Comparative analyses of tumorigenic mechanisms of Merkel cell polyomavirus T antigens

The work described in this dissertation began in 2013 and is focused on characterizing the mechanisms by which Merkel cell polyomavirus (MCV) Large T (LT) and small T (sT) antigens induce tumorigenesis through comparative analyses with oncoproteins from other tumor viruses. A peptide motif in the C-terminal region of MCV LT that bears little sequence homology with other human polyomavirus LT proteins is shown to be critical for maintaining stability of the full-length LT protein (Chapter 3). Comparison between LT antigens of MCV and SV40 demonstrate that MCV LT in direct contrast to SV40 LT is incapable of avidly binding tumor suppressor p53 and inhibiting its transactivation capabilities (Chapter 4). Lastly, promiscuous E3 ligase targeting by MCV sT through its LT-stabilization domain (LSD) results in the formation of a genomically unstable phenotype, a known hallmark of cancer (Chapter 5). Many of the features of genomic instability induced by MCV sT such as centrosome overduplication parallel what has been observed previously for human papillomavirus 16 E7 oncoprotein. Overall, these comparative analyses have not only provided greater insight into MCV biology and how its T antigens function in causing an aggressive cancer like Merkel cell carcinoma (MCC), but they have also revealed new avenues for continued study involving MCV T antigens that will continue to move the field of tumor virology forward

A systems biology analysis of brain microvascular endothelial cell lipotoxicity.

BackgroundNeurovascular inflammation is associated with a number of neurological diseases including vascular dementia and Alzheimer's disease, which are increasingly important causes of morbidity and mortality around the world. Lipotoxicity is a metabolic disorder that results from accumulation of lipids, particularly fatty acids, in non-adipose tissue leading to cellular dysfunction, lipid droplet formation, and cell death.ResultsOur studies indicate for the first time that the neurovascular circulation also can manifest lipotoxicity, which could have major effects on cognitive function. The penetration of integrative systems biology approaches is limited in this area of research, which reduces our capacity to gain an objective insight into the signal transduction and regulation dynamics at a systems level. To address this question, we treated human microvascular endothelial cells with triglyceride-rich lipoprotein (TGRL) lipolysis products and then we used genome-wide transcriptional profiling to obtain transcript abundances over four conditions. We then identified regulatory genes and their targets that have been differentially expressed through analysis of the datasets with various statistical methods. We created a functional gene network by exploiting co-expression observations through a guilt-by-association assumption. Concomitantly, we used various network inference algorithms to identify putative regulatory interactions and we integrated all predictions to construct a consensus gene regulatory network that is TGRL lipolysis product specific.ConclusionSystem biology analysis has led to the validation of putative lipid-related targets and the discovery of several genes that may be implicated in lipotoxic-related brain microvascular endothelial cell responses. Here, we report that activating transcription factors 3 (ATF3) is a principal regulator of TGRL lipolysis products-induced gene expression in human brain microvascular endothelial cell

GOPred: GO Molecular Function Prediction by Combined Classifiers

Functional protein annotation is an important matter for in vivo and in silico biology. Several computational methods have been proposed that make use of a wide range of features such as motifs, domains, homology, structure and physicochemical properties. There is no single method that performs best in all functional classification problems because information obtained using any of these features depends on the function to be assigned to the protein. In this study, we portray a novel approach that combines different methods to better represent protein function. First, we formulated the function annotation problem as a classification problem defined on 300 different Gene Ontology (GO) terms from molecular function aspect. We presented a method to form positive and negative training examples while taking into account the directed acyclic graph (DAG) structure and evidence codes of GO. We applied three different methods and their combinations. Results show that combining different methods improves prediction accuracy in most cases. The proposed method, GOPred, is available as an online computational annotation tool (http://kinaz.fen.bilkent.edu.tr/gopred)

In silico regulatory analysis for exploring human disease progression

© 2008 Holloway et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Deciphering transcriptional regulation in cancer cells and development of a new method to identify key transcriptional regulators and their target genes

