Functionally Convergent B Cell Receptor Sequences in Transgenic Rats Expressing a Human B Cell Repertoire in Response to Tetanus Toxoid and Measles Antigens.

in Frontiers in immunology (2017), 8

The identification and tracking of antigen-specific immunoglobulin (Ig) sequences within total Ig repertoires is central to high-throughput sequencing (HTS) studies of infections or vaccinations. In this ... [more ▼]

The identification and tracking of antigen-specific immunoglobulin (Ig) sequences within total Ig repertoires is central to high-throughput sequencing (HTS) studies of infections or vaccinations. In this context, public Ig sequences shared by different individuals exposed to the same antigen could be valuable markers for tracing back infections, measuring vaccine immunogenicity, and perhaps ultimately allow the reconstruction of the immunological history of an individual. Here, we immunized groups of transgenic rats expressing human Ig against tetanus toxoid (TT), Modified Vaccinia virus Ankara (MVA), measles virus hemagglutinin and fusion proteins expressed on MVA, and the environmental carcinogen benzo[a]pyrene, coupled to TT. We showed that these antigens impose a selective pressure causing the Ig heavy chain (IgH) repertoires of the rats to converge toward the expression of antibodies with highly similar IgH CDR3 amino acid sequences. We present a computational approach, similar to differential gene expression analysis, that selects for clusters of CDR3s with 80% similarity, significantly overrepresented within the different groups of immunized rats. These IgH clusters represent antigen-induced IgH signatures exhibiting stereotypic amino acid patterns including previously described TT- and measles-specific IgH sequences. Our data suggest that with the presented methodology, transgenic Ig rats can be utilized as a model to identify antigen-induced, human IgH signatures to a variety of different antigens. [less ▲]

In Silico prediction of transcription factor binding sites by probabilistic models

Doctoral thesis (2012)

The characterization of in silico detected transcription factor binding sites represents a fundamental problem in the field of regulatory gene expression analysis. Several approaches have been proposed to ... [more ▼]

The characterization of in silico detected transcription factor binding sites represents a fundamental problem in the field of regulatory gene expression analysis. Several approaches have been proposed to model DNA-protein-interactions, composed by two main classes: qualitative models considering a consensus sequence and quantitative models providing a measure of binding affinity. The latter can be further subdivided in models assuming an independent contribution of the nucleotides forming a potential binding site and more flexible ones implicating a positional interdependence. In this work the applicability of three probabilistic models to predict transcription factor binding sites has been investigated: (i) the simple position weight matrix (PWM), assuming independence, and two flexible models capturing positional interdependencies represented by a (ii) Chow-Liu Tree and (iii) Ensemble of Trees model. The training and validation of the models on the Mus musculus subset of the UniPROBE database revealed that complex models provide a better predictive power suggesting a high amount of transcription factors binding motifs being affected by positional interdependencies. Additionally, numerous transcription factors were detected, for which the Ensemble of Trees model outperformed both, the Chow-Liu Tree and PWM model. The UniPROBE-based trained models have been applied in a biological context - the prediction of differential binding profiles in five different ChIP-seq samples, followed by the detection of causative regulatory SNPs. The chosen set-up involved family trio data, meaning genotype data from a family composed of father, mother and daughter, providing internal validation. The models provide strong power to correctly classify true negatives in an independent biological sample, represented by a high specificity. The applied approach to detect causative regulatory SNPs, resulted in a candidate list of 20 SNPs. Those gain strong support by epigenetic markers and both, model-based predicted binding affinity of the comprising binding site and significant p-values, describing the effect of the nucleotide exchange. [less ▲]

Genome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages

in BMC Genomics (2012), 13(50),

Background: The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human ... [more ▼]

Background: The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human origin has not yet been provided. Results: We performed ChIP-seq in phorbol myristate acetate-differentiated THP-1 cells (macrophage-type) after stimulation with the potent synthetic LXR ligand T0901317 (T09). Microarray gene expression analysis was performed in the same cellular model. We identified 1357 genome-wide LXR locations (FDR < 1%), of which 526 were observed after T09 treatment. De novo analysis of LXR binding sequences identified a DR4-type element as the major motif. On mRNA level T09 up-regulated 1258 genes and repressed 455 genes. Our results show that LXR actions are focused on 112 genomic regions that contain up to 11 T09 target genes per region under the control of highly stringent LXR binding sites with individual constellations for each region. We could confirm that LXR controls lipid metabolism and transport and observed a strong association with apoptosis-related functions. Conclusions: This first report on genome-wide binding of LXR in a human cell line provides new insights into the transcriptional network of LXR and its target genes with their link to physiological processes, such as apoptosis. The gene expression microarray and sequence data have been submitted collectively to the NCBI Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo under accession number GSE28319. [less ▲]

Interferon-γ-induced activation of Signal Transducer and Activator of Transcription 1 (STAT1) up-regulates the tumor suppressing microRNA-29 family in melanoma cells

in Cell Communication and Signaling (2012), 10

Background: The type-II-cytokine IFN-γ is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic ... [more ▼]

Background: The type-II-cytokine IFN-γ is a pivotal player in innate immune responses but also assumes functions in controlling tumor cell growth by orchestrating cellular responses against neoplastic cells. The role of IFN-γ in melanoma is not fully understood: it is a well-known growth inhibitor of melanoma cells in vitro. On the other hand, IFN-γ may also facilitate melanoma progression. While interferon-regulated genes encoding proteins have been intensively studied since decades, the contribution of miRNAs to effects mediated by interferons is an emerging area of research.We recently described a distinct and dynamic regulation of a whole panel of microRNAs (miRNAs) after IFN-γ-stimulation. The aim of this study was to analyze the transcriptional regulation of miR-29 family members in detail, identify potential interesting target genes and thus further elucidate a potential signaling pathway IFN-γ → Jak→ P-STAT1 → miR-29 → miR-29 target genes and its implication for melanoma growth. Results: Here we show that IFN-γ induces STAT1-dependently a profound up-regulation of the miR-29 primary cluster pri-29a∼b-1 in melanoma cell lines. Furthermore, expression levels of pri-29a∼b-1 and mature miR-29a and miR-29b were elevated while the pri-29b-2∼c cluster was almost undetectable. We observed an inverse correlation between miR-29a/b expression and the proliferation rate of various melanoma cell lines. This finding could be corroborated in cells transfected with either miR-29 mimics or inhibitors. The IFN-γ-induced G1-arrest of melanoma cells involves down-regulation of CDK6, which we proved to be a direct target of miR-29 in these cells. Compared to nevi and normal skin, and metastatic melanoma samples, miR-29a and miR-29b levels were found strikingly elevated in certain patient samples derived from primary melanoma. Conclusions: Our findings reveal that the miR-29a/b1 cluster is to be included in the group of IFN- and STAT-regulated genes. The up-regulated miR-29 family members may act as effectors of cytokine signalling in melanoma and other cancer cells as well as in the immune system. © 2012 Schmitt et al.; licensee BioMed Central Ltd. [less ▲]