2016 — 2017 |
Frietze, Seth E Kaufer, Benedikt B. (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Integration and Epigenetic Regulation of the Hhv-6 Genome @ University of Vermont & St Agric College
? DESCRIPTION (provided by applicant): During primary infection, herpesviruses establish a latent infection that allows the virus to persist in the host for life. It was recently discovered hat certain herpesviruses integrate their genome into the telomeres of host chromosomes during latency, while most others maintain their genome as an episome in the nucleus. One of these viruses is human herpesvirus 6 (HHV-6), which is associated with a spectrum of diseases including seizures, encephalitis, and graft rejection in transplant patients. However, there are critical gaps in our knowledge regarding the molecular mechanisms of herpesvirus infections, including the mechanisms by which virus gene expression and DNA integration is controlled as well as the events required for virus reactivation. To address these critical gaps in our knowledge, we propose to use our in vitro infection system for HHV-6 integration, latency and reactivation coupled with functional genomics to determine the fate of the virus genome in these processes. We will first determine the viral genomic signatures during the establishment of latency and integration by measuring differential gene expression, chromatin modification and integration events during the transition from lytic to latent infection (Specific Aim 1). Next, we ill then investigate the viral and cellular determinants involved in virus reactivation. Upon treatment of latently infected cells with histone deacetylase inhibitor drugs (HDACi) in vitro, only in a fraction of the cells reactivate. We therefore hypothesize that virus-reactivating cells have a characteristic gene expression response to drug treatment, which will reveal cellular and viral genes that participate in either maintenance of latency or virus reactivation. We will define this drug response by treating cells with HDACi, isolation of reactivating and non-reactivating cells by FACS and performing the assays described in Aim 1 to determine the signature of reactivation (Specific Aim 2). Finally, we will determine the viral genomic signature of cells from iciHHV-6 patients to obtain an insight into the viral regulation in these individuals and to validae our in vitro model (Specific Aim 3). Our proposed experiments will describe, with high-resolution, the connections between transcription, chromatin regulation and viral DNA integration over the course of initial infection, establishment of latency and reactivation. Successful completion of our specific aims will provide knowledge required to understand how transcription from the integrated latent virus genome is controlled, thereby providing possible therapeutic targets to treat viral complications during transplantation.
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2018 — 2022 |
Frietze, Seth Teets, Nicholas Waters, James Axen, Heather Cahan, Sara [⬀] Cahan, Sara [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Rii Track-2 Fec: From Genome to Phenome in a Stressful World: Epigenetic Regulatory Mechanisms Mediating Thermal Plasticity in Drosophila @ University of Vermont & State Agricultural College
Non-technical description Over the last sixty years, science has made incredible advances in our understanding of the role of genetics in determining the structure and function of living organisms, with important implications for our ability to treat disease, improve agricultural resources, and conserve natural populations and communities. Yet even the same genes can have very different effects depending on the environment in which an organism develops and lives, with life-long or even multigenerational effects on physical traits when exposed to environmental stress. How these changes are accomplished at the molecular level is poorly understood, despite being critical for successful adjustment of the body to acute and chronic stress conditions. In this project, the researchers will investigate the epigenetic response to stressful temperatures in the fruit fly Drosophila melanogaster, along with closely related species living in diverse environments, as a model for uncovering the molecular mechanisms by which cells detect and respond to environmental stress. This work will provide research and training opportunities for a diverse group of graduate and undergraduate students in cutting-edge genomic sequencing technologies and bioinformatic analysis, with STEM outreach to high-school students from under-served urban and rural communities in three EPSCoR jurisdictions (Vermont, Rhode Island, and Kentucky). In addition, five new faculty members will be mentored as part of this project.
