Claire A. Rinehart

Conserved Antagonism between JMJD2A/KDM4A and HP1γ during Cell Cycle Progression

The KDM4/JMJD2 family of histone demethylases is amplified in human cancers. However, little is known about their physiologic or tumorigenic roles. We have identified a conserved and unappreciated role for the JMJD2A/KDM4A H3K9/36 tridemethylase in cell cycle progression. We demonstrate that JMJD2A protein levels are regulated in a cell cycle-dependent manner and that JMJD2A overexpression increased chromatin accessibility, S phase progression, and altered replication timing of specific genomic loci. These phenotypes depended on JMJD2A enzymatic activity. Strikingly, depletion of the only C. elegans homolog, JMJD-2, slowed DNA replication and increased ATR/p53-dependent apoptosis. Importantly, overexpression of HP1γ antagonized JMJD2A-dependent progression through S phase, and depletion of HPL-2 rescued the DNA replication-related phenotypes in jmjd-2(-/-) animals. Our findings describe a highly conserved model whereby JMJD2A regulates DNA replication by antagonizing HP1γ and controlling chromatin accessibility.

The SKP1-Cul1-F-box and Leucine-rich Repeat Protein 4 (SCF-FbxL4) Ubiquitin Ligase Regulates Lysine Demethylase 4A (KDM4A)/Jumonji Domain-containing 2A (JMJD2A) Protein

Chromatin-modifying enzymes play a fundamental role in regulating chromatin structure so that DNA replication is spatially and temporally coordinated. For example, the lysine demethylase 4A/Jumonji domain-containing 2A (KDM4A/JMJD2A) is tightly regulated during the cell cycle. Overexpression of JMJD2A leads to altered replication timing and faster S phase progression. In this study, we demonstrate that degradation of JMJD2A is regulated by the proteasome. JMJD2A turnover is coordinated through the SKP1-Cul1-F-box ubiquitin ligase complex that contains cullin 1 and the F-box and leucine-rich repeat protein 4 (FbxL4). This complex interacted with JMJD2A. Ubiquitin overexpression restored turnover and blocked the JMJD2A-dependent faster S phase progression in a cullin 1-dependent manner. Furthermore, increased ubiquitin levels decreased JMJD2A occupancy and BrdU incorporation at target sites. This study highlights a finely tuned mechanism for regulating histone demethylase levels and emphasizes the need to tightly regulate chromatin modifiers so that the cell cycle occurs properly.

Proceedings of the ninth annual UT-ORNL-KBRIN Bioinformatics Summit 2010

The University of Tennessee (UT), the Oak Ridge National Laboratory (ORNL), and the Kentucky Biomedical Research Infrastructure Network (KBRIN), have collaborated over the past eleven years to share research and educational expertise in bioinformatics. One result of this collaboration is the joint sponsorship of an annual regional summit to bring together researchers, educators and students who are interested in bioinformatics from a variety of research and educational institutions. This summit provides unique opportunities for collaboration and forging links between members of the various institutions. This year, the Eleventh Annual UT-ORNL-KBRIN Bioinformatics Summit was held at the Seelbach Hilton Hotel in Louisville, Kentucky from March 30-April 1, 2012. A total of 232 participants pre-registered for the summit, with 126 from various Kentucky institutions and 80 from various Tennessee institutions. A number of additional participants came from universities and research institutions from other states and countries, e.g. University of Arkansas Medical Sciences, Michigan State University, University of Cincinnati, Iowa State University, etc. Eighty-four registrants were faculty, with an additional 68 students, 37 staff, and 32 postdoctoral participants (with 12 undeclared). The conference program consisted of three days of presentations. The first day included a pre-summit of talks by Kentucky researchers and a workshop on Next-Generation Sequencing technologies. The next two days were dedicated to scientific presentations divided into four plenary sessions on Next-Generation Sequencing, Medical Informatics, Metagenomics, and Behavioral and Comparative Genomics. The Medical Informatics session was followed by four short talks, selected from 47 submitted poster abstracts.

Prophage-mediated defence against viral attack and viral counter-defence

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host–virus dynamics, and counter-defence promotes phage co-evolution.

Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes

ABSTRACT Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc2155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

At least four distinct circadian regulatory mechanisms are required for all phases of rhythms in mRNA amount.

