JC Virus Small t Antigen Binds Phosphatase PP2A and Rb Family Proteins and Is Required for Efficient Viral DNA Replication Activity

BACKGROUND: The human polyomavirus, JC virus (JCV) produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV's large tumor antigen (TAg) and three T' proteins, but little is known about small tumor antigen (tAg) functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. METHODOLOGY AND FINDINGS: We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A), and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. CONCLUSIONS: JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg null mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells

The USFDA approved "epigenetic drug", Decitabine, exerts its effect by hypomethylating DNA, demonstrating the pivotal role aberrant genome-wide DNA methylation patterns play in cancer ontology. Using sensitive technologies in a cellular model of Acute Myeloid Leukemia, we demonstrate that while Decitabine reduces the global levels of 5-methylcytosine (5mC), it results in paradoxical increase of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels. Hitherto, the only biological mechanism known to generate 5hmC, 5fC and 5caC, involving oxidation of 5mC by members of Ten-Eleven-Translocation (TET) dioxygenase family, was not observed to undergo any alteration during DAC treatment. Using a multi-compartmental model of DNA methylation, we show that partial selectivity of TET enzymes for hemi-methylated CpG dinucleotides could lead to such alterations in 5hmC content. Furthermore, we investigated the binding of TET1-catalytic domain (CD)-GFP to DNA by Fluorescent Correlation Spectroscopy in live cells and detected the gradual increase of the DNA bound fraction of TET1-CD-GFP after treatment with Decitabine. Our study provides novel insights on the therapeutic activity of DAC in the backdrop of the newly discovered derivatives of 5mC and suggests that 5hmC has the potential to serve as a biomarker for monitoring the clinical success of patients receiving DAC

In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position–, cell type–, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs

The Influence of Hydroxylation on Maintaining CpG Methylation Patterns: A Hidden Markov Model Approach

German Research Council (DFG) as part of the Collaborative Research Center "Physical modeling of non-equilibrium processes in biological systems" (SFB 1027) and the Cluster of Excellence on Multimodal Computing and Interaction at Saarland Universit

Microenvironmental Influences that Drive Progression from Benign Breast Disease to Invasive Breast Cancer

Invasive breast cancer represents the endpoint of a developmental process that originates in the terminal duct lobular units and is believed to progress through stages of increasing proliferation, atypical hyperplasia, and carcinoma in situ before the cancer acquires invasive and metastatic capabilities. By comparison with invasive breast cancer, which has been studied extensively, the preceding stages of benign breast disease are more poorly understood. Much less is known about the molecular changes underlying benign breast disease development and progression, as well as the transition from in situ into invasive disease. Even less focus has been given to the specific role of stroma in this progression. The reasons for lack of knowledge about these lesions often come from their small size and limited sample availability. More challenges are posed by limitations of the models used to investigate the lesions preceding invasive breast cancer. However, recent studies have identified alterations in stromal cell function that may be critical for disease progression from benign disease to invasive cancer: key functions of myoepithelial cells that maintain tissue structure are lost, while tissue fibroblasts become activated to produce proteases that degrade the extracellular matrix and trigger the invasive cellular phenotype. Gene expression profiling of stromal alterations associated with disease progression has also identified key transcriptional changes that occur early in disease development. In this review, we will summarize recent studies showing how stromal factors can facilitate progression of ductal carcinoma in situ to invasive disease. We also suggest approaches to identify processes that control earlier stages of disease progression

Nonlinear model standardization for the analysis and design of nonlinear systems with multiple equilibria

In engineering practice, a nonlinear system stable about several equilibria is often studied by linearizing the system over a small range of operation around each of these equilibria, and allowing the study of the system using linear system methods. Theoretically, for operations beyond a small range but still within the stable regime of an equilibrium, the system behaves nonlinearly, and can be described and investigated using the Volterra series approach. However, there is still no available approach that can systematically transform the model of a nonlinear system into a form that can be studied over the whole stable regime about an equilibrium so as to facilitate the system study using the Volterra series approach. This transformation is, in the present study, referred to as nonlinear model standardization, which is the extension of the well-known concept of linearization to the nonlinear case. In this paper, a novel approach to nonlinear model standardization is proposed for nonlinear systems that can be described by a Nonlinear AutoRegressive model with eXogeneous input (NARX) or a nonlinear differential equation (NDE) model. The proposed approach is then used in three case studies covering the applications in nonlinear system analysis, nonlinear system design, and nonlinearity compensation, respectively, demonstrating the significance of the proposed nonlinear model standardization in a wide range of engineering practices

A Stochastic Model for the Formation of Spatial Methylation Patterns

DNA methylation is an epigenetic mechanism whose important role in development has been widely recognized. This epigenetic modification results in heritable changes in gene expression not encoded by the DNA sequence. The underlying mechanisms controlling DNA methylation are only partly understood and recently different mechanistic models of enzyme activities responsible for DNA methylation have been proposed. Here we extend existing Hidden Markov Models (HMMs) for DNA methylation by describing the occurrence of spatial methylation patterns over time and propose several models with different neighborhood dependencies. We perform numerical analysis of the HMMs applied to bisulfite sequencing measurements and accurately predict wild-type data. In addition, we find evidence that the enzymes' activities depend on the left 5' neighborhood but not on the right 3' neighborhood.Comment: 18 pages, 7 figures, content of former appendix now included in the main part; accepted by 15th International Conference on Computational Methods in Systems Biology (CMSB), 201

Revelando o vírus, ocultando pessoas: exames de monitoramento (CD4 e CVP) e relação médico-paciente no contexto da AIDS Disclosing the virus, hiding the patients: follow-up tests (CD4 and VL) and the physician-patient relationship in the AIDS setting

O objetivo deste artigo é discutir os significados associados aos exames de contagem de linfócitos CD4 e quantificação da carga viral plasmática do HIV (CVP) para pacientes vivendo com AIDS e médicos da atenção, buscando analisar os reflexos de sua crescente utilização na relação terapêutica. Foi realizado um estudo qualitativo em dois centros de referência em HIV/AIDS com observação participante e entrevistas semi-estruturadas com 27 pacientes vivendo com AIDS e quatro médicos. A observação das consultas médicas mostrou que elas são rápidas, objetivas e centradas no resultado dos exames CD4 e CVP, o que reforça uma visão hegemônica do saber médico e uma perspectiva biomédica que instrumentaliza a sua prática. Para médicos e pacientes, os exames passam a refletir a "verdade" sobre a doença do paciente em detrimento da anamnese e do exame clínico, fato que se reflete na relação terapêutica e na desatenção por parte dos médicos à subjetividade dos pacientes. Mais do que nunca há necessidade da retomada da prática da boa clínica e do reconhecimento do papel do sujeito na prática da medicina como arte de curar.<br>The aim of this study is to discuss the meanings associated with the CD4 lymphocyte count and HIV plasma viral load (VL) for patients living with AIDS and the attending physicians, seeking to analyze the impacts of the increasing use of these tests in the treatment setting. A qualitative study was performed in two HIV/AIDS referral centers with participant observation and semi-structured interviews with 27 patients living with AIDS and four physicians. Observation of the medical consultations showed that they are quick, objective, and centered on the CD4 and VL test results, thus reinforcing a hegemonic view of medical knowledge and a biomedical perspective that instrumentalizes their practice. For physicians and patients, the tests tend to reflect the "truth" on the patient's disease, to the detriment of the patient history and clinical examination, impacting the therapeutic relationship and leading to the physician's lack of attention to the patients' subjectivity. More than ever, there is a need to reclaim good clinical practice and acknowledge the subject's role in medical practice as a healing art