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The Notch signaling pathway is crucial for mammalian heart development. It controls cell-fate decisions, coordinates patterning processes and regulates proliferation and differentiation. Critical Notch effectors are Hey bHLH transcription factors (TF) that are expressed in atrial (Hey1) and ventricular (Hey2) cardiomyocytes (CM) and in the developing endocardium (Hey1/2/L). The importance of Hey proteins for cardiac development is demonstrated by knockout (KO) mice, which suffer from lethal cardiac defects, such as ventricular septum defects (VSD), valve defects and cardiomyopathy. Despite this clear functional relevance, little is known about Hey downstream targets in the heart and the molecular mechanism by which they are regulated.
Here, I use a cell culture system with inducible Hey1, Hey2 or HeyL expression to study Hey target gene regulation in HEK293 cells, in murine embryonic stem cells (ESC) and in ESC derived CM. In HEK293 cells, I could show that genome wide binding sites largely overlap between all three Hey proteins, but HeyL has many additional binding sites that are not bound by Hey1 or Hey2. Shared binding sites are located close to transcription start sites (TSS) where Hey proteins preferentially bind to canonical E boxes, although more loosely defined modes of binding exist. Additional sites only bound by HeyL are more scattered across the genome. The ability of HeyL to bind these sites depends on the C-terminal part of the protein. Although there are genes which are differently regulated by HeyL, it is unclear whether this regulation results from binding of additional sites by HeyL.
Additionally, Hey target gene regulation was studied in ESC and differentiated CM, which are more relevant for the observed cardiac phenotypes. ESC derived CM contract in culture and are positive for typical cardiac markers by qRT PCR and staining. According to these markers differentiation is unaffected by prolonged Hey1 or Hey2 overexpression. Regulated genes are largely redundant between Hey1 and Hey2. These are mainly other TF involved in e.g. developmental processes, apoptosis, cell migration and cell cycle. Many target genes are cell type specifically regulated causing a shift in Hey repression of genes involved in cell migration in ESC to repression of genes involved in cell cycle in CM.
The number of Hey binding sites is reduced in CM and HEK293 cells compared to ESC, most likely due to more regions of dense chromatin in differentiated cells. Binding sites are enriched at the proximal promoters of down-regulated genes, compared to up-or non-regulated genes. This indicates that up-regulation primarily results from indirect effects, while down-regulation is the direct results of Hey binding to target promoters. The extent of repression generally correlates with the amount of Hey binding and subsequent recruitment of histone deacetylases (Hdac) to target promoters resulting in histone H3 deacetylation.
However, in CM the repressive effect of Hey binding on a subset of genes can be annulled, likely due to binding of cardiac specific activators like Srf, Nkx2-5 and Gata4. These factors seem not to interfere with Hey binding in CM, but they recruit histone acetylases such as p300 that may counteract Hey mediated histone H3 deacetylation. Such a scenario explains differential regulation of Hey target genes between ESC and CM resulting in gene and cell-type specific regulation.
In this work we wanted to investigate the role of NFATc1 in lymphocyte physiology and in pathological conditions (eg. psoriasis). NFATc1 is part of the signal transduction
pathways that regulates B cells activation and function. NFATc1 has different isoforms that are due to different promoters (P1 and P2), polyadenylation and alternative splicing. Moreover, we tried to elucidate the points of interactions between the NFAT and the NF-κB pathways in
activated B-cell fate. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA binding domain. We used mice which over-express a constitutive active version of NFATc1/α in their B cells with -or without- an ablated IRF4. IRF4 inhibits cell cycle progression of germinal center B cell-derived Burkitt’s lymphoma cells and
induces terminal differentiation toward plasma cells. Our experiments showed that a ‘double hit’ in factors affecting B cell activation (NFATc1 in this case) and late B cell Differentiation (IRF4 in this case) alter the development of the B cells, lead to increase in their numbers and increase in stimulation induced proliferation. Therefore, the overall picture indicates a link between these 2 genes and probable carcinogenic alterations that may occur in B cells.