Cancer cells accumulate genetic changes during carcinogenesis. The dimension of these changes range from point mutations to large chromosomal aberrations. It has been widely accepted that essential genetic programs are thereby dysregulated that normally would prevent uncontrolled cellular division and growth. Transcription factors (TFs) are key proteins of gene regulation and are frequently associated with genetic pathologies, e.g. MYCN in neuroblastomas (NBs). Research on gene regulation -in general or condition-specific- thus is a central aspect in cancer research, and it is also the focus of my work. In a carcinogenesis model of NBs without MYCN-amplification, mutations of chromosome 11q (11q-CNA) are suspected to critically influence tumor development. We were able to refine this model by means of gene expression analysis on 11q-CNA in NBs with different clinical outcome. Gene expression profiles of NBs with unfavorable progression differed significantly between tumors with and without 11q-CNA, whereas 11q-CNA in NBs with favorable outcome is apparently compensated by a yet unknown mechanism. The TF-encoding gene CAMTA1 is located on the chromosomal region 1p, which is frequently deleted in NBs. In vitro experiments with ectopic induction of CAMTA1 yielded CAMTA1-regulated genes with different gene expression profiles that were functionally associated by enrichment analyses with cell cycle regulation and neuronal differentiation. The suggested role of CAMTA1 as a tumor suppressor gene was confirmed by additional in vivo experiments. Furthermore, we studied the effect of MYC and MYCN in NBs without MYCN-amplification and found that these TF also strongly regulate a large number of common target genes according to their own gene expression in these tumors. Promoter analyses and chromatin immunoprecipitation additionally supported the regulation of the determined target genes by MYC/MYCN. The genome-wide application of promoter and enrichment analyses on gene expression data from mouse models enabled us to predict target TFs of Rage signaling. E2f1 and E2f4 were validated experimentally as components of the Rage-dependent gene regulatory network. Finally, we used our experience from gene expression analysis to develop a novel machine learning method to precisely predict TF target gene relationships in human. We combined results from a genome-wide correlation meta-analysis on 4064 microarray gene expression profiles and promoter analyses on TF binding sites with known regulatory interactions between TFs and target genes in our approach. Our method outperformed other comparable methods in human, as we improved shortcomings of other algorithms specifically for higher eukaryotes, in particular the frequently (erroneously) assumed correlation between the mRNA expression of TFs and their target genes. We made our method freely available as a software package with multiple applications like the identification of key TFs in a multiplicity of cellular systems (e.g. cancer cells)

Insight into Merkel Cell Polyomavirus Replication Through the Study of the Viral Early Proteins: Large and Small Tumor Antigens

The association of Merkel cell polyomavirus (MCPyV) and Merkel cell carcinoma (MCC) established in 2008 has opened doors for the study of virus-induced oncogenesis. Much of what is known about polyomaviruses (PyVs) stems from decades of studies on SV40. However, recent research has shown differences among PyVs that might help explain the uniqueness of MCPyV that makes it the only PyV discovered to date that is associated with a human cancer. Therefore, it is important to understand the biology of this virus and its oncogenic potential. My study focuses on two of the early proteins of MCPyV, namely the large tumor antigen (LT) and the small tumor antigen (sT). Both proteins are multi-functional, contributing to viral replication and the stimulation of cellular proliferation. MCPyV LT is the viral helicase that binds to the viral origin (Ori) to initiate unwinding and the replication of the double-stranded DNA genome. Like other PyVs, MCPyV requires numerous cellular proteins to replicate its genome. In my research, I show that, in the presence of the viral Ori, the binding of LT to the Ori forms replication factories in the nucleus. A number of cellular factors involved in the host DNA damage response (DDR) re-localize to the sites of MCPyV LT-mediated replication. Inhibition of the DNA damage response by either drug treatment or siRNA knockdown decreases MCPyV replication, suggesting that an intact host DDR pathway is essential for the optimal replication of MCPyV. Previous research has shown that MCPyV sT indirectly enhances viral replication by stabilizing LT. In my study, I discover that sT is a metalloprotein that coordinates two iron-sulfur clusters. Mutations in the highly conserved cysteines found in MCPyV sTs abolishes its ability to stimulate LT-mediated viral DNA replication, and that sT can enhance LT-mediated replication in a manner that is independent of LT stabilization. Moreover, I show that sT translocates to the nuclear replication factories formed in the presence of LT and Ori, suggesting a more direct role of sT in promoting viral DNA replication. Interestingly, upon treatment with the potent antiviral agent cidofovir, sT-mediated enhancement of MCPyV replication is robustly inhibited, while replication driven by LT alone was not affected much. This finding supports the use of cidofovir in controlling PyV infection and offers MCPyV sT as a potential drug target to dampen viral growth. In summary, my work elucidates various aspects of MCPyV replication, including the involvement of the host DDR machinery and the role of sT that could potentially be targeted by drug treatment. This study contributes to the understanding of the virology of MCPyV and opens avenues for further research

Functional Analysis of Chromodomain Helicase DNA Binding Protein 2(CHD2) mediated Genomic Stability

Histone modifying enzymes and chromatin remodeling complexes play an important regulatory role in chromatin dynamics that dictate the interaction of regulatory factors involved in processes such as DNA replication, recombination, repair and transcription, with DNA template. The CHD (Chromodomain Helicase DNA Binding Protein) family of proteins is known to be involved in the regulation of gene expression, recombination and chromatin remodeling via their chromatin specific interactions and activities. Phenotypic analysis of the Chd2 mutant mouse model developed by our laboratory indicates that the Chd2 protein plays a critical role in tumor suppression as the heterozygous mutant mice develop spontaneous lymphomas. In this study we demonstrate that mutation of Chd2 renders cells susceptible to inefficient DNA repair and genomic instability. Homozygous and heterozygous Chd2 mutant mouse embryonic fibroblast accumulates higher levels of gamma-H2AX after DNA damage. Chd2 mutant cells show inefficiency in DNA repair of DNA lesions induced by X-rays and UV irradiation as assessed by single cell gel electrophoresis assays. These cells also exhibit increased chromosomal aberrations after treatment with low doses of X-ray irradiation (2 Gy) and show increased radiosensitivity in a clonogenic survival assay. At the molecular level, endogenous CHD2 protein level is induced after exposure to X-ray radiation. In addition, we have also demonstrated in this study that CHD2 is phosphorylated after DNA damage and is a potential substrate for phosphoinositide 3-kinase-related kinases (PIKK) - ATM/ATR. Additionally, mass spectrometric analysis showed possible association of CHD2 with the paraspeckle family of proteins known to be involved in an array of cellular processes specifically in RNA processing and DNA repair. An in vivo splicing assay demonstrated that CHD2 played a role in modulation of pre-mRNA splicing event. Collectively, our findings suggest that CHD2 is a multi-functional protein working with the paraspeckle protein complex to facilitate both the pre-mRNA splicing process and the initial DNA repair process. CHD2 may also be involved in the later stages of DNA damage response pathway by influencing p53’s transcriptional activity

