable states that were either fully silenced or expressed at an intermediate level. The heritable nature of this unique silencing defect was influenced by, but not completely dependent on, changes in rDNA copy number. Therefore, this study revealed a heritable state of intermediate silencing and linked this state to a central silencing factor, Sir2.

Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

A novel allele of SIR2 reveals a heritable intermediate state of gene silencing

Genetic information acquires additional meaning through epigenetic regulation, the process by which genetically identical cells can exhibit heritable differences in gene expression and phenotype. Inheritance of epigenetic information is a critical step in maintaining cellular identity and organismal health. In Saccharomyces cerevisiae, one form of epigenetic regulation is the transcriptional silencing of two mating-type loci, HML and HMR, by the SIR-protein complex. To focus on the epigenetic dimension of this gene regulation, we conducted a forward mutagenesis screen to identify mutants exhibiting an epigenetic or metastable silencing defect. We utilized fluorescent reporters at HML and HMR, and screened yeast colonies for epigenetic silencing defects. We uncovered numerous independent sir1 alleles, a gene known to be required for stable epigenetic inheritance. More interestingly, we recovered a missense mutation within SIR2, which encodes a highly conserved histone deacetylase. In contrast to sir1Δ, which exhibits states that are either fully silenced or fully expressed, this sir2 allele exhibited heritable states that were either fully silenced or expressed at an intermediate level. The heritable nature of this unique silencing defect was influenced by, but not completely dependent on, changes in rDNA copy number. Therefore, this study revealed a heritable state of intermediate silencing and linked this state to a central silencing factor, Sir2.

Abstract 229: Searching Genetic Modifiers For bag3 -based Cardiomyopathy Using Adult Zebrafish Models

We recently developed a forward mutagenesis screening strategy in adult zebrafish to screen gene-breaking transposon (GBT) mutants, and identified four genetic modifiers for doxorubicin-induced cardiomyopathy. However, it remains unclear whether these genetic modifiers identified from an acquired cardiomyopathy model exert similar modifying effects on inherited cardiomyopathy models. To address this question, we generated BCL2-associated athanogene 3 (bag3) gene knockout in adult zebrafish, using the transcription activator-like effector nucleases (TALEN) genome editing technology. In the bag3-/- fish, progressive cardiac phenotypes reminiscent of human cardiomyopathy such as fetal gene activation, myofibril loss and cardiac dysfunction were detected. At the single myofibril level, reduced active contractility was observed, supporting the dilated cardiomyopathy (DCM)-like phenotype. Based on the ejection fraction index quantified using a newly developed ex vivo assay, different pathogenesis stages including pre-DCM, early-DCM and late DCM were defined. Next, we assessed the potential modifying effects of the four DIC-modifying mutants on bag3 -based cardiomyopathy model. Different from the other three GBT mutants, GBT0411+/- , which tags the long isoform of dnajb6 b gene, dramatically accelerated the cardiac dysfunction and fish mortality in the bag3-/- fish, suggesting dnajb6b as a sensitive genetic modifier for bag3-based cardiomyopathy. Mechanistically, we showed that Bag3 physically interacts with Dnajb6, and we hypothesize that impaired autophagy and/or endoplasmic reticulum stress convey the synergistic cardiac dysfunction and fish mortality phenotypes in the GBT0411+/-;bag3-/- double mutants. In summary, this study demonstrates that an inherited cardiomyopathy model can be established in an adult zebrafish, which can be utilized to search genetic modifiers. Future studies employing this simple vertebrate model amenable to forward mutagenesis screening promise systematic identification of genetic modifiers for different types of cardiomyopathies, a foundation for individualized medicine.

N-Ethyl-N-Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

SUMMARY In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.