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Anti-Neutrophile Cytoplasmatische Antikörper verursachen nekrotisierende Vaskulitiden kleiner Blutgefäße. Die Serinprotease PR3 ist ein ANCA-Zielantigen, welches von zirkulierenden ANCA auf der Zellmembran erkannt wird. ANCA aktivieren neutrophile Granulozyten, die dann die nekrotisierende Vaskulitis verursachen. Das Membran-PR3 Expressionsmuster ist bimodal wobei mPR3-niedrig- und mPR3-hoch-exprimierende Zellen existieren. Wir testeten die Hypothese, dass ein Membranrezeptor eine hohe mPR3-Expression vermittelt. Wir verwendeten humane neutrophile Granulozyten, neutrophil-differenzierte Stammzellen und transfizierte HEK293 Zellen. Wir identifizierten das Glykoprotein CD177 als einen mPR3-präsentierenden Rezeptor. CD177 zeigte eine spezifische Bindung von reifem PR3-Protein, nicht aber von einem unprozessierten PR3. Wir separierten die mPR3-Zellpopulationen und führten Durchflusszytometrie, Giemsa-Färbung, Western Blot-Experimente und RT-PCR für die PR3 und CD177 mRNA-Expression durch. Wir fanden, dass die mPR3hoch neutrophilen Granulozyten PR3- und CD177-Protein enthielten, während in den mPR3niedrig neutrophilen Granulozyten nur PR3, aber kein CD177 detektierbar war. Die CD177-Regulation vollzog sich auf transkriptioneller Ebene, da die Zellen, die negativ für das CD177-Protein waren auch keine mRNA transkribierten. Um die Grundlage der fehlenden CD177-Transkription zu analysieren, identifizierten wir den Transkriptionsstart von CD177 für eine anschließende Mutations- und SNP-Analyse. Die CD177-Sequenzen der proteinkodierenden Regionen und der Intron-Exon-Übergänge der beiden Zellpopulationen waren identisch. Jedoch fanden wir, dass das CD177-Gen einer monoallelischen Expression unterliegt. Es wurde dabei maternale als auch paternale monoallelische Expression detektiert. In weiterführenden Untersuchungen soll der Regulationsmechanismus der monoallelischen CD177-Expression charakterisiert werden. / Anti-Neutrophil Cytoplasmic Antibodies cause necrotizing small-vessel vasculitis. The serine protease PR3 provides a main ANCA target antigen and is recognized by circulating ANCA on the neutrophil cell surface. ANCA activate neutrophils and activated neutrophils cause vasculitis. The membrane-PR3 expression pattern is bimodal in that low and high mPR3 expressing cells can be distinguished. We tested the hypothesis that a membrane receptor mediates mPR3high expression. We studied human neutrophils, neutrophilic differentiated CD34-positive hematopoietic stem cells and transfected HEK293 cells. We identified the glycoprotein CD177 as an mPR3 presenting receptor. CD177 demonstrated specific binding of mature, but not of unprocessed pro-PR3. We separated the two mPR3 populations and performed cytometry analysis, Giemsa staining, western blot analysis and RT-PCR for PR3 and CD177 expression. We detected PR3 and CD177 protein in mPR3high expressing neutrophils, whereas only PR3, but no CD177 was found in mPR3low expressing cells. Regulation took place on a transcriptional level because cells that were negative for CD177 protein were also negative for mRNA. To further study this finding, we identified the CD177 transcription start for a subsequent mutation and SNP analysis. CD177 sequences of the protein-coding regions and the intron-exon regions did not differ in both populations. However, we found a monoallelic CD177 expression and were able to detect maternal as well as paternal allele expression. Future experiments will elucidate the mechanisms that control monoallelic CD177 gene expression.

