Genomic Binding Profiling of the Fission Yeast Stress-Activated MAPK Sty1 and the bZIP Transcriptional Activator Atf1 in Response to H2O2

10.1371/journal.pone.0011620PLoS ONE57

A Cds1-Mediated Checkpoint Protects the MBF Activator Rep2 from Ubiquitination by Anaphase-Promoting Complex/Cyclosome-Ste9 at S-Phase Arrest in Fission Yeast▿ †

Transcription of the MluI cell cycle box (MCB) motif-containing genes at G1 phase is regulated by the MCB-binding factors (MBF) (also called DSC1) in Schizosaccharomyces pombe. Upon S-phase arrest, the MBF transcriptional activity is induced through the accumulation of the MBF activator Rep2. In this study, we show that the turnover of Rep2 is attributable to ubiquitin-mediated proteolysis. Levels of Rep2 oscillate during the cell cycle, with a peak at G1 phase, coincident with the MBF activity. Furthermore, we show that Rep2 ubiquitination requires the function of the E3 ligase anaphase-promoting complex/cyclosome (APC/C). Ste9 can be phosphorylated by the checkpoint kinase Cds1 in vitro, and its inhibition/phosphorylation at S-phase arrest is dependent on the function of Cds1. Our data indicate that the Cds1-dependent stabilization of Rep2 is achieved through the inhibition/phosphorylation of APC/C-Ste9 at the onset of S-phase arrest. Stabilization of Rep2 is important for stimulating transcription of the MBF-dependent genes to ensure a sufficient supply of proteins essential for cell recovery from S-phase arrest. We propose that oscillation of Rep2 plays a role in regulation of periodic transcription of the MBF-dependent genes during cell cycle progression

Differential Regulation of FLOWERING LOCUS C Expression by Vernalization in Cabbage and Arabidopsis

Vernalization is required to induce flowering in cabbage (Brassica oleracea var Capitata L.). Since FLOWERING LOCUS C (FLC) was identified as a major repressor of flowering in the vernalization pathway in Arabidopsis (Arabidopsis thaliana), two homologs of AtFLC, BoFLC3-2 and BoFLC4-1, were isolated from cabbage to investigate the molecular mechanism of vernalization in cabbage flowering. In addition to the sequence homology, the genomic organization of cabbage FLC is similar to that of AtFLC, except that BoFLC has a relatively smaller intron 1 compared to that of AtFLC. A vernalization-mediated decrease in FLC transcript level was correlated with an increase in FT transcript level in the apex of cabbage. This observation is in agreement with the down-regulation of FT by FLC in Arabidopsis. Yet, unlike that in Arabidopsis, the accumulation of cabbage FLC transcript decreased after cold treatment of leafy plants but not imbibed seeds, which is consistent with the promotion of cabbage flowering by vernalizing adult plants rather than seeds. To further dissect the different regulation of FLC expression between seed-vernalization-responsive species (e.g. Arabidopsis) and plant-vernalization-responsive species (e.g. cabbage), the pBoFLC4-1∷BoFLC4-1∷GUS construct was introduced into Arabidopsis to examine its vernalization response. Down-regulation of the BoFLC4-1∷GUS construct by seed vernalization was unstable and incomplete; in addition, the expression of BoFLC4-1∷GUS was not suppressed by vernalization of transgenic rosette-stage Arabidopsis plants. We propose a hypothesis to illustrate the distinct mechanism by which vernalization regulates the expression of FLC in cabbage and Arabidopsis

A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

<div><p>Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.</p> </div

Predictive Factors for BRCA1 and BRCA2 Genetic Testing in an Asian Clinic-Based Population.