We also show that in splenic B cells, c-Rel (of the NF-κB canonical pathway) Support the induction of NFATc1/αA through BCR signals. We also found evidence that the lack of NFATc1 affects the expression of Rel-B (of the NF-κB non-canonical pathway). These data suggest a tight interplay between NFATc1 and NF-κB in B cells, influencing the competence of B cells and their functions in peripheral tissues.
We also used IMQ-induced psoriasis-like inflammation on mice which either lack NFATc1 from B cell. Psoriasis is a systemic chronic immunological disease characterized
primarily by abnormal accelerated proliferation of the skin keratinocytes. In psoriasis, the precipitating event leads to immune cell activation. Our experiments showed that NFATc1 is needed for the development of psoriasis. It also showed that IL-10 is the link that enables NFAT
from altering the B cell compartment (eg Bregs) in order to affect inflammation. The important role of B cell in psoriasis is supported by the flared up psoriasis-like inflammation in mice that lack B cells. Bregs is a special type of B cells that regulate other B cells and T cells; tuning the immunological response through immunomodulatory cytokines.
Peritonitis is a common disease in man, frequently caused by fungi, such as Candida albicans; however, in seldom cases opportunistic infections with Saccharomyces cerevisiae are described. Resident peritoneal macrophages (prMΦ) are the major group of phagocytic cells in the peritoneum. They express a broad range of surface pattern recognition receptors (PRR) to recognize invaders. Yeast infections are primarily detected by the Dectin-1 receptor, which triggers activation of NFAT and NF-κB pathways.
The transcription of the Nfatc1 gene is directed by the two alternative promoters, inducible P1 and relatively constitutive P2 promoter. While the role of P1-directed NFATc1α-isoforms to promote survival and proliferation of activated lymphocytes is well-established, the relevance of constitutively generated NFATc1β-isoforms, mainly expressed in resting lymphocytes, myeloid and non-lymphoid cells, remains unclear. Moreover, former work at our department indicated different roles for NFATc1α- and NFATc1β-proteins in lymphocytes.
Our data revealed the functional role of NFATc1 in peritoneal resident macrophages. We demonstrated that the expression of NFATc1β is required for a proper immune response of prMΦ during fungal infection-induced acute peritonitis. We identified Ccl2, a major chemokine produced in response to fungal infections by prMΦ, as a novel NFATc1 target gene which is cooperatively regulated through the NFAT- and canonical NF-κB pathways. Consequently, we showed that NFATc1β deficiency in prMΦ results in a decreased infiltration of inflammatory monocytes, leading to a delayed clearance of peritoneal fungal infection.
We could further show that the expression of NFATc1β-isoforms is irrelevant for homeostasis of myeloid and adaptive immune system cells and that NFATc1α- (but not β-) isoforms are required for a normal development of peritoneal B1a cells. In contrast to the situation in myeloid cells, NFATc1β deficiency is compensated by increased expression of NFATc1α-isoforms in lymphoid cells. As a consequence, NFATc1ß is dispensable for activation of the adaptive immune system.
Taken together our results illustrate the redundancy and indispensability of NFATc1-isoforms in the adaptive and innate immune system, indicating a complex regulatory system for Nfatc1 gene expression in different compartments of the immune system and likely beyond that.