African trypanosomes are a major plague in sub-Saharan Africa. They cause sleeping sickness in humans and nagana in cattle. These parasites are transmitted between their mammalian hosts by tsetse flies. They are adapting to their different environments through differentiation processes. These processes involve, amongst other things, the expression of different surface coats. These coats are made of procyclin protein at the insect midgut procyclic stage and of variant surface glycoprotein (VSG) at the mammalian bloodstream stage. At a given time, one VSG is expressed from a single VSG gene out of a repertoire of more than 1500 VSG genes present in the trypanosomes genome. The expressed VSG gene is always located at one of fifteen telomeric polycistronic transcription units called expression sites (ES). The VSG coat is changed regularly in a process called antigenic variation allowing trypanosomes to escape the immune response. The exact mechanism controlling the selection of the active ES is not yet known and controversies have been raised concerning the ES transcription control. Although several molecular factors involved in the ES monoallelic-expression have been identified, none of them seems to be a critical regulator.<p><p>Thus during my thesis we decided to explore two aspects of ES expression: (A) deciphering the level at which this expression is controlled and (B) fishing for new protein factors controlling this expression.<p>A) It is not even clear at which level the ES transcription control takes place. In particular, there has been debate on whether it is taking place at the transcription initiation or elongation level. Previous experiments generated contradictory conclusions and gave rise to two different models. The first model suggested that transcription initiation takes place in all ESs simultaneously. The second model suggested that transcription is initiated in only two ESs, one being fully active and a second being pre-active. These two models were equally able to account for the finding of transcripts from different ES within a trypanosome population provided the pre-active ES differs between individual cells. In order to decide if a single or multiple ES promoters can initiate transcription in a given cell, single cell RT-PCR targeting the beginning of the ES was required. Thus single cell RT-PCR was performed and an analysis of the obtained transcripts showed that transcription initiation is taking place on many ES while only one VSG is transcribed. This permitted the unambiguous conclusion that the monoallelic expression of VSG is exerted by controls operating downstream from transcription initiation, suggesting transcription elongation or RNA processing as critical control steps. <p>B) We have characterized a new nuclear protein, Tb alba3, involved in the repression of silent VSGs. Its invalidation lead to chromatin opening in the silent expression sites and to a raise in their expression. As this protein is cytoplasmic and binding procyclin mRNAs at the procyclic stage, it could be a new versatile factor, shuttling between the cytoplasm and the nucleus and involved both in the inverse regulation of major surface antigens at different differentiation stages and the control of antigenic variation.<p><p>These results enhance our understanding of ES transcription control and of ES monoallelic expression. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

X-Chromosom-Inaktivierung (XCI) ist der Mechanismus, den Säuger zur Dosiskompensierung zwischen weiblichen und männlichen Zellen verwenden. XCI wird ausgelöst durch die monoallelische Hochregulation der langen nicht-kodierenden RNA Xist von einem der zwei X-Chromosomen in weiblichen Zellen. Die Xist RNA vermittelt dann das Ausschalten der Gene auf diesem X-Chromosom. Das wirft einige interessante Fragen auf: Wie zählen Zellen ihre X-Chromosomen und stellen sicher, dass genau eines aktiv bleibt? Wie entscheiden sie, welches X-Chromosom aktiv bleibt und welches ausgeschaltet wird? Und wie erinnern sie sich an diese Entscheidung und behalten sie stabil bei durch alle weiteren Zellteilungen? Mithilfe eines stochastischen Modells zeigen wir, dass diese XCI Regulation prinzipiell durch nur zwei Regulatoren erklärt werden kann: Ein global (in trans) agierender XCI Aktivator und ein lokal (in cis) agierender XCI Repressor. Dieses Netzwerk aus nur zwei Regulatoren kann die Xist Expressionsmuster in verschiedenen Säugerspezies reproduzieren, von der Maus bis zum Mensch. Es sagt außerdem voraus, dass Zellen in der Lage sind, biallelische zu monoallelischer Xist Expression zu korrigieren, eine Vorhersage, für die wir tatsächlich experimentelle Belege finden. Mit einem mechanistischen Modell zeigen wir, dass das cis-Gedächtnis über den Xist Expressionszustand durch Antisense-Transkription zustande kommen könnte. Auf dieser Hypothese aufbauend untersucht der zweite Teil der Arbeit das Potential von Antisense-Transkription, ein lokales Gedächtnis über den Expressionszustand eines Gens zu generieren, genauer. Diese Analyse sagt vorher, dass Antisense-Repression den Expressionszustand eines Lokus tatsächlich für einige Tage stabil erhalten kann. / X-chromosome inactivation (XCI) is the mechanism for dosage compensation between the sexes in mammals. It is initiated through monoallelic upregulation of the long non-coding RNA Xist from one X chromosome, which mediates almost complete transcriptional silencing of this X chromosome. XCI regulation raises intriguing and thus far unanswered questions: How do cells count their X chromosomes and ensure that exactly one stays active? How do they make a mutually exclusive choice for one inactive X chromosome, and how do they then stably maintain this choice throughout subsequent cell divisions? Using stochastic modeling, we show that XCI onset only requires two regulators: A trans-acting Xist activator that ensures female specificity and a cis-acting Xist repressor that allows stable maintenance of alternative Xist expression states. This two-regulator network can recapitulate Xist expression patterns across different species and makes a novel prediction that is validated experimentally: Cells are able to revert biallelic Xist expression to monoallelic expression. With a mechanistic stochastic model we show that Xist's antisense transcript Tsix might be the cis-acting Xist repressor, uncovering the molecular mechanism behind the stabilization of the alternative Xist expression states. Building upon Tsix' possible functional role in stabilizing alternative Xist expression states on the active and inactive X chromosome, the second part of this thesis investigates the potential of antisense transcription to maintain a transient transcriptional memory. We find that mutual repression between a pair of antisense genes can allow the locus to remember the transcription state it has acquired due to a past signal for several days.