No description available.

No description available.

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No description available.

MexCD-OprJ is an RND-type multidrug efflux pump present in P. aeruginosa and is capable of exporting, and as such providing resistance to, several clinically important antimicrobials including fluoroquinolones, cephems, macrolides, and several biocides including chlorhexidine (CHX). Expression of mexCD-oprJ is negatively regulated by NfxB, a LacI-type repressor. The promoter region of mexCD-oprJ was identified and included two inverted repeat operator sites, B1 and B2, both of which are required in order for NfxB to bind, thereby repressing mexCD-oprJ. NfxB oligomerizes into a tetramer in solution and likely functions as a dimer of NfxB homodimers. In addition to being derepressed by loss of NfxB, MexCD-OprJ is inducible by a variety of non-antibiotic membrane-damaging agents (MDAs) such as CHX. A homologue of NfxB, PA4596, was found to be induced in response to CHX-promoted envelope stress in an AlgU-dependent manner and is directly repressed by NfxB. Loss of PA4596 resulted in increased resistance to the biocide CHX, shown to be a result of increased CHX-dependent expression of mexCD-oprJ. Susceptibility to CHX was restored upon expression of PA4596 from the plasmid pAK1900 as was decreased expression of mexCD-oprJ in the presence of CHX, indicating that PA4596 contributes to mexCD-oprJ repression in the presence of CHX. PA4596 was found to form oligomers in solution, likely dimers and tetramers. In the absence of NfxB, PA4596 is unable to contribute to repression of mexCD-oprJ. However, NfxB and PA4596 interact and together form a repressor capable of regulating mexCD-oprJ expression. Screening of transposon mutants for increased resistance to erythromycin potentially indicative of increased mexCD-oprJ expression lead to the identification of several novel genes including PA0479, cupA3, faoA, PA3259, mucD, and clpA whose loss generated a multidrug resistance profile consistent with increased production of MexCD-OprJ. However, further studies are required to determine how each of these genes may be affecting expression of mexCD-oprJ. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-06-12 12:07:28.67

<p>Diversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear. </p><p>We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.</p><p>Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.</p> / Dissertation

<p>Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.</p> / Dissertation

One of the most important outstanding questions in biology involves the precise spatial and temporal regulation of gene activity, which enables different cell types to express the specific set of genes required for their function and is therefore a cornerstone for complex biological life. Cis-regulatory elements, such as gene promoters and enhancers, play a key role in controlling gene activity. These elements physically interact with the genes they regulate within structural chromatin domains. The organisation of chromosomes into these domains is critical for specific regulation of gene expression and disruption of these structures underlies common human disease. However, it is not understood how chromatin domains form, how interactions between the cis-regulatory elements contained within them are established, or how such interactions influence gene expression. The major hurdles in addressing these questions are to determine chromatin structures with high resolution and sensitivity and to examine their dynamic nature within single cells. To overcome these, I have developed Tri-C, a new chromosome conformation capture assay that can analyse concurrent chromatin interactions at single alleles at high resolution. By combining Tri-C with conventional chromosome conformation capture techniques, I have analysed the three-dimensional regulatory landscapes of the well-characterised murine globin loci at unprecedented depth. Additionally, to examine the roles of cis-regulatory elements in establishing chromatin architecture, I have analysed how engineered deletions in enhancers and CTCF-binding elements in the murine alpha-globin locus disrupt its chromatin landscape. These analyses reveal that the chromatin domains containing the globin genes represent compartmentalised structures, which are delimited by CTCF boundaries. The heterogeneity of interactions in these domains between individual cells is indicative for a dynamic process of loop extrusion underlying their formation. Within chromatin domains, preferential structures are formed in which multiple enhancers and promoters interact simultaneously. These complexes provide a structural basis for understanding how multiple cis-regulatory elements cooperate to establish robust regulation of gene expression. Importantly, these complex, tissue-specific structures, cannot be explained by loop extrusion alone and indicate other, independent mechanisms contributing to chromosome architecture, likely involving interactions mediated by multi-protein complexes. Together, these analyses show that the current view of genome organisation, in which chromosomes are organised by stable loops and domains that self-assemble into hierarchical structures, is not correct. Rather, chromatin architecture reflects a complex interplay between distinct molecular mechanisms contributing to the formation of regulatory landscapes that facilitate precise, robust control of gene expression.

