Differences In Promoter Activity Research Articles

A haplotype variant of the human chromogranin A gene (CHGA) promoter increases CHGA expression and the risk for cardiometabolic disorders

The acidic glycoprotein chromogranin A (CHGA) is co-stored/co-secreted with catecholamines and crucial for secretory vesicle biogenesis in neuronal/neuroendocrine cells. CHGA is dysregulated in several cardiovascular diseases, but the underlying mechanisms are not well established. Here, we sought to identify common polymorphisms in the CHGA promoter and to explore the mechanistic basis of their plausible contribution to regulating CHGA protein levels in circulation. Resequencing of the CHGA promoter in an Indian population (n = 769) yielded nine single-nucleotide polymorphisms (SNPs): G-1106A, A-1018T, T-1014C, T-988G, G-513A, G-462A, T-415C, C-89A, and C-57T. Linkage disequilibrium (LD) analysis indicated strong LD among SNPs at the -1014, -988, -462, and -89 bp positions and between the -1018 and -57 bp positions. Haplotype analysis predicted five major promoter haplotypes that displayed differential promoter activities in neuronal cells; specifically, haplotype 2 (containing variant T alleles at -1018 and -57 bp) exhibited the highest promoter activity. Systematic computational and experimental analyses revealed that transcription factor c-Rel has a role in activating the CHGA promoter haplotype 2 under basal and pathophysiological conditions (viz. inflammation and hypoxia). Consistent with the higher in vitro CHGA promoter activity of haplotype 2, individuals carrying this haplotype had higher plasma CHGA levels, plasma glucose levels, diastolic blood pressure, and body mass index. In conclusion, these results suggest a functional role of the CHGA promoter haplotype 2 (occurring in a large proportion of the world population) in enhancing CHGA expression in haplotype 2 carriers who may be at higher risk for cardiovascular/metabolic disorders.

Cell-type specific differences in promoter activity of the ALS-linked C9orf72 mouse ortholog

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Both loss-of-function and gain-of-function mechanisms have been proposed to underlie this disease, but the pathogenic pathways are not fully understood. To better understand the involvement of different cell types in the pathogenesis of ALS, we systematically analyzed the distribution of promoter activity of the mouse ortholog of C9orf72 in the central nervous system. We demonstrate that C9orf72 promoter activity is widespread in both excitatory and inhibitory neurons as well as in oligodendrocytes and oligodendrocyte precursor cells. In contrast, few microglia and astrocytes exhibit detectable C9orf72 promoter activity. Although at a gross level, the distribution of C9orf72 promoter activity largely follows overall cellular density, we found that it is selectively enriched in subsets of neurons and glial cells that degenerate in ALS. Specifically, we show that C9orf72 promoter activity is enriched in corticospinal and spinal motor neurons as well as in oligodendrocytes in brain regions that are affected in ALS. These results suggest that cell autonomous changes in both neurons and glia may contribute to C9orf72-mediated disease, as has been shown for mutations in superoxide dismutase-1 (SOD1).

Monitoring Promoter Activity by Flow Cytometry.

Genetic reporters have become invaluable tools for indirectly monitoring promoter activities. The quantitative measurement of promoter activities using reporter gene systems is fundamental for pharmaceutical, biomedical, and molecular biology research. Genetic reporters are used not only for measuring promoter activities but also for understanding the mechanisms controlling gene transcription and in theidentification, and characterization of cis-acting regulatory elements. Fluorescent reporter proteins including enhanced green fluorescent protein (EGFP ) are reliable for monitoring quantitative underlying differences in promoter activities. The emitted fluorescence intensity of the expressed reporter is measured at the single-cell level by flow cytometry and represents a readout for the promoter activities. In this chapter, the protocol for measurement and analyzing of transfected cells expressing the reporter gene EGFP is thoroughly described and fully illustrated.

Existence of Two Distinct Infectious Endogenous Retroviruses in Domestic Cats and Their Different Strategies for Adaptation to Transcriptional Regulation.

Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells. Previous work identified one of the youngest feline ERV groups, ERV-DC, and reported that two ERV-DC loci, ERV-DC10 and ERV-DC18 (ERV-DC10/DC18), can replicate in cultured cells. Here, we identified another replication-competent provirus, ERV-DC14, on chromosome C1q32. ERV-DC14 differs from ERV-DC10/DC18 in its phylogeny, receptor usage, and, most notably, transcriptional activities; although ERV-DC14 can replicate in cultured cells, it cannot establish a persistent infection owing to its low transcriptional activity. Furthermore, we examined ERV-DC transcription and its regulation in feline tissues. Quantitative reverse transcription-PCR (RT-PCR) detected extremely low ERV-DC10 expression levels in feline tissues, and bisulfite sequencing showed that 5' long terminal repeats (LTRs) of ERV-DC10/DC18 are significantly hypermethylated in feline blood cells. Reporter assays found that the 5'-LTR promoter activities of ERV-DC10/DC18 are high, whereas that of ERV-DC14 is low. This difference in promoter activity is due to a single substitution from A to T in the LTR, and reverse mutation at this nucleotide in ERV-DC14 enhanced its replication and enabled it to persistently infect cultured cells. Therefore, ERV-DC LTRs can be divided into two types based on this nucleotide, the A type or T type, which have strong or attenuated promoter activity, respectively. Notably, ERV-DCs with T-type LTRs, such as ERV-DC14, have expanded in the cat genome significantly more than A-type ERV-DCs, despite their low promoter activities. Our results provide insights into how the host controls potentially infectious ERVs and, conversely, how ERVs adapt to and invade the host genome. The domestic cat genome contains many endogenous retroviruses, including ERV-DCs. These ERV-DCs have been acquired through germ cell infections with exogenous retroviruses. Some of these ERV-DCs are still capable of producing infectious virions. Hosts must tightly control these ERVs because replication-competent viruses in the genome pose a risk to the host. Here, we investigated how ERV-DCs are adapted by their hosts. Replication-competent viruses with strong promoter activity, such as ERV-DC10 and ERV-DC18, were suppressed by promoter methylation in LTRs. On the other hand, replication-competent viruses with weak promoter activity, such as ERV-DC14, seemed to escape strict control via promoter methylation by the host. Interestingly, ERV-DCs with weak promoter activity, such as ERV-DC14, have expanded in the cat genome significantly more than ERV-DCs with strong promoter activity. Our results improve the understanding of the host-virus conflict and how ERVs adapt in their hosts over time.

Abstract B12: Polymorphisms in the Arntl2 promoter affect metastatic susceptibility in breast cancer

Abstract Metastasis accounts for the majority of cancer-related deaths. Although exact mechanisms are unknown, studies in our laboratory have demonstrated that germline polymorphisms could alter tumorigenesis and metastasis. Our lab developed a mouse genetics approach to investigate the effect of inherited single nucleotide polymorphisms (SNPs) on metastasis by crossing inbred mice of various backgrounds and subsequent mapping of quantitative trait loci (QTL) responsible for the phenotype. Using this approach, we crossed a highly metastatic (FVB) and low metastatic (MOLF) mouse strain and identified Arntl2, a circadian rhythm transcription factor, as a gene with differential expression that predicted distant metastasis-free survival in breast cancer patients. shRNA-mediated reduction in Arntl2 expression suppressed lung metastases, establishing Arntl2 as a metastasis modifier. Initial analysis revealed no polymorphisms in coding regions that could account for the differential expression in FVB and MOLF. Therefore, we performed whole genome sequencing of MOLF to identify SNP differences between MOLF and FVB. Overlapping this analysis with DNAse hypersensitivity sites in mouse mammary tumor cell lines revealed 13 polymorphisms in the predicted promoter region of Arntl2. FVB and MOLF promoters exhibited differential promoter activity in vitro, suggesting that these SNPs could explain the difference in Arntl2 expression between these two strains. In order to validate the causative role of the SNPs in vivo, metastatic cell lines were engineered using CRISPR technology to specifically replace the FVB with the MOLF promoter. Substitution of this promoter reduced Arntl2 transcript levels and subsequently decreased pulmonary metastases, as expected. Since Arntl2 is a transcription factor, current studies are focused on identifying Arntl2-regulated genes to investigate the pathways that are involved in modulating metastasis. This study will have important implications regarding the function of circadian rhythm in cancer progression and provide a potential molecular mechanism to explain the increased risk of breast cancers in nightshift workers. Furthermore, to the best of our knowledge this would be the first example of genome editing to validate polymorphisms in transcriptional control elements associated with inherited susceptibility for metastasis. Citation Format: Ngoc-Han Ha, Kent W. Hunter. Polymorphisms in the Arntl2 promoter affect metastatic susceptibility in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B12.