Transcriptional Regulation Of MicroRNA Genes And The Regulatory Networks In Which They Participate

MicroRNA genes are short, non-coding RNAs that function as post-transcriptional gene regulators. Although they have been implicated in organismal development as well as a variety of human diseases, there is still surprisingly little known about their transcriptional regulation. The understanding of microRNA transcription is very important for determining their regulators as well as the specific role they may play in signaling cascades. This dissertation focused on the comparison of mammalian microRNA promoters and upstream sequences to those of known protein coding genes. This dissertation is also focused on determining potential regulatory networks that microRNA genes may participate in, particularly those networks involved in the TGFβ / SMAD signaling pathway. The comparison of intergenic microRNA upstream sequences to those of protein coding genes revealed that the former are up to twice as conserved as the latter, except in the first 500 base pairs where the conservation is similar. Further investigation of the upstream sequences by RNA Polymerase II ChIP-chip revealed the transcription start site for 35 primary-microRNA transcripts. The identification of features capable of distinguishing core promoter regions from background sequences using a support vector machine approach revealed that the transcription start site of primary-microRNA genes share the same sequence features as protein coding genes. These results suggest that in fact microRNA genes are transcribed by the same mechanism by which protein coding genes are transcribed. This information allowed us to then identify the regulatory elements of microRNA genes in the same manner in which we use for protein coding genes. Identification of a SMAD family transcription factor binding site upstream of the human let-7d microRNA revealed a feed-forward regulatory circuit involved in epithelial mesenchymal transition. This provided the first evidence of a direct link between a growth factor and the expression of a microRNA gene. The understanding of microRNA transcriptional regulation has great public health significance. The ability to understand how these post-transcriptional gene regulators function in cellular networks may provide new molecular targets for cures or therapies to a variety of human diseases

dbOGAP - An Integrated Bioinformatics Resource for Protein O-GlcNAcylation

<p>Abstract</p> <p>Background</p> <p>Protein O-GlcNAcylation (or O-GlcNAc-ylation) is an O-linked glycosylation involving the transfer of β-<it>N</it>-acetylglucosamine to the hydroxyl group of serine or threonine residues of proteins. Growing evidences suggest that protein O-GlcNAcylation is common and is analogous to phosphorylation in modulating broad ranges of biological processes. However, compared to phosphorylation, the amount of protein O-GlcNAcylation data is relatively limited and its annotation in databases is scarce. Furthermore, a bioinformatics resource for O-GlcNAcylation is lacking, and an O-GlcNAcylation site prediction tool is much needed.</p> <p>Description</p> <p>We developed a database of O-GlcNAcylated proteins and sites, dbOGAP, primarily based on literature published since O-GlcNAcylation was first described in 1984. The database currently contains ~800 proteins with experimental O-GlcNAcylation information, of which ~61% are of humans, and 172 proteins have a total of ~400 O-GlcNAcylation sites identified. The O-GlcNAcylated proteins are primarily nucleocytoplasmic, including membrane- and non-membrane bounded organelle-associated proteins. The known O-GlcNAcylated proteins exert a broad range of functions including transcriptional regulation, macromolecular complex assembly, intracellular transport, translation, and regulation of cell growth or death. The database also contains ~365 potential O-GlcNAcylated proteins inferred from known O-GlcNAcylated orthologs. Additional annotations, including other protein posttranslational modifications, biological pathways and disease information are integrated into the database. We developed an O-GlcNAcylation site prediction system, OGlcNAcScan, based on Support Vector Machine and trained using protein sequences with known O-GlcNAcylation sites from dbOGAP. The site prediction system achieved an area under ROC curve of 74.3% in five-fold cross-validation. The dbOGAP website was developed to allow for performing search and query on O-GlcNAcylated proteins and associated literature, as well as for browsing by gene names, organisms or pathways, and downloading of the database. Also available from the website, the OGlcNAcScan tool presents a list of predicted O-GlcNAcylation sites for given protein sequences.</p> <p>Conclusions</p> <p>dbOGAP is the first public bioinformatics resource to allow systematic access to the O-GlcNAcylated proteins, and related functional information and bibliography, as well as to an O-GlcNAcylation site prediction tool. The resource will facilitate research on O-GlcNAcylation and its proteomic identification.</p