Analysis of Haplotype Structure in the Bovine Major Histocompatibility Complex

The goal of this project was to identify and characterize polymorphic markers spanning regions of the bovine major histocompatibility complex (BoLA) to analyze patterns of genetic variation and haplotype structure across diverse cattle breeds with various breed histories and selection pressures. Genetic markers that demonstrated sufficient levels of polymorphism, locus specificity, Mendelian inheritance, and the accurate typing of alleles across diverse haplotypes were chosen to define separate haplotype structures for the BoLA IIb and BoLA IIa-III-I regions and to evaluate breakpoints in linkage disequilibrium within the regions surrounding BoLA IIa-III-I. A total of 23 microsatellites, two SNPSTRs, 62 SNPs, and the alleles of three class IIa genes were selected for use in this study. These markers revealed eleven recombination events, low levels of recombination in BoLA IIa-III-I, a sharp break in haplotype structure in the region centromeric to class IIa, prolonged linkage disequilibrium in the extended class I region, strong conservation of BoLA IIa-III-I haplotype structure, BoLA IIa-III-I homozygous haplotype identity across seven different breeds of cattle, and a small number of common BoLA IIa-III-I haplotypes within the Angus and Holstein breeds. This work demonstrated that 52 SNPs from the Illumina 50K SNPchip could accurately predict BoLA IIa-III-I haplotypes. These 52 SNPs represent tagSNPs that can predict BoLA IIa-III-I genetic variation and could offer a cost-effective means for screening large sample sizes for haplotype/disease association studies in the future

Mammalian comparative genomics and epigenomics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references.The human genome sequence can be thought of as an instruction manual for our species, written and rewritten over more than a billion of years of evolution. Taking a complete inventory of our genome, dissecting its genes and their functional components, and elucidating how these genes are selectively used to establish and maintain cell types with markedly different behaviors, are key challenges of modern biology. In this thesis we present contributions to our understanding of the structure, function and evolution of the human genome. We rely on two complementary approaches. First, we study signatures of evolutionary processes that have acted on the genome using comparative sequence analysis. We generate high quality draft genome sequences of the chimpanzee, the dog and the opossum. These species share a last common ancestor with humans approximately 6 million, 80 million and 140 million years ago, respectively, and therefore provide distinct perspectives on our evolutionary history. We apply computational methods to explore the functional organization of the genome and to identify genes that contribute to shared and species-specific traits. Second, we study how the genome is bound by proteins and packaged into chromatin in distinct cell types. We develop new methods to map protein-DNA interactions and DNA methylation using single-molecule based sequencing technology. We apply these methods to identify new functional sequence elements based on characteristic chromatin signatures, and to explore the relationship between DNA sequence, chromatin and cellular state.by Tarjei Sigurd Mikkelsen.Ph.D

Expression of G protein-coupled receptor 19 in human lung cancer cells is triggered by entry into S phase and supports G2/M cell cycle progression