Effects of stem cell transcription factor-expressing vaccinia viruses in oncolytic virotherapy
(2012)
Cancer remains the second leading cause of death in the industrialized. The data from many different studies investigating the nature of cancer-initiating cells coined the description ‘cancer stem cells’ and has major implications on conventional cancer therapy. Thus, to improve the outcome of cancer treatment and to lower negative side effects, the development of novel therapeutic regimens is indispensable. It has been demonstrated in many preclinical studies that oncolytic virotherapy using vaccinia virus may provide a powerful and well-tolerable new tool in cancer therapy which is currently investigated in several clinical trials (Phase I & II) as stand-alone treatment or in combination with conventional cancer therapy. Cancer-initiating cells and stem cells share a variety of characteristics like the ability to self-renew, differentiation potential, quiescence, drug and radiation resistance, activation and inhibition of similar signaling pathways as well as expression of cell surface markers and stem cell-related genes. In this work, two new recombinant vaccinia viruses expressing the transcription factors Nanog (GLV-1h205) and Oct4 (GLV-1h208) were engineered to provide deeper insight of these stem cell master regulators in their significance of cancer-initiation and their impact on oncolytic virotherapy. Both viruses were analyzed for their replication potential in A549 and PC-3 human cancer cells. Marker gene expression was assessed by RT-PCR, SDS-PAGE and Western blotting, ELISA or immunocytochemistry.Furthermore, the effect of GLV-1h205 infection on the cell cycle in A549 cells was analyzed. Next, the effects of virus-mediated expression of stem cell transcription factors on therapeutic efficacy and survival rates in A549 xenograft mouse models was analyzed. A non-functional Nanog mutant-expressing virus strain (GLV-1h321) was engineered to analyze whether the observed therapeutic benefits were promoter- or payload-driven. Furthermore, this study analyzed the potential of GLV-1h68 to infect, replicate in, and lyse colorectal cancer cell lines to study whether oncolytic vaccinia viruses can be potential new and less invasive treatment regimens for late stage colorectal cancer. Marker gene expression was assessed by fluorescence microscopy and FACS. The transcription factor Klf4 is highly expressed in quiescent, terminally differentiated cells in the colonic epithelium whereas it is dramatically downregulated in colon cancers. Klf4 expression leads to cell growth arrest and inhibits Wnt signaling by binding to beta-catenin. To further improve the treatment of colorectal cancers, new recombinant vaccinia viruses (GLV-1h290-292) mediating the expression of differing amounts of the tumor suppressor Klf4 by using different promoter strengths were engineered. Initial characterization of recombinant vaccinia viruses expressing Klf4 by replication assay, cell viability assay, SDS-PAGE and Western blotting, immuncytochemistry and analysis of protein functionality by qPCR and ELISA analysis for cellular beta-catenin expression, demonstrated promoter strength-dependent expression of and impact of Klf4. To further boost the effects of tumor suppressor Klf4, a vaccinia virus strain expressing Klf4 with a C-terminal fusion of the TAT transduction domain (GLV-1h391) was engineered. Treatment of HT-29 non-responder tumors in vivo with GLV-1h291 and GLV-1h391 led to significant tumor growth inhibition and improved overall survival compared to GLV-1h68. This makes the Klf4-TAT expressing GLV-1h391 a promising candidate for the treatment of colorectal cancer in man.
