Evolution der Runt-Gene mit Fokus auf die Skelettbildung und T-Zellentwicklung

Das Ziel dieser Arbeit war, die Evolution der Runt-Gene in der Stammesgeschichte der Chordata zu rekonstruieren, sowie die Runt-Genfunktion bei der Skelettbildung und T-Zellentwicklung besser zu verstehen. Im Rahmen dieser Arbeit wurde gezeigt, dass in der Stammart der Chordata nur ein Runt-Gen vorhanden war und der Runt-Lokus in der Evolution der Vertebraten tripliziert wurde. Beim Lanzettfischchen als Vertreter der Chordata mit ursprünglichen Merkmalen war ein molekulares Netzwerk für Skelettbildung, unter Beteiligung von SoxE, Hedgehog- und Runt-Genen, im Kiemendarm exprimiert. Dieses molekulare Netzwerk wurde in der Evolution der Vertebraten nach Genduplikationen diversifiziert und diente als molekulares Entwicklungsmodul für Knorpel, Knochen, Placoidschuppen und Zähne. In diesem Netzwerk ist Runx2 essentiell für die Knochenbildung und im Rahmen dieser Habilitation konnten durch ein Expressionsscreening in einem Runx2-Knockout Mausmodell bereits bekannte und neue Transkripte mit Relevanz für die Knochenbildung entdeckt werden. Für eines dieser Gene mit damals unbekannter Funktion, TMEM119 (Transmembrane Protein 119) wurde inzwischen eine wichtige Rolle bei der Differenzierung von Osteoblasten beschrieben. Des Weiteren wurde die Bedeutung von Runx1 in der T-Zellentwicklung anhand eines Runx1-Knockout Mausmodells erforscht. Dabei lag der Schwerpunkt auf der Hochdurchsatzsequenzierung von TCR-Genumlagerungen. Zur zuverlässigen Herstellung der Amplikon-Libraries wurde eine zweistufige PCR-Methodik entwickelt, welche durch einen eingebauten Kontaminationsschutz Kreuzkontaminationen von der ersten zur zweiten PCR-Stufe verhindert. Unsere Analysen zeigten, dass aufgrund des Runx1-Knockouts TCR-Genumlagerung nur in sehr reduziertem Umfang stattfanden und wir konnten Veränderungen der V(D)J-Struktur nachweisen. Die T-Zellentwicklung war stark reduziert und die Thymusstruktur (Cortex und Medulla) ging verloren. Weiterhin konnten wir zeigen, dass Runx1 an Runx1-Bindungsstellen an der Initiationsstelle der TCR-Genumlagerungen bindet. Zusammen mit publizierten Daten, die eine direkte Bindung von Runx1 an das Rekombinations-aktivierende Protein 1 in sich entwickelnden T-Zellen zeigten, spricht dies dafür, dass Runx1 neben der Rolle als Transkriptionsfaktor auch eine Rolle als Rekombinase-Kofaktor hat. Unsere Analysen sprechen weiterhin dafür, dass RUNX1 nicht nur bei TCR-Genumlagerungen (physiologischen Deletionen), sondern auch bei pathologischen Deletionen als Rekombinase-Kofaktor beteiligt ist. Denn RUNX1-Bindungsstellen sind an rekurrenten Deletionsrändern bei der ALL mit einer ETV6-RUNX1 Translokation signifikant angereichert und wir konnten zeigen, dass eine RUNX1-Bindungsstelle im CDKN2A/B Bruchpunkt funktionell relevant ist. Dies zeigt, dass Mechanismen wie TCR-Genumlagerungen, welche in der Evolution der Vertebraten zu einem sehr effektiven Immunsystem führten, bei einer fehlgeleiteten Rekombinationsmaschinerie Risiken mit sich bringen, z.B. bei der Entstehung von pathologischen Deletionen bei der ETV6-RUNX1 ALL

A new method to prevent carry-over contaminations in two-step PCR NGS library preparations

Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross- contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR- based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy

Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep

BACKGROUND: The sheep is an important model animal for testing novel fracture treatments and other medical applications. Despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and DNA sequences are available for only a small number of sheep genes. RESULTS: In this work we have sequenced over 47 thousand expressed sequence tags (ESTs) from libraries developed from healing bone in a sheep model of fracture healing. These ESTs were clustered with the previously available 10 thousand sheep ESTs to a total of 19087 contigs with an average length of 603 nucleotides. We used the newly identified sequences to develop RT-PCR assays for 78 sheep genes and measured differential expression during the course of fracture healing between days 7 and 42 postfracture. All genes showed significant shifts at one or more time points. 23 of the genes were differentially expressed between postfracture days 7 and 10, which could reflect an important role for these genes for the initiation of osteogenesis. CONCLUSION: The sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for Ovis aries, with partial or complete sequences being made available for over 5,800 previously unsequenced sheep genes

Deep Sequencing of MYC DNA-Binding Sites in Burkitt Lymphoma

BACKGROUND: MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far. METHODOLOGY/PRINCIPAL FINDINGS: ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing. CONCLUSION/SIGNIFICANCE: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology

Evolution of a Core Gene Network for Skeletogenesis in Chordates

The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1–3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module

Identification of immunorelevant genes from greater wax moth (Galleria mellonella) by a subtractive hybridization approach

In this study we have analyzed bacterial lipopolysaccharide (LPS) induced genes in hemocytes of the Lepidopteran species Galleria mellonella using subtractive hybridization, followed by suppressive PCR. We have found genes that show homologies to molecules, such as gloverin, peptidoglycan recognition proteins and transferrin known to be involved in immunomodulation after bacterial infection in other species. In addition, a few molecules previously not described in the innate immune reactions were detected, such as a RNA binding molecule and tyrosine hydroxylase. Furthermore, the full-length cDNA of a LPS-induced molecule with six toxin-2-like domains is described to be a promising candidate to further elucidate the relationship between toxin- and defensin-like domains in arthropod host defense