Identification of Set1 Target Genes

The Set1 complex, a histone methyltransferase complex found in S. cerevisiae (budding yeast), is the only histone methyltransferase responsible for catalyzing methylation of histone H3 at Lysine 4. It possesses homologues in other species, humans included. While yeast only have the Set1 complex, the human homologues of the yeast Set1 complex include mixed-lineage leukemia family (MLL1-4), Set1 A, Set1 B, among others. MLL1-4 has been shown to play a role in transcription, cell type specification, and the development of leukemia. One application of characterizing the role of a protein is that the information gained can provide insight into the function of its homologues; possibly leading to treatments for mutations in those homologues. This ongoing discovery-based project aims to characterize the role of Set1 in Saccharomyces cerevisiae, a model organism frequently used in genetics research commonly known as budding yeast. By utilizing a high-copy plasmid suppression screen, we aim to identify specific genes that are regulated by Set1. Set1’s methylation (and activation of certain genes) has been shown by other studies to be necessary for cell wall integrity, and therefore colony growth, at high temperatures. This sensitivity was the basis of selection for this experiment. Plasmids from a library were transformed into the SET1 deletes to observe which, if any, rescued the cells. Plasmids from the library were extracted from E. coli using a standard miniprep protocol. Yeast was transformed using a long transformation protocol. The transformed cells were incubated for four to five days at 39.5oC

Determining the role of epigenetic factors in antifungal drug resistance

Epigenetic factors are proteins that regulate gene expression by altering transcriptional machinery access to nucleosomes, DNA wrapped around histone proteins. Two classes of epigenetic factors are ATP-dependent chromatin remodelers and histone modifiers such as histone methyltransferases (HMTs), proteins that add methyl groups to histone tails. This study focuses on AIF4 (Antifungal-Induced Factor 4), a possible HMT induced upon neutral lipid depletion that we hypothesize is regulating antifungal drug resistance genes. Overexpression of AIF4 results in hypersensitivity to antifungal drugs. Studying epigenetic factors in the yeast Saccharomyces cerevisiae, including AIF4, can lead to better understanding of cell adaptation to their environments and insight into antifungal drug resistance of pathogenic yeast. This project will focus on identifying suppressors of AIF4’s hypersensitive phenotype and exploring whether genes in the neutral lipid pathway are necessary for AIF4 expression. To support our hypothesis, I will grow yeast colonies with overexpressed AIF4 on media containing antifungal drugs. Overexpressing AIF4 strains exposed to antifungal drugs over time suppressed the grow defect. Re-plating the suppressor colonies showed drug resistance, suggesting that a genetic mutation(s) occurred. Suppressor colonies will be analyzed for AIF4 expression and genome-wide sequencing to identify the suppressor mutation(s). In addition, I have generated deletions for genes that encode neutral lipid production enzymes, and I will determine if AIF4 expression is affected. Single and double deletions will determine if a particular neutral lipid is required for the expression of AIF4. Overall, my work will help to characterize a pathway required for AIF4 expression and drug resistance

H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity

Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: A versatile and rapid ChIP procedure

© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana

Overview of Heat Addition and Efficiency Predictions for an Advanced Stirling Convertor

The U.S. Department of Energy (DOE) and Lockheed Martin Space Systems Company (LMSSC) have been developing the Advanced Stirling Radioisotope Generator (ASRG) for use as a power system for space science missions. This generator would use two high-efficiency Advanced Stirling Convertors (ASCs), developed by Sunpower Inc. and NASA Glenn Research Center (GRC). The ASCs convert thermal energy from a radioisotope heat source into electricity. As part of ground testing of these ASCs, different operating conditions are used to simulate expected mission conditions. These conditions require achieving a particular operating frequency, hot end and cold end temperatures, and specified electrical power output for a given net heat input. Microporous bulk insulation is used in the ground support test hardware to minimize the loss of thermal energy from the electric heat source to the environment. The insulation package is characterized before operation to predict how much heat will be absorbed by the convertor and how much will be lost to the environment during operation. In an effort to validate these predictions, numerous tasks have been performed, which provided a more accurate value for net heat input into the ASCs. This test and modeling effort included: (a) making thermophysical property measurements of test setup materials to provide inputs to the numerical models, (b) acquiring additional test data that was collected during convertor tests to provide numerical models with temperature profiles of the test setup via thermocouple and infrared measurements, (c) using multidimensional numerical models (computational fluid dynamics code) to predict net heat input of an operating convertor, and (d) using validation test hardware to provide direct comparison of numerical results and validate the multidimensional numerical models used to predict convertor net heat input. This effort produced high fidelity ASC net heat input predictions, which were successfully validated using specially designed test hardware enabling measurement of heat transferred through a simulated Stirling cycle. The overall effort and results are discussed

Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression

Histone H3 lysine 4 (H3K4) methyltransferases are conserved from yeast to humans, assemble in multisubunit complexes, and are needed to regulate gene expression. The yeast H3K4 methyltransferase complex, Set1 complex or complex of proteins associated with Set1 (COMPASS), consists of Set1 and conserved Set1-associated proteins: Swd1, Swd2, Swd3, Spp1, Bre2, Sdc1, and Shg1. The removal of the WD40 domain-containing subunits Swd1 and Swd3 leads to a loss of Set1 protein and consequently a complete loss ofH3K4methylation. However, until now, how these WD40 domain-containing proteins interact with Set1 and contribute to the stability of Set1 and H3K4 methylation has not been determined. In this study, we identified small basic and acidic patches that mediate protein interactions between theC terminus of Swd1 and the nSET domain of Set1. Absence of either the basic or acidic patches of Set1 and Swd1, respectively, disrupts the interaction between Set1 and Swd1, diminishes Set1 protein levels, and abolishesH3K4methylation. Moreover, these basic and acidic patches are also important for cell growth, telomere silencing, and gene expression. We also show that the basic and acidic patches of Set1 and Swd1 are conserved in their human counter-parts SET1A/B and RBBP5, respectively, and are needed for the protein interaction between SET1A and RBBP5. Therefore, this charge-based interaction is likely important for maintaining the protein stability of the human SET1A/B methyltransferase complexes so that proper H3K4 methylation, cell growth, and gene expression can also occur in mammals. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Overview of Heat Addition and Efficiency Predictions for an Advanced Stirling Convertor

Past methods of predicting net heat input needed to be validated. Validation effort pursued with several paths including improving model inputs, using test hardware to provide validation data, and validating high fidelity models. Validation test hardware provided direct measurement of net heat input for comparison to predicted values. Predicted value of net heat input was 1.7 percent less than measured value and initial calculations of measurement uncertainty were 2.1 percent (under review). Lessons learned during validation effort were incorporated into convertor modeling approach which improved predictions of convertor efficiency

Data file : sediments of the East Atlantic continental margin, northwest Africa : sample collection and analysis

The petrology, provenance, and history of sediments from the continental shelf and upper continental slope of western Africa have been studied in some detail by scientists from the Woods Hole Oceanographic Institution as part of a long-term investigation of the marine geology of the Eastern Atlantic Continental Margin (funded by the National Science Foundation through the Office of the International Decade of Ocean Exploration in a grant to Dr. K.O. Emery- GX-28193). In this data file we present the analytical data and other information relating to all of the readily available samples (1178) of sediment from northwestern Africa (off the coasts of Morocco and what was recently called Spanish Sahara). These data have been described and interpreted in a recent article in the scientific literature (Summerhayes and others, 1976). The data file contains sample locations, shipboard descriptions, size data, sand fraction composition, clay mineral composition, carbonate assemblage, and carbonate, nitrogen, and carbon contents. The object of the data file is to make these data readily available to other research groups interested in African margin sediments.Prepared for the National Science Foundation (IDOE) under Grant No. GX-28193

Resolving the Radio Source Background: Deeper Understanding Through Confusion

We used the Karl G. Jansky Very Large Array (VLA) to image one primary beam area at 3 GHz with 8 arcsec FWHM resolution and 1.0 microJy/beam rms noise near the pointing center. The P(D) distribution from the central 10 arcmin of this confusion-limited image constrains the count of discrete sources in the 1 < S(microJy/beam) < 10 range. At this level the brightness-weighted differential count S^2 n(S) is converging rapidly, as predicted by evolutionary models in which the faintest radio sources are star-forming galaxies; and ~96$% of the background originating in galaxies has been resolved into discrete sources. About 63% of the radio background is produced by AGNs, and the remaining 37% comes from star-forming galaxies that obey the far-infrared (FIR) / radio correlation and account for most of the FIR background at lambda = 160 microns. Our new data confirm that radio sources powered by AGNs and star formation evolve at about the same rate, a result consistent with AGN feedback and the rough correlation of black hole and bulge stellar masses. The confusion at centimeter wavelengths is low enough that neither the planned SKA nor its pathfinder ASKAP EMU survey should be confusion limited, and the ultimate source detection limit imposed by "natural" confusion is < 0.01 microJy at 1.4 GHz. If discrete sources dominate the bright extragalactic background reported by ARCADE2 at 3.3 GHz, they cannot be located in or near galaxies and most are < 0.03 microJy at 1.4 GHz.Comment: 28 pages including 16 figures. ApJ accepted for publicatio