University Of Tennessee Knoxville

NIH Research Projects · FY 2025 · 2019-09

Project Summary/Abstract: The 3D folding of human chromosomes inside the nucleus affects critical biological processes, including gene regulation and DNA repair, while also influencing the physical properties of the nucleus. To make progress toward our overarching goal of understanding the principles of chromosome folding and their importance in health and disease, my research program investigates the responses of 3D genome structure to physical perturbations. We examine alterations of chromosome structure in the context of DNA damage, nucleus structure disruption by lamin mutation, cell migration through narrow constrictions, and externally applied forces. Our recent work has revealed a striking robustness of chromosome structure to many transient perturbations, but has shown stable alterations in the 3D genome and cell phenotype can result from chronic or repeated stresses. By comparing complementary systems, we have identified different levels of the chromosome structure hierarchy that respond to different stresses: DNA damage results in strengthening of the local loop and topologically associating domain (TAD) structure while physical deformations of the nucleus associated with altered spatial segregation of heterochromatin and euchromatin. New research in a variety of systems is revealing the important role of nucleus mechanosensation in cell fate decisions. We are now well- positioned to connect cellular and imaging observations of such phenomena with the 3D genome changes that accompany them. In this next funding period, our research will examine what molecular mechanisms contribute to stable 3D genome and phenotype changes after cancer cells pass through multiple rounds of constricted migration. We will contrast this constricted migration system with responses elicited by externally applied forces on the cell. We will examine how disruption of nucleus architecture by a lamin mutation affects genome structure reprogramming during differentiation and contributes to the patient phenotypes of the premature aging disease Progeria. As many of these stressors also induce DNA damage, we will finally investigate the effects of chromosome structure alterations after DNA damage on DNA repair, gene regulation, and cellular response to subsequent stresses. Observations across our experimental systems combined with computational analysis and modeling will clarify the relationship between changes in chromosome structure and gene expression and reveal how microscopically observed alterations in chromosome conformation relate to changes in contact patterns detected by chromosome conformation capture (Hi-C) family techniques. Our previous innovations in single cell genomic data analysis and integration position us to investigate how average shifts in cell population behavior connect to the heterogeneity and dynamics of chromosome structure and gene expression at a single cell level. Defining connections between physical perturbations, genome architecture, and cell fate will inform future diagnostics and treatments for diseases such as cancer, premature aging, cardiomyopathies, and any condition where the physical state of the cell influences cell function.

NIH Research Projects · FY 2025 · 2018-09

PROJECT SUMMARY The goal of this proposal is to provide a mechanistic understanding of how cell division in bacteria is controlled at a molecular level. Understanding these mechanisms, which are broadly conserved among the bacteria, is important because it can reveal potential targets for new antibacterial agents that inhibit cell division and stop cell propagation. The division process in bacteria involves two stages. In the first distinct step, FtsZ protofilament assembly, the Z-ring, forms. In Escherichia coli, the model organism for this study, the first step occurs early in the cell cycle. Only after a significant delay, which can last half of the cell cycle, does the second stage of cell division start. In this later step, septal peptidoglycan synthesis begins, and the cell constricts. In this stage, more than two dozen proteins are involved, most of them in a complex referred to as the divisome. It is not yet understood what determines the onset of either the first or the second stage of the division, both of which are critical for the cell's survival. This significant gap in our knowledge exists even though many proteins involved in cell division are known and their pairwise binding interactions mapped out. The difficulty in understanding processes controlling cell division arises from the presence of a large number of different interactions within the divisome and of many pathways that are partially redundant. Moreover, the protein assemblies, which in most studies are viewed as static, are highly dynamic, turning over in a matter of seconds in an energy-dependent process such as treadmilling. The complexity of the problem requires not only further experiments but the integration of the existing experimental results into a comprehensive modeling framework. Accordingly, we combine state-of-the-art experimental methods with stochastic cell cycle simulations and 3D modeling of assembly reactions of proteins involved in cell division. On the latter front, we leverage our previous work and ongoing collaborations. In the experimental work, we use molecular biology and genetic methods alongside high throughput and super-resolution microscopy, and we develop novel microfluidic devices for this research. These techniques have already generated large amounts of information-rich data that, among other findings, have shed new light on processes leading to the assembly of Z-ring from individual FtsZ protofilaments and determining the role of DNA replication over the control of the progression of the second stage of the division. The proposed work aims to consolidate these past findings into a single mechanistic framework. The knowledge gained will enhance our understanding of fundamental cellular processes in bacteria and provide a basis for designing effective antibacterial therapies that target bacterial cell division.

NIH Research Projects · FY 2025 · 2017-07

Project Summary/Abstract In the United States, Ixodes scapularis ticks harbor and transmit several pathogens including human anaplasmosis bacterial agent Anaplasma phagocytophilum. This bacterium is transmitted to the vertebrate host by an infected tick bite. Several studies have addressed molecular mechanisms that A. phagocytophilum uses to survive in the mammalian host. Relatively, few studies have clearly defined the molecular strategies that this bacterium uses to survive in ticks. In the previous funding period, we performed a comprehensive molecular analysis on I. scapularis organic anion transporting polypeptides 4056 (IsOATP4056) and genes involved in the tryptophan metabolism pathway in A. phagocytophilum-tick interactions. We made substantial progress and have published several research articles showing importance of IsOATP4056 and the tryptophan pathway in the survival and transmission of A. phagocytophilum from ticks to the naïve vertebrate host. Passive Immunization with IsOATP4056 antibodies impaired A. phagocytophilum transmission from ticks to the vertebrate host. Anaplasma phagocytophilum upregulates IsOATP4056 and activates the tryptophan pathway leading to the increased synthesis of endogenous levels of tryptophan metabolite, Xanthurenic acid (XA). The increased XA prevents the build-up of reactive oxygen species facilitating bacterial survival in tick cells. In addition, A. phagocytophilum activates the XA-mediated p38-MAPK pathway to inhibit tick cell death thereby facilitating the survival of both the bacterial agent and its vector host. We also reported a novel role for XA in the transcriptional activation of isoatp4056 gene expression. Furthermore, A. phagocytophilum downregulates microRNA (miR133) that targets isoatp4056 for its survival and transmission from vector to the vertebrate host. These findings from our previous funding period provide a strong rationale for the proposed aims for this R01 (competing renewal) application. In Aim 1, we propose to understand the mechanism of how passive immunization affects the transmission of A. phagocytophilum from ticks to the vertebrate host. In Aim 2, we will analyze whether targeting IsOATP4056 via active immunization or OATP inhibitor treatment affects the transmission of A. phagocytophilum from ticks to the vertebrate host. In Aim 3, we will characterize XA-mediated regulation of miRNAs that are important in regulating gene expression during A. phagocytophilum survival and transmission from infected ticks to the naïve host. Based on the success in the previous funding period and the aims proposed in this renewal application, we believe that this could be a transformative study that not only serves as a model to study intimate relationships established by pathogens with their arthropod vectors but may also lead in the development of new strategies to interrupt the transmission of this and perhaps other rickettsial bacteria of medical importance.