A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression

Single nucleotide polymorphisms (SNPs) within the MIR137 gene locus have been shown to confer risk for schizophrenia through genome-wide association studies (GWAS). The expression levels of microRNA-137 (miR-137) and its validated gene targets have also been shown to be disrupted in several neuropsychiatric conditions, including schizophrenia. Regulation of miR-137 expression is thus imperative for normal neuronal functioning. We previously characterized an internal promoter domain within the MIR137 gene that contained a variable number tandem repeat (VNTR) polymorphism and could alter the in vitro levels of miR-137 in a stimulus-induced and allele-specific manner. We now demonstrate that haplotype tagging-SNP analysis linked the rs1625579 GWAS SNP for schizophrenia to this internal MIR137 promoter through a proxy SNP rs2660304 located at this domain. We postulated that the rs2660304 promoter SNP may act as predisposing factor for schizophrenia through altering the levels of miR-137 expression in a genotype-dependent manner. Reporter gene analysis of the internal MIR137 promoter containing the common VNTR variant demonstrated genotype-dependent differences in promoter activity with respect to rs2660304. In line with previous reports, the major allele of the rs2660304 proxy SNP, which has previously been linked with schizophrenia risk through genetic association, resulted in downregulation of reporter gene expression in a tissue culture model. The genetic influence of the rs2660304 proxy SNP on the transcriptional activity of the internal MIR137 promoter, and thus the levels of miR-137 expression, therefore offers a distinct regulatory mechanism to explain the functional significance of the rs1625579 GWAS SNP for schizophrenia risk.

Expression of Rice CYP450-Like Gene (Os08g01480) in Arabidopsis Modulates Regulatory Network Leading to Heavy Metal and Other Abiotic Stress Tolerance.

Heavy metal (HM) toxicity has become a grave problem in the world since it leads to hazardous effects on living organisms. Transcriptomic/proteomic studies in plants have identified a large number of metal-responsive gene families. Of these, cytochrome-P450 (CYPs) family members are composed of enzymes carrying out detoxification of exogenous molecules. Here, we report a CYP-like protein encoded by Os08g01480 locus in rice that helps the plant to combat HM and other abiotic stresses. To functionally characterize CYP-like gene, cDNA and promoter were isolated from rice to develop Arabidopsis transgenic lines. Heterologous expression of Os08g01480 in Arabidopsis provided significant tolerance towards abiotic stresses. In silico analysis reveals that Os08g01480 might help plants to combat environmental stress via modulating auxin metabolism. Transgenic lines expressing reporter gene under control of Os08g01480 promoter demonstrated differential promoter activity in different tissues during environmental stresses. These studies indicated that differential expression of Os08g01480 might be modulating response of plants towards environmental stresses as well as in different developmental stages.

Differential promoter activity by nucleotide substitution at a type 2 diabetes genome-wide association study signal upstream of the wolframin gene.