The National Comprehensive Cancer Network (NCCN) has proposed guidelines for the genetic testing of the BRCA1 and BRCA2 genes, based on studies in western populations. This current study assessed potential predictive factors for BRCA mutation probability, in an Asian population.A total of 359 breast cancer patients, who presented with either a family history (FH) of breast and/or ovarian cancer or early onset breast cancer, were accrued at the National Cancer Center Singapore (NCCS). The relationships between clinico-pathological features and mutational status were calculated using the Chi-squared test and binary logistic regression analysis.Of 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in BRCA1 and/or BRCA2. BRCA1 mutations were more likely to be found in ER-negative than ER-positive breast cancer patients (P=0.01). Moreover, ER-negative patients with BRCA mutations were diagnosed at an earlier age (40 vs. 48 years, P=0.008). Similarly, triple-negative breast cancer (TNBC) patients were more likely to have BRCA1 mutations (P=0.001) and that these patients were diagnosed at a relatively younger age than non-TNBC patients (38 vs. 46 years, P=0.028). Our analysis has confirmed that ER-negative status, TNBC status and a FH of hereditary breast and ovarian cancer (HBOC) are strong factors predicting the likelihood of having BRCA mutations.Our study provides evidence that TNBC or ER-negative patients may benefit from BRCA genetic testing, particularly younger patients (<40 years) or those with a strong FH of HBOC, in Asian patients

Microfluidic device for automated NGS library preparation.

<p>(a) Automated multi-column device mounted on a plastic carrier that provides wells for loading samples and reagents and for pressurized operation of the device. The wells used to load reagents for NGS library preparation are labeled. Chromatography columns for the selective binding and release of DNA were formed either with ChargeSwitch beads or with carboxylated beads. Reagents that were used exclusively with the carboxylated beads are labeled in green. (b) Schematic of single reactor unit for reaction mixing and DNA purification. The regions denoted in the reaction circuit are as follows: Green, Sample; Orange, Buffer; Blue, Enzyme. Red solid rectangular boxes represent activated valves that partition the individual circuits. (c) Parallelization of 16 reactors on chip for preparation of up to 16 independent libraries. Layout of the entire device without the valve map showing reagent inlets and the design for multiplex library generation. The serpentine metering channel designed to ensure reliable column packing is highlighted in orange. (d) Schematics showing cross-sections of purification columns loaded with either 1) ChargeSwitch beads, which are held in place with a frit layer and a cap layer formed by larger beads, or 2) carboxylated beads.</p

Quantification of <i>E. coli</i> strain DH10B library DNA after size selection.

<p>(a) Illumina libraries; (b) Ion Torrent libraries. Asterisks indicate sample modules where buffer was loaded instead of genomic DNA. (c) Efficiency of library preparation reactions on the AMCC chip. The percentage of <i>E. coli</i> DNA fragments with Illumina sequencing adapters ligated onto both ends was estimated by RT-qPCR. The amount of <i>E. coli</i> genomic DNA present was determined by RT-qPCR with primer pairs recognizing six regions of the <i>E. coli</i> genome, and the amount of library DNA with adapters ligated onto both ends was determined by RT-qPCR with a primer pair recognizing the Illumina sequencing adapters. RT-qPCR data were converted to nanograms of DNA using standard curves (Materials and Methods). The estimated amounts of <i>E. coli</i> genomic DNA present in each library varied somewhat between the six locus-specific RT-qPCR reactions, so the bar graph indicates the mean values, and the error bars indicate the standard error of the mean.</p

Coverage depth from a sequencing run using an Illumina U-2 OS osteosarcoma cell line library prepared on the AMCC chip.

<p>The library was run on the MiSeq using the 2x150 bp paired-end sequencing protocol. (a) Distribution of sequencing reads across the reference human genome, which has been divided into 1 million bins to assess coverage uniformity. (b) Average coverage across different chromosomes. (c) Average sequencing depth across different chromosomes. The Y chromosome is absent from the U-2 OS osteosarcoma cell line.</p

Association between ER status, TNBC status, with <i>BRCA</i> mutation status.

<p>*<i>P</i>-values that were statistically significant are indicated in <b>bold</b>.</p><p>Association between ER status, TNBC status, with <i>BRCA</i> mutation status.</p

Potential predictive factors for <i>BRCA1</i> and <i>BRCA2</i> mutations in patients stratified by ER status and TNBC status.

<p>*<i>P</i>-values that were statistically significant are indicated in <b>bold</b>.</p><p>Potential predictive factors for <i>BRCA1</i> and <i>BRCA2</i> mutations in patients stratified by ER status and TNBC status.</p