Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Transcriptional regulation by the orphan nuclear receptor ERRgamma

The nuclear receptor (NR) superfamily is comprised of receptors for small lipopfilic ligands such as steroid hormones, thyroid hormone, retinoids, and vitamin D. NRs are ligand-inducible transcription factors capable of both activating and repressing their target gene expression. They control a wide range of biological functions connected to growth, development, and homeostasis. In addition to the ligand-regulated receptors, the family includes a large group of receptors whose physiological ligands are unknown. These receptors are referred to as orphan NRs. Estrogen-related receptor gamma (ERRgamma) belongs to the ERR subfamily of orphan NRs together with the related ERRalpha and ERRbeta. ERRs share amino acid sequence homology with the classical estrogen receptors (ERs) but they are unable to bind natural estrogenic ligands. ERRgamma is expressed in several embryonic and adult tissues but its biological role is still largely unknown. ERRgamma activates reporter gene expression in transfected cells independently of added hormones implying that ERRgamma harbors constitutive activity. However, the intrinsic activity of ERRgamma can be inhibited by synthetic compounds such as the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT). Ligands of NRs can act as agonists that activate transcription, as antagonists that prevent activation of transcription, or as inverse agonists that antagonize the constitutive transcriptional activity of receptor. Most of the synthetic ERRgamma ligands act as inverse agonists but recently, a synthetic ERRgamma agonist GSK4716 was identified. This demonstrates that it is possible to design and identify agonists for ERRgamma. Prior to this thesis work, the structural and functional characteristics of ERRgamma were largely unknown. The aim of this study was to define the functional requirements for ERRgamma-mediated transcriptional regulation and to examine the cross-talk between ERRgamma and other NRs. Due to the fact that natural physiological ligands of ERRgamma are unknown, another aim of this study was to seek new natural compounds that may affect transcriptional activity of ERRgamma. Plant-derived phytoestrogens have previously been shown to act as ligands for ERs and ERRalpha, and therefore the effects of these compounds were also studied on ERRgamma-mediated transcriptional regulation. This work demonstrated that ERRgamma-mediated transcriptional regulation was dependent on DNA-binding, dimerization and activation function-2. Heterodimerization with ERRalpha inhibited the transcriptional activity of ERRgamma. In addition to 4-OHT, another anti-estrogen, 4-hydroxytoremifene (4-OHtor), was identified as an inverse agonist of ERRgamma. Interestingly, ERRgamma activated transcription in the presence of 4-OHT and 4-OHtor on activator protein-1 binding sites. ERRgamma was found to interact with another orphan NR Nurr1 by repressing the ability of Nurr1 to activate transcription of the osteopontin gene. Transcriptional activity of ERRgamma was shown to be stimulated by the phytoestrogen equol. Structural model analysis and mutational experiments indicated that equol was able to bind to the ligand binding domain of ERRgamma. The growth inhibitory effect of ERRgamma on prostate cancer cells was found to be enhanced by equol. In summary, this study demonstrates that despite the absence of an endogenous physiological ligand, the activity of ERRgamma can be modulated in other ways such as dimerization with related receptors or by cross-talk with other transcription factors as well as by binding some synthetic or natural compounds.

Latent TGF-β binding proteins -3 and -4 : transcriptional control and extracellular matrix targeting