The transcription factor NFATc1 has been shown to regulate the activation and differentiation of T-cells and B-cells, of DCs and megakaryocytes. Dysregulation of NFAT signaling was shown to be associated with the generation of autoimmune diseases, malignant transformation and the development of cancer [71]. The primary goal of this work was to gain insights on Nfatc1 induction and regulation in lymphocytes and to find new direct NFATc1 target genes. Three new BAC -transgenic reporter mouse strains (tgNfatc1/Egfp, tgNfatc1/DE1 and tgNfatc1/DE2) were applied to analyze Nfatc1 induction and regulation in primary murine B- and T-cells. As a result, we were able to show the persistent requirement of immunoreceptor-signaling for constant Nfatc1 induction, particularly, for NFATc1/αA expression. Furthermore, we showed that NF-κB inducing agents, such as LPS, CpG or CD40 receptor engagement, in combination with primary receptor-signals, positively contributed to Nfact1 induction in B-cells [137]. We sought to establish a new system which could help to identify direct NFATc1 target genes by means of ChIP and NGS in genom-wide approaches. We were able to successfully generate a new BAC-transgene encoding a biotinylatable short isoform of NFATc1, which is currently injected into mice oocyte at the TFM in Mainz. In addition, in vivo biotinylatable NFATc1–isoforms were cloned and stably expressed in the murine B-cell lymphoma line WEHI-231. The successful use of these cells stably overexpressing either the short NFATc1/αA or the long NFATc1/βC isoform along with the bacterial BirA biotin ligase was confirmed by intracellular stainings, FACS analysis, confocal microscopy and protein IP. By NGS, we detected 2185 genes which are specifically controlled by NFATc1/αA, and 1306 genes which are exclusively controlled by NFATc1/βC. This shows that the Nfatc1 locus encodes “two genes” which exhibit alternate, in part opposite functions. Studies on the induction of apoptosis and cell-death revealed opposed roles for the highly inducible short isoform NFATc1/αA and the constantly expressed long isoform NFATc1/βC. These findings were confirmed by whole transcriptome-sequencing performed with cells overexpressing NFATc1/αA and NFATc1/βC. Several thousand genes were found to be significantly altered in their expression profile, preferentially genes involved in apoptosis and PCD for NFATc1/βC or genes involved in transcriptional regulation and cell-cycle processes for NFATc1/αA. In addition we were able to perform ChIP-seq for NFATc1/αA and NFATc1/βC in an ab-independent approach. We found potential new target-sites, but further studies will have to address this ambitious goal in the future. In individual ChIP assays, we showed direct binding of NFATc1/αA and NFATc1/βC to the Prdm1 and Aicda promoter regions which are individually controlled by the NFATc1 isoforms.
Pluripotency describes the ability of stem cells to form every cell type of the body.. Pluripotent stem cells are e.g. embryonic stem cells (ESCs), but also the so called induced pluripotent stem cells (IPS cells), that are generated by reprogramming differentiated somatic cells into a pluripotent state. Furthermore, it has been shown that spermatogonia (SG) derived from adult testes of mouse or human are pluripotent. Because of their ability to differentiate into every somatic cell type, pluripotent stem cells have a unique status in research and regenerative medicine. For the latter, they offer a valuable opportunity to replace destroyed tissues or organs. For basic research, stem cells represent a useful system to study differentiation or developmental processes that are difficult to access in the physiological situation e.g. during embryogenesis. Both applications, however, require methods that allow efficient and directed differentiation of stem cells into defined specialized cell types. This study first aims to investigate the differentiation potential of SG derived from the teleost fish medaka (Oryzias latipes). My results demonstrate that medaka SG are able to form different somatic cell types, namely adipocytes, melanocytes, osteoblasts, and neurons. This indicates that medake SG have retained a broad differentiation potential suggesting that pluripotency is not restricted to mouse and human SG but might be conserved among vertebrates. Next, I wanted to establish a differentiation method that is solely based on ectopic expression of genes known to be essential for the formation of certain somatic cell types – so called master regulators (MRs). My findings show that ectopic expression of the melanocyte-specific transcription factor mitf-m that has previously been shown to induce differentiation of medaka ESCs into pigment cells resulted in the formation of the same cell type in medaka SG. This approach could be used to generate other somatic cell types. Thus, ectopic expression of the MRs cbfa1 and mash1 