Antagonistic Gcn5-Hda1 interactions revealed by mutations to the Anaphase Promoting Complex in yeast

<p>Abstract</p> <p>Background</p> <p>Histone post-translational modifications are critical for gene expression and cell viability. A broad spectrum of histone lysine residues have been identified in yeast that are targeted by a variety of modifying enzymes. However, the regulation and interaction of these enzymes remains relatively uncharacterized. Previously we demonstrated that deletion of either the histone acetyltransferase (HAT) <it>GCN5 </it>or the histone deacetylase (HDAC) <it>HDA1 </it>exacerbated the temperature sensitive (<it>ts</it>) mutant phenotype of the Anaphase Promoting Complex (APC) <it>apc5^CA</it>allele. Here, the <it>apc5^CA</it>mutant background is used to study a previously uncharacterized functional antagonistic genetic interaction between Gcn5 and Hda1 that is not detected in <it>APC5 </it>cells.</p> <p>Results</p> <p>Using Northerns, Westerns, reverse transcriptase PCR (rtPCR), chromatin immunoprecipitation (ChIP), and mutant phenotype suppression analysis, we observed that Hda1 and Gcn5 appear to compete for recruitment to promoters. We observed that the presence of Hda1 can partially occlude the binding of Gcn5 to the same promoter. Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1. Using sequential ChIP we show that Hda1 and Tup1 likely form complexes at these promoters, and that complex formation can be increased by deleting <it>GCN5</it>.</p> <p>Conclusions</p> <p>Our data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex. We predict that in <it>apc5^CA</it>cells the accumulation of an APC target may compensate for the loss of both <it>GCN5 </it>and <it>HDA1</it>.</p

The Role of Host Genetics in Susceptibility to Influenza: A Systematic Review

Background: The World Health Organization has identified studies of the role of host genetics on susceptibility to severe influenza as a priority. A systematic review was conducted to summarize the current state of evidence on the role of host genetics in susceptibility to influenza (PROSPERO registration number: CRD42011001380). Methods and Findings: PubMed, Web of Science, the Cochrane Library, and OpenSIGLE were searched using a pre-defined strategy for all entries up to the date of the search. Two reviewers independently screened the title and abstract of 1,371 unique articles, and 72 full text publications were selected for inclusion. Mouse models clearly demonstrate that host genetics plays a critical role in susceptibility to a range of human and avian influenza viruses. The Mx genes encoding interferon inducible proteins are the best studied but their relevance to susceptibility in humans is unknown. Although the MxA gene should be considered a candidate gene for further study in humans, over 100 other candidate genes have been proposed. There are however no data associating any of these candidate genes to susceptibility in humans, with the only published study in humans being under-powered. One genealogy study presents moderate evidence of a heritable component to the risk of influenza-associated death, and while the marked familial aggregation of H5N1 cases is suggestive of host genetic factors, this remains unproven. Conclusion: The fundamental question ‘‘Is susceptibility to severe influenza in humans heritable?’ ’ remains unanswered. No

The Yeast Forkhead Transcription Factors Fkh1 and Fkh2 Regulate Lifespan and Stress Response Together with the Anaphase-Promoting Complex

Forkhead box O (FOXO) transcription factors have a conserved function in regulating metazoan lifespan. A key function in this process involves the regulation of the cell cycle and stress responses including free radical scavenging. We employed yeast chronological and replicative lifespan assays, as well as oxidative stress assays, to explore the potential evolutionary conservation of function between the FOXOs and the yeast forkhead box transcription factors FKH1 and FKH2. We report that the deletion of both FKH genes impedes normal lifespan and stress resistance, particularly in stationary phase cells, which are non-responsive to caloric restriction. Conversely, increased expression of the FKHs leads to extended lifespan and improved stress response. Here we show the Anaphase-Promoting Complex (APC) genetically interacts with the Fkh pathway, likely working in a linear pathway under normal conditions, as fkh1D fkh2D post-mitotic survival is epistatic to that observed in apc5 CA mutants. However, under stress conditions, post-mitotic survival is dramatically impaired in apc5 CA fkh1D fkh2D, while increased expression of either FKH rescues APC mutant growth defects. This study establishes the FKHs role as evolutionarily conserved regulators of lifespan in yeast and identifies the APC as a novel component of this mechanis

Biodistribution of a Radiolabeled Antibody in Mice as an Approach to Evaluating Antibody Pharmacokinetics