Extracellular matrix (ECM) is a complex network of various proteins and proteoglycans which provides tissues with structural strength and resilience. By harvesting signaling molecules like growth factors ECM has the capacity to control cellular functions including proliferation, differentiation and cell survival. Latent transforming growth factor β (TGF-β) binding proteins (LTBPs) associate fibrillar structures of the ECM and mediate the efficient secretion and ECM deposition of latent TGF-β. The current work was conducted to determine the regulatory regions of LTBP-3 and -4 genes to gain insight into their tissue-specific expression which also has impact on TGF-β biology. Furthermore, the current research aimed at defining the ECM targeting of the N-terminal variants of LTBP-4 (LTBP-4S and -4L), which is required to understand their functions in tissues and to gain insight into conditions in which TGF-β is activated. To characterize the regulatory regions of LTBP-3 and -4 genes in silico and functional promoter analysis techniques were employed. It was found that the expression of LTBP-4S and -4L are under control of two independent promoters. This finding was in accordance with the observed expression patterns of LTBP-4S and -4L in human tissues. All promoter regions characterized in this study were TATAless, GC-rich and highly conserved between human and mouse species. Putative binding sites for Sp1 and GATA family of transcription factors were recognized in all of these regulatory regions. It is possible that these transcription factors control the basal expression of LTBP-3 and -4 genes. Smad binding element was found within the LTBP-3 and -4S promoter regions, but it was not present in LTBP-4L promoter. Although this element important for TGF-β signaling was present in LTBP-4S promoter, TGF-β did not induce its transcriptional activity. LTBP-3 promoter activity and mRNA expression instead were stimulated by TGF-β1 in osteosarcoma cells. It was found that the stimulatory effect of TGF-β was mediated by Smad and Erk MAPK signaling pathways. The current work explored the ECM targeting of LTBP-4S and identified binding partners of this protein. It was found that the N-terminal end of LTBP-4S possesses fibronectin (FN) binding sites which are critical for its ECM targeting. FN deficient fibroblasts incorporated LTBP-4S into their ECM only after addition of exogenous FN. Furthermore, LTBP-4S was found to have heparin binding regions, of which the C-terminal binding site mediated fibroblast adhesion. Soluble heparin prevented the ECM association of LTBP-4S in fibroblast cultures. In the current work it was observed that there are significant differences in the secretion, processing and ECM targeting of LTBP-4S and -4L. Interestingly, it was observed that most of the secreted LTBP-4L was associated with latent TGF-β1, whereas LTBP-4S was mainly secreted as a free form from CHO cells. This thesis provides information on transcriptional regulation of LTBP-3 and -4 genes, which is required for the deeper understanding of their tissue-specific functions. Further, the current work elucidates the structural variability of LTBPs, which appears to have impact on secretion and ECM targeting of TGF-β. These findings may advance understanding the abnormal activation of TGF-β which is associated with connective tissue disorders and cancer.

Functional Analysis of MrpL55, Vig and Mat1 Acting at the Crossroad of Cell Cycle, Transcription and Metabolism Regulation

Cell proliferation, transcription and metabolism are regulated by complex partly overlapping signaling networks involving proteins in various subcellular compartments. The objective of this study was to increase our knowledge on such regulatory networks and their interrelationships through analysis of MrpL55, Vig, and Mat1 representing three gene products implicated in regulation of cell cycle, transcription, and metabolism. Genome-wide and biochemical in vitro studies have previously revealed MrpL55 as a component of the large subunit of the mitochondrial ribosome and demonstrated a possible role for the protein in cell cycle regulation. Vig has been implicated in heterochromatin formation and identified as a constituent of the RNAi-induced silencing complex (RISC) involved in cell cycle regulation and RNAi-directed transcriptional gene silencing (TGS) coupled to RNA polymerase II (RNAPII) transcription. Mat1 has been characterized as a regulatory subunit of cyclin-dependent kinase 7 (Cdk7) complex phosphorylating and regulating critical targets involved in cell cycle progression, energy metabolism and transcription by RNAPII. The first part of the study explored whether mRpL55 is required for cell viability or involved in a regulation of energy metabolism and cell proliferation. The results revealed a dynamic requirement of the essential Drosophila mRpL55 gene during development and suggested a function of MrpL55 in cell cycle control either at the G1/S or G2/M transition prior to cell differentiation. This first in vivo characterization of a metazoan-specific constituent of the large subunit of mitochondrial ribosome also demonstrated forth compelling evidence of the interconnection of nuclear and mitochondrial genomes as well as complex functions of the evolutionarily young metazoan-specific mitochondrial ribosomal proteins. In studies on the Drosophila RISC complex regulation, it was noted that Vig, a protein involved in heterochromatin formation, unlike other analyzed RISC associated proteins Argonaute2 and R2D2, is dynamically phosphorylated in a dsRNA-independent manner. Vig displays similarity with a known in vivo substrate for protein kinase C (PKC), human chromatin remodeling factor Ki-1/57, and is efficiently phosphorylated by PKC on multiple sites in vitro. These results suggest that function of the RISC complex protein Vig in RNAi-directed TGS and chromatin modification may be regulated through dsRNA-independent phosphorylation by PKC. In the third part of this study the role of Mat1 in regulating RNAPII transcription was investigated using cultured murine immortal fibroblasts with a conditional allele of Mat1. The results demonstrated that phosphorylation of the carboxy-terminal domain (CTD) of the large subunit of RNAPII in the heptapeptide YSPTSPS repeat in Mat-/- cells was over 10-fold reduced on Serine-5 and subsequently on Serine-2. Occupancy of the hypophosphorylated RNAPII in gene bodies was detectably decreased, whereas capping, splicing, histone methylation and mRNA levels were generally not affected. However, a subset of transcripts in absence of Mat1 was repressed and associated with decreased occupancy of RNAPII at promoters as well as defective capping. The results identify the Cdk7-CycH-Mat1 kinase submodule of TFIIH as a stimulatory non-essential regulator of transcriptional elongation and a genespecific essential factor for stable binding of RNAPII at the promoter region and capping. The results of these studies suggest important roles for both MrpL55 and Mat1 in cell cycle progression and their possible interplay at the G2/M stage in undifferentiated cells. The identified function of Mat1 and of TFIIH kinase complex in gene-specific transcriptional repression is challenging for further studies in regard to a possible link to Vig and RISC-mediated transcriptional gene silencing.

Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.

Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

Hematopoietic Stem Cell Development and Transcriptional Regulation

The continuous production of blood cells, a process termed hematopoiesis, is sustained throughout the lifetime of an individual by a relatively small population of cells known as hematopoietic stem cells (HSCs). HSCs are unique cells characterized by their ability to self-renew and give rise to all types of mature blood cells. Given their high proliferative potential, HSCs need to be tightly regulated on the cellular and molecular levels or could otherwise turn malignant. On the other hand, the tight regulatory control of HSC function also translates into difficulties in culturing and expanding HSCs in vitro. In fact, it is currently not possible to maintain or expand HSCs ex vivo without rapid loss of self-renewal. Increased knowledge of the unique features of important HSC niches and of key transcriptional regulatory programs that govern HSC behavior is thus needed. Additional insight in the mechanisms of stem cell formation could enable us to recapitulate the processes of HSC formation and self-renewal/expansion ex vivo with the ultimate goal of creating an unlimited supply of HSCs from e.g. human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPS) to be used in therapy. We thus asked: How are hematopoietic stem cells formed and in what cellular niches does this happen (Papers I, II)? What are the molecular mechanisms that govern hematopoietic stem cell development and differentiation (Papers III, IV)? Importantly, we could show that placenta is a major fetal hematopoietic niche that harbors a large number of HSCs during midgestation (Paper I)(Gekas et al., 2005). In order to address whether the HSCs found in placenta were formed there we utilized the Runx1-LacZ knock-in and Ncx1 knockout mouse models (Paper II). Importantly, we could show that HSCs emerge de novo in the placental vasculature in the absence of circulation (Rhodes et al., 2008). Furthermore, we could identify defined microenvironmental niches within the placenta with distinct roles in hematopoiesis: the large vessels of the chorioallantoic mesenchyme serve as sites of HSC generation whereas the placental labyrinth is a niche supporting HSC expansion (Rhodes et al., 2008). Overall, these studies illustrate the importance of distinct milieus in the emergence and subsequent maturation of HSCs. To ensure proper function of HSCs several regulatory mechanisms are in place. The microenvironment in which HSCs reside provides soluble factors and cell-cell interactions. In the cell-nucleus, these cell-extrinsic cues are interpreted in the context of cell-intrinsic developmental programs which are governed by transcription factors. An essential transcription factor for initiation of hematopoiesis is Scl/Tal1 (stem cell leukemia gene/T-cell acute leukemia gene 1). Loss of Scl results in early embryonic death and total lack of all blood cells, yet deactivation of Scl in the adult does not affect HSC function (Mikkola et al., 2003b. In order to define the temporal window of Scl requirement during fetal hematopoietic development, we deactivated Scl in all hematopoietic lineages shortly after hematopoietic specification in the embryo . Interestingly, maturation, expansion and function of fetal HSCs was unaffected, and, as in the adult, red blood cell and platelet differentiation was impaired (Paper III)(Schlaeger et al., 2005). These findings highlight that, once specified, the hematopoietic fate is stable even in the absence of Scl and is maintained through mechanisms that are distinct from those required for the initial fate choice. As the critical downstream targets of Scl remain unknown, we sought to identify and characterize target genes of Scl (Paper IV). We could identify transcription factor Mef2C (myocyte enhancer factor 2 C) as a novel direct target gene of Scl specifically in the megakaryocyte lineage which largely explains the megakaryocyte defect observed in Scl deficient mice. In addition, we observed an Scl-independent requirement of Mef2C in the B-cell compartment, as loss of Mef2C leads to accelerated B-cell aging (Gekas et al. Submitted). Taken together, these studies identify key extracellular microenvironments and intracellular transcriptional regulators that dictate different stages of HSC development, from emergence to lineage choice to aging.