in MF-SG was sufficient to induce differentiation into osteoblasts and neurons, respectively. Interestingly, these differentiation processes included the activation of genes that are expressed earlier during embryogenesis than the differentiation-inducing MR. Furthermore, my findings show that the approach of MR-induced differentiation can be transferred to mammalian stem cell systems. Ectopic expression of the neural transcription factor ngn2 was sufficient to induce efficient and rapid differentiation of neurons in mouse ESCs. This differentiation process also included the induction of genes that in vivo are activated at earlier stages that ngn2. By generating a transgenic cell line allowing induction of ectopic ngn2 expression, it was possible to obtain a relatively pure culture of functional neurons. Ngn2-induced differentiation did not require any additional signals and occurred even under pluripotency promoting conditions. Moreover, ectopic expression of ngn2 did also induce the formation of cells with neuronal morphology in IPS cells indicating that MR-induced differentiation is operative in different stem cell types. Furthermore, protein transduction of Ngn2 into mouse ESCs also resulted in a neuronal differentiation process up to the appearance of neural precursor cells. Last, my results show that MR-induced differentiation can also be used to generate other cell types than neurons from mouse ESCs. Myoblasts and macrophage-like cells were generated by ectopic expression of the MRs myoD and cebpa, respectively. Using transgenic cell lines enabling induction of MR expression it was possible to obtain mixed cultures with two different differentiation processes occurring in parallel. Altogether this study shows that ectopic expression of single genes is sufficient to induce directed differentiation of stem cells into defined cell types. The feasibility of this approach was demonstrated for different MRs and consequently different somatic cell types. Furthermore, MR induced differentiation was operative in different stem cell types from fish and mouse. Thus, one can conclude that certain genes are able to define cell fates in in vitro stem cell systems and that this cell fate defining potential appears to be a conserved feature in vertebrates. These findings therefore provide new insights in the role of MRs in cell commitment and differentiation processes. Furthermore, this study presents a new method to induce directed differentiation of stem cells that offers several advantages regarding efficiency, rapidness, and reproducibility. MR-induced differentiation therefore represents a promising tool for both stem cell research and regenerative medicine.
SUMMARY GABP is a heterodymeric member of Ets-family transcription factors. It consists of two subunits – GABPa which contains DNA binding domain and GABPb, which provides transcriptional activation domain and nuclear localization signal. GABPa/b complex is essential for transcriptional activation of multiple lineage-restricted and housekeeping genes, several viral genes, and in some cases might function as transcriptional repressor. Large variety of data indicates involvement of GABP in the complex regulation of cell growth, specified by quiescence, stimulation/proliferation, apoptosis and senescence. Expression level of GABPa subunit is rapidly increased when resting cells enter S-phase, and GABPa/b complex is critical to promote the continuity of the cell cycle. Conditional inactivation of GABPa expression in mouse embryonic fibroblasts results in a complete block of proliferation and acquisition of senescence-like phenotype. However, the influence of GABP on the other cell growth determinant – the apoptosis – remains largely obscure. Therefore we aimed to investigate the influence of GABPa/b expression level on the cell growth in vitro. Using siRNA approach we achieved efficient but only transient down-regulation of GABPa expression which precluded further cell growth studies. Persistent increase of the expression of GABPb subunit only resulted in a positive effect on the cell growth speed. Simultaneous conditional overexpression of both GABPa and GABPb subunits though, strongly reduced the growth of the affected cell cultures in reversible and in expression level dependent manner. Interestingly, GABPa/b overexpressing cells did show neither cell cycle arrest nor massive induction of apoptosis. However, more detailed analyses revealed that dampened apoptotic processes were taking place in GABPa/b−overexpressing cells, starting with a prominent activation of caspase-12. Interestingly, activation of downstream effector caspases was rather suppressed explaining a weak increase of apoptotic cells in GABPa/b overexpressing cultures. This effect suggests that the activation of caspase-12 by elevated amounts of exogenous GABPa/b reflects the normal physiological mechanism of caspase-12 regulation.