Functional knowledge of most genetic variants identified from genome-wide association studies (GWAS) for type 2 diabetes (T2D) is limited. A recent T2D GWAS revealed an association signal (rs4689388) upstream of the gene encoding Wolfram syndrome 1 (WFS1) whose intrinsic nucleotide variants had been previously associated with T2D in several candidate gene analyses. The aim of the present study was to identify functional variants of the GWAS signal. Promoter activity of luciferase reporter constructs was compared with haplotypes including variants composing a linkage disequilibrium block in the vicinity of rs4689388 in HEK293 and HepG2 cells. Promoter activity was highest with the most frequent haplotype (H1; ATCGT) and lowest with second most frequent haplotype (H2; GATCG), whose nucleotide alleles were all complementary to those of H1. Further analysis with artificial haplotypes revealed differential transcriptional activity by nucleotide substitution of rs4320200, rs13107806, or rs13127445 (P < 0.05). This concurred with changes in predicted transcription factor binding site by their allele substitutions. The previously reported GWAS signal for T2D may be identified by the differential promoter activity of rs4320200, rs13107806, and rs13127445 in the promoter of WFS1 by nucleotide substitution.

Promoter-Autonomous Functioning in a Controlled Environment using Single Molecule FISH.

Transcription is a highly regulated biological process, initiated through the assembly of complexes at the promoter that contain both the general transcriptional machinery and promoter-specific factors. Despite the abundance of studies focusing on transcription, certain questions have remained unanswered. It is not clear how the transcriptional profile of a promoter is affected by genomic context. Also, there is no single cell method to directly compare transcriptional profiles independent of gene length and sequence. In this work, we employ a single genetic site for isolating the transcriptional kinetics of yeast promoters. Utilizing single molecule FISH, we directly compare the transcriptional activity of different promoters, considering both synthesis and cell-to-cell variability. With this approach, we provide evidence suggesting promoters autonomously encode their associated transcriptional profiles, independent of genomic locus, gene length and gene sequence.

Transcriptional Downregulation by Nucleotide Substitution with the Minor Allele of rs3760776 Located in the Promoter of FUT6 Gene.

We examined the promoter activity of an association signal in an upstream region of the gene encoding fucosyltransferae 6 (FUT6) identified from a recent genomewide association study for the N-glycan level. The luciferase assay using reporter constructs with T and C alleles at rs3760776 revealed differential promoter activity. The amount of luciferin expressed with the C allele corresponded to that without the reporter construct (P > 0.05). On the other hand, the expression was dramatically reduced with the T allele (P < 0.05). The difference in transcriptional activity between the two alleles was confirmed by an electrophoretic mobility shift assay. It demonstrated that the promoter with a T allele had a stronger binding affinity to nuclear factors than that with the C allele. We concluded that the T allele of rs3760776 might repress the transcription of the FUT6 gene. Further studies are warranted to understand its underlying mechanism and its influence on susceptibility to potential diseases.

Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter.

Profile of differential promoter activity by nucleotide substitution at GWAS signals for multiple sclerosis.

This experimental study was conducted with completely randomized design. Genome-wide association studies (GWAS) have revealed a large number of genetic associations of nucleotide sequence variants with susceptibility to multiple sclerosis (MS). Nevertheless, studies to identify the functional relevance of these variants lag far behind identification of the GWAS signals. Expression quantitative trait loci (eQTLs) analysis and promoter activity analysis with the variants filtered by GWAS were conducted to identify their functional alleles and haplotypes. The promoter activity was assayed with reporter constructs containing variants at 8 MS GWAS signals resulted from 18 GWAS. The promoter activity differed by alternative sequence variants at upstream regions of the CYP24A1, CYP27B1, SYK, RAD21L1, PVR, ODF3B, and RGS14 genes (P<0.05). The transcriptional regulations of sequence variants were also found by identifications of eQTLs for their corresponding genes with lymphoblastoid cells in the current study (SYK, ODF3B, RGS14, and PVR, P<8.33×10⁻³) and with dendritic cells in a previous study (CYP27B1, P=1.84×10⁻⁶). This study identified regulatory nucleotide sequences in the promoters of the CYP24A1, CYP27B1, SYK, RAD21L1, PVR, ODF3B, and RGS14 genes, and their variants differentially affected gene expression. This might result in their associations with MS susceptibility in previous GWAS. Further functional studies are required to understand the process of transcriptional regulation of the variants identified in the current study and the mechanisms underlying susceptibility to MS.