Since the advent of techniques to investigate gene expression on a large scale, numerous circadian rhythms in mRNA amount have been reported. These rhythms generally differ in amplitude and phase. The authors investigated how far a parameter not regulated by the circadian clock can influence the phase of a rhythm in RNA amount arising from a circadian rhythm of transcription. Using a discrete-time approach, they modeled a sinusoidal rhythm in transcription with various constant exponential RNA decay rates. They found that the slower the RNA degradation, the later the phase of the RNA amount rhythm compared with the phase of the transcriptional rhythm. However, they also found that the phase of the RNA amount rhythm is limited to a timeframe spanning the first quarter of the period following the phase of the transcriptional rhythm. This finding is independent of the amplitude and vertical shift of the transcriptional rhythm or even of the way constant RNA degradation is modeled. The authors confirmed their results with a continuous-time model, which allowed them to derive a simple formula relating the phase of the RNA amount rhythm solely to the phase and period of its sinusoidal transcriptional rhythm and its constant RNA half-life. This simple formula even holds true for the best sinusoidal approximations of a nonsinusoidal rhythm of transcription and RNA amount. When expanding the model to include additional events with constant exponential kinetics, such as RNA processing, they found that each event expands the phase limit by another quarter of the period when it occurs in sequence but not when it occurs as a competing process. However, the limit expansion comes at the price of minuscule amplitudes. When using a discrete-time approach to model constant rates of transcription with a sinusoidal RNA half-life, the authors found that the phase of the RNA amount rhythm is unaffected by changes in the constant rate of transcription. In summary, their data show that at least 4 distinct circadian regulatory mechanisms are required to allow for all phases in rhythms of RNA amount, one for each quarter of the period.

Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas.

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii.

Comprehensive study of excess phosphate response reveals ethylene mediated signaling that negatively regulates plant growth and development

Excess Phosphorus (P) in agriculture is causing serious environmental problems like eutrophication of lakes and rivers. Unlike the enormous information available for phosphate starvation response (P0), very few information is available for the effect of excess phosphate Pi on plants. Characterization of Excess Phosphate Response (EPiR) is essential for designing strategies to increase phosphate accumulation and tolerance. We show a significant modulation in the root developmental plasticity under the increasing supply of excess Pi. An excess supply of 20 mM Pi (P20) produces a shallow root system architecture (RSA), reduces primary root growth, root apical meristem size, and meristematic activity in Arabidopsis. The inhibition of primary root growth and development is indeterminate in nature and caused by the decrease in number of meristematic cortical cells due to EPiR. Significant changes occurred in metal nutrients level due to excess Pi supply. A comparative microarray investigation of the EPiR response reveals a modulation in ethylene biosynthesis and signaling, metal ions deficiency response, and root development related genes. We used ethylene-insensitive or sensitive mutants to provide more evidence for ethylene-mediated signaling. A new role of EPiR in regulating the developmental responses of plants mediated by ethylene has been demonstrated.

Statistical analysis of microarray gene expression data from a mouse model of toxoplasmosis

Background Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii is a major cause of morbidity and mortality in patients with AIDS and an important cause of miscarriage, stillbirth and congenital disease in newborns. Previous studies have provided evidence that dietary supplementation with vitamin E and selenium is harmful during experimental toxoplasmosis in mice, whereas a diet deficient in vitamin E and selenium results in decreased numbers of tissue cysts in the brain and dramatically reduced brain pathology. The overall goal of the present study was to determine the impact of dietary supplementation with antioxidants on gene expression in the brains of non-infected mice and in mice infected with T. gondii using microarray analysis. RNA was isolated from the brains of C57BL/6 mice, and an Agilent Oligo Whole Mouse Genome Microarray (Agilent Technologies, Inc.) was performed. A total of 48 chips were normalized by Z ratios and the Data Driven Harr Fisch Normalization methods. Differentially expressed genes were identified by applying thresholds to identify significant values and the results were compared between the normalization methods. These differentially expressed genes and their respective fold change ratios were used in Ingenuity Pathway Analysis (IPA) software to analyze the pathways involved with these genes.

The HHMI National Genomics Research Initiative at Western Kentucky University

Background Western Kentucky University is a member of a select group of colleges and universities chosen to participate in a national science experiment designed to improve undergraduate science education. To introduce freshman students to authentic research, the Howard Hughes Medical Institute’s Science Education Alliance (HHMI SEA) has implemented an innovative program to introduce genomics into the classroom. The program draws on themes and techniques from biology, microbiology, molecular biology, genomics and bioinformatics. The WKU course is called “Genome Discovery and Exploration” (GDE); WKU Genome Discovery and Exploration Program [http://bioweb.wku.edu/asp/wkungri/]. During the fall semester, students isolate bacterial viruses from the environment and characterize them using a variety of techniques including DNA analysis and electron microscopy. Over the winter break, the complete DNA sequence of one of the viruses isolated by the GDE students is determined. During the spring semester, GDE students learn how use computer programs (e.g. Consed, Glimmer, Genemark, BLAST and Phamerator) to identify all the viral genes and to make genome wide comparisons to identify evolutionary relationships. All course participants and the SEA staff are connected via the internet to share and analyze data, troubleshoot, and discuss results. Near the end of the spring semester, HHMI hosts a research symposium for course faculty and one student from each institution to report on their discoveries and experiences. As unique phages are characterized on campuses nationwide, students will have the opportunity to publish their research for the broader scientific community. This hands-on experience with the tools of Bioinformatics is an outstanding training opportunity and this unique program has helped the WKU Biology Department create an introductory pipeline to research experiences that complements our existing and planned instructional programs and allows freshman to engage in the thrill of discovery.