The yeast Candida albicans is a member of the normal microflora on the mucosal surfaces of the gastrointestinal and urogenital tract in healthy persons. However, it is an opportunistic pathogen that can cause a range of infections from superficial to disseminated, in response to perturbation of the normal microflora or alterations in the host immunity. C. albicans exhibits a variety of characteristics such as adhesion, morphogenetic switching and secreted aspartic protease production that contribute to its virulence. Expression of many of these virulence factors is controlled by the availability of essential element, nitrogen. C. albicans undergoes morphogenetic transition to form filaments under nitrogen starvation conditions and this switch is controlled by the ammonium permease Mep2p. However, little is known about how this signaling function of Mep2p is regulated. Mutational analysis of Mep2p was carried out to identify the residues that confer signaling activity to this permease. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is dispensable for ammonium transport but essential for the signaling activity of Mep2p. In this work, progressive C-terminal truncations analysis demonstrated that a MEP2DC433 allele was still able to induce filamentation while nitrogen starvation-induced filamentous growth was abolished in cells expressing a MEP2DC432 allele. Therefore, tyrosine at position 433 (Y433) is the last amino acid in Mep2p that is essential for signaling. To gain insights into how the signaling activity of Mep2p is regulated by ammonium availability and transport, conserved residues that have been implicated in ammonium binding or uptake were mutated. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is predicted to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filamentation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate along with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filamentation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filamentation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. An important aspect in the ability of Mep2p to stimulate filamentation in response to nitrogen limitation is its high expression levels. The cis-acting sequences and trans-acting regulators that mediate MEP2 induction in response to nitrogen limitation were identified. Promoter analysis revealed that two putative binding sites for GATA transcription factors have a central role in MEP2 expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To elucidate the roles of the GATA transcription factors GLN3 and GAT1 in regulating MEP2 expression, mutants lacking one or both of these transcription factors were constructed. Mep2p expression was strongly reduced in gln3D and gat1D single mutants and virtually abolished in gln3D gat1D double mutants. Deletion of GLN3 strongly inhibited filamentous growth under limiting nitrogen conditions, which could be rescued by constitutive expression of MEP2 from the ADH1 promoter. In contrast, inactivation of GAT1 had no effect on filamentation. Surprisingly, filamentation became partially independent of the presence of a functional MEP2 gene in the gat1D mutants, indicating that the loss of GAT1 function results in the activation of other pathways that induce filamentous growth. These findings demonstrated that the GATA transcription factors Gln3p and Gat1p control expression of the MEP2 ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced filamentous growth in C. albicans. C. albicans mutants lacking both the GATA transcription factors Gln3p and Gat1p were unable to grow in a medium containing an alternative nitrogen source, bovine serum albumin (BSA) as the sole nitrogen source. The ability to utilize proteins as sole source of nitrogen for growth of C. albicans is conferred by the secreted aspartic protease Sap2p, which degrades the proteins, and oligopeptide transporters that mediate uptake of the proteolytic products into cell. The growth defect of gln3D gat1D mutants was mainly caused by their inability to express the SAP2 gene, as SAP2 expression from the constitutive ADH1 promoter restored the ability of the mutants to grow on BSA. Expression of STP1, which encodes a transcription factor that is required for SAP2 induction in the presence of proteins, was regulated by Gln3p and Gat1p. Forced expression of STP1 from a tetracycline-inducible promoter bypassed the requirement of the GATA transcription factors for growth of C. albicans on proteins. When preferred nitrogen sources are available, SAP2 is repressed and this nitrogen catabolite repression of SAP2 was correlated with downregulation of STP1 under these conditions. Tetracycline-induced STP1 expression abolished nitrogen catabolite repression of SAP2, demonstrating that regulation of STP1 expression levels by the GATA transcription factors is a key aspect of both positive and negative regulation of SAP2 expression. Therefore, by using a regulatory cascade in which expression of the specific transcription factor Stp1p is controlled by the general regulators Gln3p and Gat1p, C. albicans places SAP2 expression under nitrogen control and ensures proper expression of this virulence determinant. In summary, the present study illustrated how GATA factors, Gln3p and Gat1p, play partially overlapping, but distinct roles, in mediating the appropriate responses of C. albicans to the availability of different nitrogen sources. These responses are also determinants of pathogenicity of the fungus. The relative contributions of Gln3p and Gat1p vary with their target genes and the availability of nitrogen source. Overall, these findings provide us with a better understanding of the molecular basis of some of the important processes that help in adaptation of C. albicans to various environmental conditions. The