Every living organism possesses a genome that contains within it a unique set of genes, a substantial number of which encode proteins. Over the last 20 years, it has become apparent that organismal complexity arises not from the specific complement of genes per se, but rather from interactions between the gene products - in particular, interactions between proteins. As an inevitable consequence of the crowded cellular interior, most protein-protein interactions are fleeting. However, many are significantly more long-lived and result in stable protein complexes, in which the constituent subunits are obligately dependent on their binding partners. Despite the abundance of protein complexes and their critical importance to the cell, we currently have an incomplete understanding of the mechanisms by which the cell ensures their correct assembly. In the chapters that follow, I have attempted to improve our understanding of the regulatory systems underlying assembly of protein complexes, and the way in which assembly as a whole affects the behaviour of the cell. The thesis opens with an extended literature review covering the currently available methods for characterising protein complexes. After this introduction, chapters 2-4 are concerned with regulatory mechanisms and biological implications common to the assembly of all protein complexes. Chapter 5 diverges from this work, and describes a family of evolutionarily related proteins that regulate the behaviour of condensins and cohesins. Bacterial and archaeal genomes contain far less non-coding DNA than eukaryotes, and coding genes are often packaged into discrete units known as operons. The proteins encoded within operons are usually functionally related, either through participation in metabolic pathways or as subunits of heteromeric protein complexes. Since protein complexes assemble via ordered pathways, we reasoned that there might be a signature of assembly order present in operons, the genes of which are translated in sequential order. By comparing computationally predicted assembly pathways with gene order in operons, we demonstrated this to be the case for the large majority of operon-encoded complexes. Within operons, gene order follows assembly order, and adjacent genes are substantially more likely to share a physical interface than those further apart. This work demonstrates that efficient assembly of complexes is of sufficient importance as to have placed major constraints on the evolution of operon gene order. Following this study of bacterial operons, I present results from research investigating how patterns of protein degradation in eukaryotes are influenced by the formation of protein complexes. This showed that, whilst most proteins display exponential degradation kinetics, a sizeable minority deviate considerably from this pattern, instead being more consistent with a two-step degradation process. These proteins are predominantly members of heteromeric complexes, and their two-step decay profiles can be explained using a model under which bound and unbound subunits are degraded at different rates. Within individual complexes, we find that non-exponentially decaying proteins tend to form larger interfaces, assemble earlier, and show a higher degree of coexpression, consistent with the idea that bound subunits are degraded at a slower rate than unbound or peripheral subunits. This model also explains the behaviour of proteins in aneuploid cells where one or more chromosomes have been duplicated. In general, protein abundance scales with gene copy number, so that the immediate effect of duplicating a chromosome is to double the abundance of the proteins encoded on it. However, previous analyses of mass spectrometry data, as well as my own, have shown that the abundance of many proteins on duplicated chromosomes is significantly attenuated compared to what one would expect. These proteins, like those with non-exponential degradation patterns, are very often members of larger complexes. Since the overall concentration of a protein complex is constrained by that of its least abundant members, duplicating a single subunit will predominantly increase the unbound, unstable fraction of that subunit. The results from this work strongly suggest that the apparent attenuation of many proteins observed in aneuploid cells is indeed a consequence of the failure of these proteins to assemble into complexes. Finally, I present a study concerning an important, universally conserved family of protein complexes, namely the SMC-kleisins. Two members of this family, condensin and cohesin, are responsible for two hallmarks of eukaryotic chromatin organisation: the formation of condensed, linear chromosomes, and sister chromatid cohesion during cell division. Unlike other SMC-kleisins, condensin and cohesin possess a number of regulators containing HEAT repeats. By developing a computational pipeline for searching and clustering paralogous repeat proteins, I was able to demonstrate that these regulators form a distinct sub-family within the larger class of HEAT repeat proteins. Furthermore, these regulators arose very early in eukaryotic history, hinting at a possible role in the origin of modern condensins and cohesins.

Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is a fungal pathogen of maize that causes ear rots and stalk rots worldwide. In addition, it produces a group of mycotoxins called fumonisins when the fungus colonizes maize and maize-based products. Fumonisin B1 (FB1), the predominant form occurring in nature, can cause detrimental health effects in animals and humans. Several efforts were made to study the host and pathogen factors that contribute to the production of fumonisins. Using the available genomic resources, three genes with a potential role in FB1 regulation and development were identified. The genes are GBP1, GBB1 and GAP1. This research describes molecular characterization of these genes with respect to regulation of FB1 and development in F. verticillioides. GBP1 is a monomeric GTP binding protein with similarity to DRG and Obg sub-classes of G-proteins. GBB1 encodes heterotrimeric GTP binding protein β subunit. GAP1 is a GPI (Glycophosphotidylinositol) anchored protein, which belongs to a family of cell wall proteins. Targeted deletion and complementation studies indicated that GBP1 is negatively associated with FB1 biosynthesis but had no effect on conidiation in F. verticillioides. GBB1 plays an important role in regulation of FB1 biosynthesis, conidiation and hyphal growth, but not virulence. GAP1 is associated with growth, development and conidiation but not in positive regulation of FB1 or pathogenicity. The outcome of this study revealed new molecular genetic components that will help scientists better understand signal transduction pathways that regulate FB1 biosynthesis and conidiation in F. verticillioides.