Technical description In this project, a team of researchers from VT, RI, and KY will work collaboratively to test the hypothesis that epigenetic regulators act as an intermediary between environmental sensors and protein production, altering the set of genes available for transcription at the level of chromatin accessibility and then fine-tuning expression through the action of epitranscriptomic molecules. The primary objectives are to determine: 1) whether and how epigenetic mechanisms mediate plastic changes in thermal tolerance; 2) the extent to which epigenetic variation underlies natural segregating variation in phenotypic plasticity; and 3) the role of epigenetic divergence in shifting capacity for acclimation over evolutionary time. To identify epigenetic mechanisms driving plasticity, the project team will characterize changes in chromatin accessibility, post-translational histone modification, miRNA and lncRNA associated with developmental acclimation, adult-reversible acclimation, and rapid hardening in response to high and low temperatures in Drosophila melanogaster. Functional genetic manipulations will be used to validate candidate causal epigenetic mechanisms. Genome-wide association mapping and experimental evolution approaches will be employed to evaluate the genetic architecture of thermal plasticity. Finally, to test whether niche transitions to colder or warmer habitats are accompanied by evolutionary gains or losses of these plastic responses, the researchers will reconstruct the history of evolutionary shifts in capacity for thermal plasticity in species across New World species of Drosophila. The project will establish comparative and experimental models for understanding the evolutionary history and molecular mechanisms of thermal plasticity that are ideally suited to address long-standing hypotheses concerning the drivers of plasticity, and investigate the ecological and evolutionary role of plasticity in promoting organismal resilience in the face of rapid, progressive shifts in climate. This project will involve five junior faculty members with different areas of expertise. Two junior faculty members are from Primarily Undergraduate Institutions (PUIs), and mentoring programs that target these junior faculty members are in place. Three post-doctoral research associates will be involved in the project, who will become familiar with the experiences of faculty members at both PUIs and PhD granting institutions.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
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0.915 |
2019 — 2021 |
Frietze, Seth E Glass, Karen Champagne |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Deciphering the Molecular Mechanisms of Histone Code Recognition by Atad2/B @ University of Vermont & St Agric College
ATAD2 is an important co-activator of the estrogen and androgen receptors. ATAD2 is known to be up- regulated in multiple different types of cancer including breast, lung, gastric, endometrial, renal, and prostate. Up-regulation of ATAD2 is often correlated with poor patient outcomes, and can be used as prognostic marker. Furthermore, silencing the expression of ATAD2 through RNA interference inhibits cell proliferation and promotes apoptosis in ovarian carcinoma, and inhibits migration and invasion of hepatocellular carcinoma and colorectal cancer cells. ATAD2B, is a poorly studied paralog of the ATAD2 gene, and although ATAD2 and ATAD2B are highly conserved, there is little known about the function of ATAD2B or its role in oncogenesis. Both the ATAD2/B proteins contain two conserved domains: an AAA ATPase domain and a bromodomain. The overall objective of the proposed research is to determine how di-acetyllysine recognition by the ATAD2/B bromodomains regulates the cellular function of these proteins. This proposal aims to: (1) characterize how cross-talk between histone modifications modulate acetyllysine recognition by the ATAD2/B bromodomains; (2) outline the molecular mechanism(s) of di-acetylated histone recognition by the ATAD2/B bromodomains; (3) determine the functional significance of di-acetyllysine recognition by the ATAD2/B bromodomains. A unique combination of in vitro biochemical, biophysical, and structural biology studies on the ATAD2/B bromodomains will be coupled with in vivo functional genomic investigations using a breast cancer progression model to characterize the biological roles of the ATAD2/B bromodomains. We will evaluate the impact of neighboring histone modifications on histone H4 tail recognition using peptide array assays in combination with isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) chemical shift perturbation techniques. We will determine the structural features of ATAD2/B bromodomains required for recognition of di-acetylated histone tail ligands using NMR and/or X-ray crystallography. To characterize the binding mode of the ATAD2/B bromodomains with their histone ligands we will carry out analytical ultracentrifugation, size-exclusion chromatography, ITC, and NMR T1/T2 relaxation experiments. Site-directed mutagenesis coupled with NMR and ITC will be used to measure the effects on ligand binding, and identify differences in the binding pockets of the ATAD2/B bromodomains. We will compare the genome-wide associations of ATAD2/B with histone H4 acetylation patterns in a breast cancer progression model to determine the impact of ATAD2/B on breast cancer cell phenotypes using ChIP-seq and RNA-seq, followed by cellular migration and invasion assays. Our multi-faceted approach will correlate specific histone modifications with ATAD2/B binding and action, which will allow us to connect histone H4 acetylation marks to bromodomain function in cancer cell proliferation. Overall, our integrated biochemical, biophysical, structural biology and functional genomics approach will reveal the biological roles of ATAD2/B and facilitate the discovery of novel drug targets to help overcome cancer.
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