Characterization Of Higher Order Chromatin Structures And Chromatin States In Cell Models Of Human Herpesvirus Infection

Human herpesviruses are ubiquitous pathogens worldwide with 90% of the global population infected with one or more Human herpesviruses (HHV’s) by adulthood. All herpesviruses have three unique life cycle stages. Upon resolution of a primary acute stage infection, they can establish a latent stage infection within the host cell nucleus. This stage is characterized primarily by transcriptional quiescence of the viral genome. Specific physiological conditions (e.g., cell stress) can cause the latent virus to enter the reactivation stage, often many years after resolution of the acute infection, in which the virus becomes replicationally active again. HHV’s are known to cause disease in humans in all three stages of their lifecycle and chronic infection is becoming increasingly associated with a wide range of human morbidities. The field has been met with challenges in establishing tractable cell models to study HHV infection in vitro and the exact mechanisms regulating the maintenance of, and transition between, the life cycle stages are largely undefined; however, chromatin structure and function are known to play a role. In this dissertation I employ both molecular and computational approaches to study the higher order chromatin structures and chromatin states in cell models of infection for two Human herpesviruses at specific stages in their life cycles. In the first part of this dissertation, I successfully demonstrate the establishment of a latent Human betaherpesvirus 6A (HHV-6A) infection, which requires integration of its genome into the host cell genome, in an in vitro-derived HEK-293 cell model. I then employ unbiased epigenomic methods along with bioinformatics techniques to identify three-dimensional virus-host contact regions and characterize the chromatin states of the latent virus. I then demonstrate the above in both a patient-derived cell model and in infected primary cells. Finally, I developed a novel and innovative computational approach to identify the sites of HHV-6A integration in host cell chromosomes using next generation sequencing data: to my knowledge, a first in the field. In the second part of this work, I employ a human fetal lung fibroblast (HFL) cell model of acute infection to study Varicella Zoster Virus (VZV) chromatin biology - specifically with regards to the transcription factor CTCF. CTCF is a key organizer of chromatin three-dimensional structure and plays a role in chromatin organization and transcriptional regulation in certain HHV’s. Herein, I present evidence that CTCF is likely involved in virus-host structural interactions – an important finding that is countervailing to the current suppositions of the Human alphaherpesvirus field. Overall, the work presented herein offers novel insight into the complex and dynamic relationship between HHV’s and host cell chromatin. With a more comprehensive characterization of the higher order chromatin structures and chromatin states that define this relationship, we can better define the HHV life cycle. Such knowledge will allow for the development of improved treatments against these insidious infections

ENSO Controls Interannual Fire Activity in Southeast Australia

El Niño–Southern Oscillation (ENSO) is the main mode controlling the variability in the ocean-atmosphere system in the South Pacific. While the ENSO influence on rainfall regimes in the South Pacific is well documented, its role in driving spatiotemporal trends in fire activity in this region has not been rigorously investigated. This is particularly the case for the highly flammable and densely populated southeast Australian sector, where ENSO is a major control over climatic variability. Here we conduct the first region-wide analysis of how ENSO controls fire activity in southeast Australia. We identify a significant relationship between ENSO and both fire frequency and area burnt. Critically, wavelet analyses reveal that despite substantial temporal variability in the ENSO system, ENSO exerts a persistent and significant influence on southeast Australian fire activity. Our analysis has direct application for developing robust predictive capacity for the increasingly important efforts at fire management

The Southern Annular Mode determines inter-annual and centennial-scale fire activity in temperate southwest Tasmania, Australia

Southern Annular Mode (SAM) is the primary mode of atmospheric variability in the Southern Hemisphere. While it is well established that the current anthropogenic‐driven trend in SAM is responsible for decreased rainfall in southern Australia, its role in driving fire regimes in this region has not been explored. We examined the connection between fire activity and SAM in southwest Tasmania, which lies in the latitudinal band of strongest correlation between SAM and rainfall in the Southern Hemisphere. We reveal that fire activity during a fire season is significantly correlated with the phase of SAM in the preceding year using superposed epoch analysis. We then synthesized new 14 charcoal records from southwest Tasmania spanning the last 1000 years, revealing a tight coupling between fire activity and SAM at centennial timescales, observing a multicentury increase in fire activity over the last 500 years and a spike in fire activity in the 21st century in response to natural and anthropogenic SAM trends

Chromatin Profiles of Chromosomally Integrated Human Herpesvirus-6A

Human herpesvirus-6A (HHV-6A) and 6B (HHV-6B) are two closely related betaherpesviruses that are associated with various diseases including seizures and encephalitis. The HHV-6A/B genomes have been shown to be present in an integrated state in the telomeres of latently infected cells. In addition, integration of HHV-6A/B in germ cells has resulted in individuals harboring this inherited chromosomally integrated HHV-6A/B (iciHHV-6) in every cell of their body. Until now, the viral transcriptome and the epigenetic modifications that contribute to the silencing of the integrated virus genome remain elusive. In the current study, we used a patient-derived iciHHV-6A cell line to assess the global viral gene expression profile by RNA-seq, and the chromatin profiles by MNase-seq and ChIP-seq analyses. In addition, we investigated an in vitro generated cell line (293-HHV-6A) that expresses GFP upon the addition of agents commonly used to induce herpesvirus reactivation such as TPA. No viral gene expression including miRNAs was detected from the HHV-6A genomes, indicating that the integrated virus is transcriptionally silent. Intriguingly, upon stimulation of the 293-HHV-6A cell line with TPA, only foreign promoters in the virus genome were activated, while all HHV-6A promoters remained completely silenced. The transcriptional silencing of latent HHV-6A was further supported by MNase-seq results, which demonstrate that the latent viral genome resides in a highly condensed nucleosome-associated state. We further explored the enrichment profiles of histone modifications via ChIP-seq analysis. Our results indicated that the HHV-6 genome is modestly enriched with the repressive histone marks H3K9me3/H3K27me3 and does not possess the active histone modifications H3K27ac/H3K4me3. Overall, these results indicate that HHV-6 genomes reside in a condensed chromatin state, providing insight into the epigenetic mechanisms associated with the silencing of the integrated HHV-6A genome