Differential Promoter Usage and Regulation Of The Human Interleukin-5 Receptor α (IL-5Rα) Gene In Developing Eosinophil Progenitors

IL-5Rα is a critical component of the IL-5 signaling pathway responsible for regulating the expansion of the eosinophil lineage for development of eosinophilia in allergic diseases, including asthma. IL-5Rα has two promoters, P1 and P2, both functionally active in eosinophilic cell lines. However, their differential roles and regulation during the development of authentic eosinophil progenitors remain unexplored. Human umbilical cord blood-derived CD34+progenitors under IL-5-induced eosinophil differentiation were transfected with IL-5Rα-P1 or -P2 promoter luciferase reporter vectors at various time points to assess their differential promoter activity. Chromatin immunoprecipitation (ChIP) was performed at corresponding time points to determine the dynamic occupancy of these promoters by transcription factors implicated in IL-5Rα regulation during eosinophil development. The IL-5Rα-P1 promoter is active throughout IL-5-induced eosinophil differentiation of CD34+/IL-5Rα+progenitors. However, P2 promoter activity is suddenly induced on day 7, overtaking P1 activity, before being attenuated during continued eosinophil development. Peak P2 activity coincides with a dramatic induction in expression of the predominant soluble IL-5Rα isoform mRNA. Correspondingly, quantitative ChIPs revealed that the two promoters exhibit differential occupancy by transcription factors GATA-1, PU.1 and/or the C/EBP family of transcription factors (α, β and ε). Human IL-5Rα-P1 and -P2 promoters are under differential regulation, mediated in part through dynamic and combinatorial interactions of stage-specific transcription factors. The coincidental peak in P2 promoter activity and IL-5Rα soluble isoform mRNA suggest that the IL-5-binding soluble receptor isoform is induced during eosinophil development to regulate or prevent excessive expansion of the eosinophil lineage in parasitic and allergic diseases.

Microsatellite Instability within the CD30 Gene Promoter Distinguishes between Normal and Neoplastic Cells in Lymphoma

Aims:As differences in promoter activity and CD30 surface expression between CD30+ lymphoid cell-lines and peripheral blood leukocytes have been shown previously to be independent of the size of the CD30 promoter microsatellite, in this study we investigate the instability within the region in a range of neoplasms including malignant lymphoma, breast and colon carcinoma and lung adenocarcinoma. Study Design:A representative sample of CD30+ and CD30lymphomas and cell lines as well as non-haemopoietic malignancies and normal tissues were typed for CD30 microsatellite length and compared. Place and Duration of Study: Department of Anatomical Pathology, Pathwest Laboratory Original Research Article British Journal of Medicine & Medical Research, 4(2): 755-765, 2014 756 Medicine WA, Perth, Australia and School of Chemistry & Biochemistry, University of Western Australia, 2000-2012. Methodology: DNA was prepared from archived biopsy specimens and used to PCR ampify the CD30 microsatellite region prior to size determination using an Genescan instrument (Applied Biosystems). Results: This study has identified instability within the CD30 promoter microsatellite region in DNA from the tumour tissue of cases of malignant lymphoma, colon carcinoma and lung adenocarcinoma but not in DNA from benign lymphoid cells or normal tissues. Conclusion: These findings indicate that variability in the length of the CD30 microsatellite may be a general characteristic of the neoplastic phenotype and may reflect defects in the mismatch repair system in these malignancies rather than a specific feature of CD30positive neoplasms. Our results suggest that the CD30-MS may be a useful marker to distinguish between normal lymphoid tissue and lymphoid malignancy.