yeast Candida albicans is a member of the normal microflora on the mucosal surfaces of the gastrointestinal and urogenital tract in healthy persons. However, it is an opportunistic pathogen that can cause a range of infections from superficial to disseminated, in response to perturbation of the normal microflora or alterations in the host immunity. C. albicans exhibits a variety of characteristics such as adhesion, morphogenetic switching and secreted aspartic protease production that contribute to its virulence. Expression of many of these virulence factors is controlled by the availability of essential element, nitrogen. C. albicans undergoes morphogenetic transition to form filaments under nitrogen starvation conditions and this switch is controlled by the ammonium permease Mep2p. However, little is known about how this signaling function of Mep2p is regulated. Mutational analysis of Mep2p was carried out to identify the residues that confer signaling activity to this permease. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is dispensable for ammonium transport but essential for the signaling activity of Mep2p. In this work, progressive C-terminal truncations analysis demonstrated that a MEP2DC433 allele was still able to induce filamentation while nitrogen starvation-induced filamentous growth was abolished in cells expressing a MEP2DC432 allele. Therefore, tyrosine at position 433 (Y433) is the last amino acid in Mep2p that is essential for signaling. To gain insights into how the signaling activity of Mep2p is regulated by ammonium availability and transport, conserved residues that have been implicated in ammonium binding or uptake were mutated. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is predicted to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filamentation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate along with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filamentation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filamentation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. An important aspect in the ability of Mep2p to stimulate filamentation in response to nitrogen limitation is its high expression levels. The cis-acting sequences and trans-acting regulators that mediate MEP2 induction in response to nitrogen limitation were identified. Promoter analysis revealed that two putative binding sites for GATA transcription factors have a central role in MEP2 expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To elucidate the roles of the GATA transcription factors GLN3 and GAT1 in regulating MEP2 expression, mutants lacking one or both of these transcription factors were constructed. Mep2p expression was strongly reduced in gln3D and gat1D single mutants and virtually abolished in gln3D gat1D double mutants. Deletion of GLN3 strongly inhibited filamentous growth under limiting nitrogen conditions, which could be rescued by constitutive expression of MEP2 from the ADH1 promoter. In contrast, inactivation of GAT1 had no effect on filamentation. Surprisingly, filamentation became partially independent of the presence of a functional MEP2 gene in the gat1D mutants, indicating that the loss of GAT1 function results in the activation of other pathways that induce filamentous growth. These findings demonstrated that the GATA transcription factors Gln3p and Gat1p control expression of the MEP2 ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced filamentous growth in C. albicans. C. albicans mutants lacking both the GATA transcription factors Gln3p and Gat1p were unable to grow in a medium containing an alternative nitrogen source, bovine serum albumin (BSA) as the sole nitrogen source. The ability to utilize proteins as sole source of nitrogen for growth of C. albicans is conferred by the secreted aspartic protease Sap2p, which degrades the proteins, and oligopeptide transporters that mediate uptake of the proteolytic products into cell. The growth defect of gln3D gat1D mutants was mainly caused by their inability to express the SAP2 gene, as SAP2 expression from the constitutive ADH1 promoter restored the ability of the mutants to grow on BSA. Expression of STP1, which encodes a transcription factor that is required for SAP2 induction in the presence of proteins, was regulated by Gln3p and Gat1p. Forced expression of STP1 from a tetracycline-inducible promoter bypassed the requirement of the GATA transcription factors for growth of C. albicans on proteins. When preferred nitrogen sources are available, SAP2 is repressed and this nitrogen catabolite repression of SAP2 was correlated with downregulation of STP1 under these conditions. Tetracycline-induced STP1 expression abolished nitrogen catabolite repression of SAP2, demonstrating that regulation of STP1 expression levels by the GATA transcription factors is a key aspect of both positive and negative regulation of SAP2 expression. Therefore, by using a regulatory cascade in which expression of the specific transcription factor Stp1p is controlled by the general regulators Gln3p and Gat1p, C. albicans places SAP2 expression under nitrogen control and ensures proper expression of this virulence determinant. In summary, the present study illustrated how GATA factors, Gln3p and Gat1p, play partially overlapping, but distinct roles, in mediating the appropriate responses of C. albicans to the availability of different nitrogen sources. These responses are also determinants of pathogenicity of the fungus. The relative contributions of Gln3p and Gat1p vary with their target genes and the availability of nitrogen source. Overall, these findings provide us with a better understanding of the molecular basis of some of the important processes that help in adaptation of C. albicans to various environmental conditions.