Background concentrations of mercury in Australian freshwater sediments: the role of catchment’s physico-chemical parameters on mercury deposition

Waterways in the Australian continent are facing increasing levels of mercury contamination due to industrialisation, agricultural intensification, energy production, urbanisation and mining. Mercury contamination undermines the use of waterways as a source of potable water and also has a deleterious effect on aquatic organisms. When developing management strategies to reduce mercury levels in waterways, it is crucial to set appropriate targets for mitigation of these contaminated waterways. These mitigation targets could be (1) trigger values or default guideline values provided by water and sediment quality guidelines or (2) background (pre-industrialisation) levels of mercury in the waterway. The aims of this study were to: (1) quantify the differences between existing environmental guideline values for mercury in aquatic systems, and background mercury concentrations, and (2) determine key factors affecting the spatial differences in background mercury concentrations in freshwater lake systems in Australia. Mercury concentrations were measured in background sediments from 21 lakes in Australia. Organic matter and precipitation were the main factors to explain mercury concentrations in sediments of lakes. These data indicate that background mercury concentrations in lake sediments can vary significantly across the continent, and the background concentrations are up to nine times lower than current sediment quality guidelines in Australia and New Zealand. This indicates that if waterway managers are aiming to restore systems to ‘pre-industrialisation’ mercury levels, it is highly important to quantify the site-specific background mercury concentration. We found that the geology of the lake catchment correlates to the background mercury concentration of lake sediments, with the highest mercury background levels being identified in lakes in igneous mafic intrusive regions and the lowest in areas underlain by regolith. Taking into account these findings, we provide a preliminary map of predicted background mercury sediment concentrations across Australia that could be used by waterway managers for determining management targets

Objective Response to Radiation Therapy and Long-Term Survival of Patients with WHO Grade II Astrocytic Gliomas with Known LOH 1p/19q Status

Background:: WHO grade II gliomas are often approached by radiation therapy (RT). However, little is known about tumor response and its potential impact on long-term survival. Patients and Methods:: Patients subjected to RT were selected from the own database of WHO grade II gliomas diagnosed between 1991 and 2000. The volumetric tumor response after RT was assessed based on magnetic resonance imaging and graded according to standard criteria as complete, partial (PR, ≥ 50%), or minor (MR, 25% to < 50%). Results:: There were 24 astrocytomas and three oligoastrocytomas. 21 patients (78%) were dead at follow-up (mean survival 74 months). None of the patients had chemotherapy. Objective response occurred in 14 patients (52%, five PR and nine MR) but was not associated with overall survival. The vast majority of the tumors had no loss of heterozygosity (LOH) 1p and/or 19q (86%). Conclusion:: Approximately 50% of patients with astrocytic WHO grade II gliomas respond to RT despite the absence of LOH for 1p/19q. The potential predictive factors for response and the impact of response on overall survival remain unclea

Higher-Order Chromatin Structures of Chromosomally Integrated HHV-6A Predict Integration Sites

Human herpesvirus -6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes. Viral integration can occur in several cell types, including germinal cells, resulting in individuals that harbor the viral genome in every cell of their body. The integrated genome is efficiently silenced but can sporadically reactivate resulting in various clinical symptoms. To date, the integration mechanism and the subsequent silencing of HHV-6A/B genes remains poorly understood. Here we investigate the genome-wide chromatin contacts of the integrated HHV-6A in latently-infected cells. We show that HHV-6A becomes transcriptionally silent upon infection of these cells over the course of seven days. In addition, we established an HHV-6-specific 4C-seq approach, revealing that the HHV-6A 3D interactome is associated with quiescent chromatin states in cells harboring integrated virus. Furthermore, we observed that the majority of virus chromatin interactions occur toward the distal ends of specific human chromosomes. Exploiting this finding, we established a 4C-seq method that accurately detects the chromosomal integration sites. We further implement long-read minION sequencing in the 4C-seq assay and developed a method to identify HHV-6A/B integration sites in clinical samples

Diverse peptide hormones affecting root growth identified in the Medicago truncatula secreted peptidome

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP (C-TERMINALLY ENCODED PEPTIDE), two CLE (CLV3/ENDOSPERM SURROUNDING REGION RELATED) and six XAP (XYLEM SAP ASSOCIATED PEPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N- and C-terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N-terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species.This work was supported by ARC grants to MAD: DP150104050 and LP150100826. NP was partly supported by an Endeavor Fellowship. NAMR was supported by an ANU Ph.D. scholarship supported by DP120101893. AI was supported by an Australian Post-graduate Award and an AW Howard Memorial Award. LC was supported by the Bruce-Veness Chandler and the John A. Lamberton research scholarship