(1) Background: Monoclonal antibodies are used in the treatment of multiple conditions including cancer, autoimmune disorders, and infectious diseases. One of the initial steps in the selection of an antibody candidate for further pre-clinical development is determining its pharmacokinetics in small animal models. The use of mass spectrometry and other techniques to determine the fate of these antibodies is laborious and expensive. Here we describe a straightforward and highly reproducible methodology for utilizing radiolabeled antibodies for pharmacokinetics studies. (2) Methods: Commercially available bifunctional linker CHXA&#8222; and 111Indium radionuclide were used. A melanin-specific chimeric antibody A1 and an isotype matching irrelevant control A2 were conjugated with the CHXA&#8222;, and then radiolabeled with 111In. The biodistribution was performed at 4 and 24 h time points in melanoma tumor-bearing and healthy C57BL/6 female mice. (3) The biodistribution of the melanin-binding antibody showed the significant uptake in the tumor, which increased with time, and very low uptake in healthy melanin-containing tissues such as the retina of the eye and melanized skin. This biodistribution pattern in healthy tissues was very close to that of the isotype matching control antibody. (4) Conclusions: The biodistribution experiment allows us to assess the pharmacokinetics of both antibodies side by side and to make a conclusion regarding the suitability of specific antibodies for further development

Effects of Melanized Bacteria and Soluble Melanin on the Intestinal Homeostasis and Microbiome In Vivo

Radiation damage is associated with inflammation and immunity in the intestinal mucosa, including gut microbiota. Melanin has a unique capacity to coordinate a biological reaction in response to environmental stimuli, such as radiation exposure. Thus, melanin and melanized microbes have potential to be used for mitigation of injury induced by radiation. The purpose of the current study is to examine the safety of these agents for future targeting gut microbiome to prevent radiation-induced injury. We administered mice with soluble allomelanin and observed its effect on the intestinal physiology and body weight. We then established a melanized bacterial strain in probiotic E. coli Nissle. We measured the body weight of the mice treated with melanized E. coli Nissle. We showed the enhanced bacterial abundance and colonization of the melanized bacteria E. coli Nissle in the intestine. Melanized E. coli Nissle colonized the colon in less than 3 h and showed consistent colonization over 24 h post one oral gavage. We did not find significant changes of bodyweight in the mice treated with melanized bacteria. We did not observe any inflammation in the intestine. These results demonstrate the safety of soluble melanin and melanin-producing bacteria and will support the future studies to treat radiation-induced injuries and restore dysbiosis

<i>FKH1</i> and <i>FKH2</i> encode redundant determinants of lifespan and stress response.

<p>(A) and (B) The cells shown were grown in CM (2% glucose) to stationary phase. The cells were either left in (A) depleted CM (DM), or (B) washed and resuspended in H₂0 for the remainder of the experiment. Colony counts were performed every other day throughout the experiment. The day when the colony count peaked was considered Day 1. Standard error is shown for at least 3 replicates. (C) Acute oxidative stress in stationary phase cells. WT and <i>fkh1Δ fkh2Δ</i> cells were grown to day 5 of stationary phase with maintenance in either DM or H₂O. 100 mM H₂O₂ was then added to one half of each sample and incubated for 60 minutes at 30°C. Diluted cells were then plated on YPD media and the colony forming units were counted. Survival was determined by dividing the colony forming units following H₂O₂ treated by untreated samples. Standard error of at least 3 replicates is shown. (D) Chronic oxidative stress in mitotically active and stationary phase cells. WT and <i>fkh1Δ fkh2Δ</i> cells from overnight log phase cultures or day 5 stationary phase cultures were treated with 100 mM H₂O₂ at 30° for 1 hour, as above, then spot diluted onto YPD plates in the absence of stress. The plates were grown at 30°C for 3 days.</p

Increased expression of the <i>FKH</i> genes increases lifespan and stress response.

<p>(A) The <i>FKH</i> OE cells were grown to stationary phase, then either maintained in DM, or 0.05% galactose was added. The cells were incubated for an additional 5 days, then split, with one half treated with 100 mM H₂O₂ for 1 hour. The other half served as the untreated control. Following the 1 hour incubation, the cells were diluted and plated onto YPD until colony forming units formed. Survival was determined by dividing the treated cells by the untreated cells. Standard error is shown for at least 3 replicates. (B) CLS was determined for the OE strains when maintained solely in DM (left panel) or in DM supplemented with 0.05% galactose (right panel). Standard error is shown for at least 3 replicates. (C) RLS was determined for the OE strains on 2% sucrose plates or sucrose plates supplemented with 0.1% galactose. Typical results are shown.</p