Regulation of B lymphocyte terminal differentiation and death by the transcription factor Blimp-1
(2005)
B lymphocyte induced maturation protein-1 (Blimp-1) and X-box-binding protein-1 (XBP-1) are indispensible transcription factors required for B lymphocyte terminal differentiation into Ig secreting plasma cells. Occurrence of an unfolded protein response (UPR) and XBP-1 splicing, due to elevated Ig levels, are critical events during plasma cell generation. However, the upstream molecule sufficient to trigger these events remain elusive. Because ectopic expression of Blimp-1 in B cells is sufficient to generate plasma cells, it is plausible that Blimp-1 might be the upstream molecule, sufficient for the induction of UPR and XBP-1 splicing. The results from the current study indicate that ectopic expression of Blimp-1 or its N-terminal domain, in B cells, is sufficient to induce XBP-1 splicing, UPR and Ig (immunoglobulin) secretion. Further more Blimp-1 is able to directly repress the antiapoptotic gene A1, by binding to specific DNA elements in A1 promoter. This repression of A1 by Blimp-1 seems to be an important prerequisite for Plasma cell differentiation because ectopic expression of A1 in primary B cells resulted in reduced immunoglobulin secretion.
We examined the regulation of NFATc1 in different lymphomas and observed an inversed correlation between the methylation status and expression of NFATc1. Our data demonstrate that aberrant DNA methylation associated with chromatin remodeling within nfatc1 locus is a major mechanism for the repression of NFATc1 expression, suggesting that the DNA methylation-mediated transcriptional silencing of NFATc1 may be a critical event in the tumorogenesis of ALCLs and cHLs. Furthermore, the DNA methylation of human nfatc1 promoter region could be used as a novel biomarker of tumor progression. Our results indicate a close link between the loss of immunoreceptor signaling and NFATc1 expression in human lymphomas. For both ALCLs and cHLs, defects in immunoreceptor signaling have been described which result in a loss of receptor-mediated gene expression programs (Schwering et al., 2003; Bonzheim et al., 2004; Marafioti et al., 2004). In T cells, one indicator gene of these programs appears to be the nfatc1 gene whose expression is controlled by TCR signals (Chuvpilo et al., 2002a). In contrast, in T cells NFATc1 expression is unaffected by TCR signals, and NFATc2 was found to be expressed at normal levels in ALCLs and cHLs (L.K., unpubl. data). Moreover, the activity of NF-kappaB factors which can bind to certain NFAT binding sites and share a distantly-related DNA binding domain with NFATs is strongly elevated in cHL cells (Bargou et al., 1997; Hinz et al., 2001; Hinz et al., 2002) suggesting that NFATs and NF-kappaBs exert very different effects on generation and maintenance of Hodgkin’s lymhomas. However, it should be mentioned that in Burkitt’s and further B cell lymphomas in which NFATc1 proteins are strongly expressed and controlled by receptor signals (Kondo et al., 2003), they could exert a promoting function in tumor development. The genes of p53 family members p63 and p73 are prominent examples for mammalian genes whose products can act both as oncoproteins and tumor suppressor genes (Hibi et al., 2000; Stiewe and Putzer, 2002), and it is likely that more genes exist which encode both tumor suppressors and oncoproteins. It remains to be shown whether the nfatc1 gene is one of them.