G Protein-gekoppelte Rezeptoren (GPCRs) stellen die größte Familie zellulärer Oberflächenrezeptoren dar. Jedoch sind nur ein Teil der mehr als 800 Rezeptoren in solchem Ausmaß charakterisiert, dass deren physiologische Rolle gut verstanden wird. Tatsächlich werden in der heutigen Pharmakotherapie lediglich etwa 60 Vertreter dieser Rezeptoren mit einer Vielzahl von Substanzen angegriffen, welche etwa 30% aller verfügbaren Medikamente ausmachen. Es ist schon lange bekannt, dass GPCRs eine ungewöhnliche Expression in Krebszellen aufweisen können. An vielen Stellen der Karzinogenese tragen sonst normale, physiologische Funktionen von GPCRs mutmaßlich zur Krebsprogression bei, wenn diese unkontrolliert und missbräuchlich in einer Zelle verwendet werden. Das Hauptziel dieser Arbeit besteht darin, GPCRs vermehrt in Bezug zu malignen Erkrankungen zu stellen. Konkret wurde nach GPCRs gesucht, die eine hohe Expression im kleinzelligen Lungenkarzinom aufweisen. Hierbei zeigte sich, dass die den G Protein-gekoppelten Rezeptor 19 (GPR19) kodierende mRNA in Proben von Patienten mit kleinzelligem Lungenkarzinom verglichen mit Proben anderer Lungenkarzinome und von untransformiertem Lungengewebe überexprimiert war. Desweiteren deuten einige Beobachtungen darauf hin, dass eine Gpr19 Überexpression in von humanen Lungenkarzinomen abgeleiteten Zelllinien diesen einen spezifischen Vorteil im Durchschreiten des Zellzykluses bietet. Die Abschwächung der Gpr19 Genexpression mittels RNA Interferenz hemmte Lungenkarzinomszelllinien in ihrer Proliferation und führte letztlich zu deren Zelltod. Das Fortschreiten des Zellzykluses über die G2/M Phase hinweg war gestört, was mit einer erhöhten Präsenz an für diese Phasen charakteristischen Proteinmarkern (Zyklin B1, phosphoryliertes Histon H3) einherging. In den untersuchten Lungenkarzinomszelllinien war die Expression der Gpr19 mRNA abhängig vom Zellzyklus. Nachdem die Zellen in verschiedenen Phasen des Zellzykluses zunächst angehalten und danach aus dem Arrest entlassen wurden, konnte ein Höchststand der Gpr19 Expression während der S Phase konstatiert werden. Zudem zeigte sich mit Hilfe von Chromatin-Immunpräzipitation, dass die Gpr19 mRNA Expression in diesen Zelllinien anscheinend unter der Kontrolle von Transkriptionsfaktoren der E2F Familie steht. Mitglieder dieser Familie sind bekannt dafür, dass sie die Genexpression vieler am Zellzyklus beteiligter Gene kontrollieren. So konnte die Gpr19 Promotorregion bei der Anwendung von gegen E2F-1 bis 4 gerichteten Antikörpern nachgewiesen werden. Und die Zerstörung mutmaßlicher E2F Bindestellen in der Gpr19 Promotorregion schwächte die Expression eines Luciferase Reporterkonstrukts ab. Diese Arbeit offenbart zum ersten Mal die vom Zellzyklus abhängige mRNA Expression eines Vertreters der GPCR Familie, GPR19. Desweiteren stellt sie eine funktionelle Verbindung zwischen dieser differenziellen Expression, einem ungehinderten Durchschreiten des Zellzykluses und der Proliferation von humanen Lungenkarzinomszelllinien her, wenn diese eine erhöhte Gpr19 Grundexpression aufweisen. Folglich validieren diese Daten GPR19 als mögliches Angriffsmolekül bei einer Überexpression im Lungenkarzinom.G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors. However, only a fraction of the more than 800 receptors have been characterized to an extent that their physiological role is reasonably well understood. In fact, current pharmacotherapy only addresses some 60 receptors with a large collection of compounds that represent about 30% of the available drugs. It has long been known that GPCRs are subject to illegitimate expression in cancer cells. Presumably, hijacking the normal physiological functions of GPCRs contributes to all biological capabilities acquired during the multistep development of human cancers. With the goal of linking G protein-coupled receptors to malignant diseases, GPCRs were searched for that revealed high expression levels in small cell lung cancer (SCLC): The mRNA encoding orphan G protein-coupled receptor 19 (GPR19) was found to be frequently overexpressed in tissue samples obtained from patients with SCLC in contrast to samples derived from non-SCLC or normal lung. Several observations indicate that overexpression of Gpr19 confers a specific advantage to human lung cancer-derived cells regarding the transition through the cell cycle. Knockdown of Gpr19 mRNA by RNA interference reduced cell growth of human lung cancer cell lines and led to cell death. Cell cycle progression through G2/M phase was impaired and this was associated with increased protein levels of cyclin B1 and phosphorylated histone H3. Gpr19 exhibited a cell cycle-dependent expression pattern in lung cancer cell lines. When cell cycle distribution profiles of cells released from cell cycle arrest were related to Gpr19 mRNA levels, a peak Gpr19 expression was detected during S phase. The control of Gpr19 expression by E2F transcription factors, which drive gene expression of many genes important for cell cycle progression, was verified by chromatin immunoprecipitation: Antibodies directed against E2F-1 to E2F-4 allowed for the recovery of the Gpr19 promoter in lung cancer-derived cell lines. E2F binding site deletion in the Gpr19 promoter resulted in diminished luciferase reporter gene expression. This is the first example of a G protein-coupled receptor showing cell cycle phase specific mRNA expression. Further results point to a functional link between the cell cycle-dependent expression of Gpr19, a correct cell cycle passage, and cellular proliferation in human lung cancer-derived cells that exhibit higher than normal basal levels of Gpr19 mRNA. Hence, the data also validate GPR19 as a candidate target when overexpressed in lung cancer