Cell-Specific Fine-Tuning of Neuronal Excitability by Differential Expression of Modulator Protein Isoforms

SLOB (SLOWPOKE-binding protein) modulates the Drosophila SLOWPOKE calcium-activated potassium channel. We have shown previously that SLOB deletion or RNAi knockdown decreases excitability of neurosecretory pars intercerebralis (PI) neurons in the adult Drosophila brain. In contrast, we found that SLOB deletion/knockdown enhances neurotransmitter release from motor neurons at the fly larval neuromuscular junction, suggesting an increase in excitability. Because two prominent SLOB isoforms, SLOB57 and SLOB71, modulate SLOWPOKE channels in opposite directions in vitro, we investigated whether divergent expression patterns of these two isoforms might underlie the differential modulation of excitability in PI and motor neurons. By performing detailed in vitro and in vivo analysis, we found strikingly different modes of regulatory control by the slob57 and slob71 promoters. The slob71, but not slob57, promoter contains binding sites for the Hunchback and Mirror transcriptional repressors. Furthermore, several core promoter elements that are absent in the slob57 promoter coordinately drive robust expression of a luciferase vector by the slob71 promoter in vitro. In addition, we visualized the expression patterns of the slob57 and slob71 promoters in vivo and found clear spatiotemporal differences in promoter activity. SLOB57 is expressed prominently in adult PI neurons, whereas larval motor neurons exclusively express SLOB71. In contrast, at the larval neuromuscular junction, SLOB57 expression appears to be restricted mainly to a subset of glial cells. Our results illustrate how the use of alternative transcriptional start sites within an ion channel modulator locus coupled with functionally relevant alternative splicing can be used to fine-tune neuronal excitability in a cell-specific manner.

Human Dopamine Transporter Gene: Differential Regulation of 18-kb Haplotypes

Since previous functional studies of short haplotypes and polymorphic sites of SLC6A3 have shown variant-dependent and drug-sensitive promoter activity, this study aimed to understand whether a large SLC6A3 regulatory region, containing these small haplotypes and polymorphic sites, can display haplotype-dependent promoter activity in a drug-sensitive and pathway-related manner. By creating and using a single copy number luciferase-reporter vector, we examined regulation of two different SLC6A3 haplotypes (A and B) of the 5´ 18-kb promoter and two known downstream regulatory variable number tandem repeats by 17 drugs in four different cellular models. The two regulatory haplotypes displayed up to 3.2-fold difference in promoter activity. The regulations were drug selective (37.5% of the drugs showed effects), and both haplotype and cell type dependent. Pathway analysis revealed at least 13 main signaling hubs targeting SLC6A3, including histone deacetylation, AKT, PKC and CK2 α-chains. SLC6A3 may be regulated via either its promoter or the variable number tandem repeats independently by specific signaling pathways and in a haplotype-dependent manner. Furthermore, we have developed the first pathway map for SLC6A3 regulation. These findings provide a framework for understanding complex and variant-dependent regulations of SLC6A3.

The use of allelic imbalance to ascertain cis-regulation for human UGT2B7 in vivo

The use of allelic imbalance to ascertain cis-regulation for human UGT2B7 in vivo

Molecular Regulation of Trypanosoma congolense-Induced Nitric Oxide Production in Macrophages

BALB/c mice are highly susceptible while C57BL/6 mice are relatively resistant to experimental Trypanosoma congolense infection. Several reports show that an early interferon-gamma (IFN-γ) response in infected mice is critically important for resistance via the activation of macrophages and production of nitric oxide (NO). NO is a pivotal effector molecule and possesses both cytostatic and cytolytic properties for the parasite. However, the molecular mechanisms leading to T. congolense (TC)-induced NO release from macrophages are not known. In this study, we investigated the signaling pathways induced by trypanosomes in immortalized macrophage cell lines from the highly susceptible BALB/c (BALB.BM) and relatively resistant C57Bl/6 (ANA-1) mice. We found that T. congolense whole cell extract (TC-WCE) induces significantly higher levels of NO production in IFN-γ-primed ANA-1 than BALB.BM cells, which was further confirmed in primary bone marrow-derived macrophage (BMDM) cultures. NO production was dependent on mitogen-activated protein kinase (MAPK, including p38, Erk1/2, and JNK) phosphorylation and was significantly inhibited by specific MAPK inhibitors in BALB.BM, but not in ANA-1 cells. In addition, T. congolense- and IFN-γ-induced NO production in ANA-1 and BALB.BM cells was dependent on STAT1 phosphorylation and was totally suppressed by the use of fludarabine (a specific STAT1 inhibitor). We further show that T. congolense induces differential iNOS transcriptional promoter activation in IFN-γ-primed cells, which is dependent on the activation of both GAS1 and GAS2 transcription factors in BALB.BM but only on GAS1 in ANA-1 cells. Taken together, our findings show the existence of differential signalling events that lead to NO production in macrophages from the highly susceptible and relatively resistant mice following treatment with IFN-γ and T. congolense. Understanding these pathways may help identify immunomodulatory mechanisms that regulate the outcome of infection during Trypanosome infections.

Further Characterization of Functional Domains of PerA, Role of Amino and Carboxy Terminal Domains in DNA Binding

PerA is a key regulator of virulence genes in enteropathogenic E. coli. PerA is a member of the AraC/XylS family of transcriptional regulators that directly regulates the expression of the bfp and per operons in response to different environmental cues. Here, we characterized mutants in both the amino (NTD) and carboxy (CTD) terminal domains of PerA that affect its ability to activate the expression of the bfp and per promoters. Mutants at residues predicted to be important for DNA binding within the CTD had a significant defect in their ability to bind to the regulatory regions of the bfp and per operons and, consequently, in transcriptional activation. Notably, mutants in specific NTD residues were also impaired to bind to DNA suggesting that this domain is involved in structuring the protein for correct DNA recognition. Mutations in residues E116 and D168, located in the vicinity of the putative linker region, significantly affected the activation of the perA promoter, without affecting PerA binding to the per or bfp regulatory sequences. Overall these results provide additional evidence of the importance of the N-terminal domain in PerA activity and suggest that the activation of these promoters involves differential interactions with the transcriptional machinery. This study further contributes to the characterization of the functional domains of PerA by identifying critical residues involved in DNA binding, differential promoter activation and, potentially, in the possible response to environmental cues.

Thymic epithelial requirement for γδ T cell development revealed in the cell ablation transgenic system with TSCOT promoter.

In order to investigate the role of thymic epithelial cell (TEC) subsets during T-cell development, we established a new transgenic system, enabling inducible cell-specific ablation as well as marking the TEC subsets using bicistronic bacterial nitroreductase and EGFP genes. Two different lengths of the TSCOT promoter in transgenic mice, named 3.1T-NE and 9.1T-NE, drive EGFP expression into TECs. In adult life, EGFP expression was located in the medulla with a smaller 3.1 kb TSCOT promoter, while it was maintained in the cortex with a 9.1 kb promoter, suggesting putative TEC specific as well as compartment specific cis elements within two promoters. Nitroreductase induced cell death was specific without bystander killing upon the treatment of prodrugs such as nitrofurantoin and metronidazol. The degree of cell death was dependent on the dose of the prodrug in the cell and the fetal thymic organ cultures (FTOCs). Fetal thymic stromal populations were analyzed based on the expression levels of EpCAM, MHCII, CDR1 and/or UEA-1. EGFP expression patterns varied among subsets indicating the differential TSCOT promoter activity in each TEC subset. Prodrug treatment in FTOCs reduced the numbers of total and subsets of thymocytes. A CD4(+)CD8(+) double positive cell population was highly susceptible in both transgenic lines. Surprisingly, there was a distinct reduction in γδ T cell population only in the 9.1T-NE thymus, indicating that they require a NTREGFP expressing TEC population. Therefore, these results support a division of labor within TEC subsets for the αβ and γδ lineage specification.