Lernassoziierte Genexpression im murinen Hippokampus

Die bei Lern- und Gedächtnisvorgängen ablaufenden neurobiologischen Prozesse sind in ihrer Funktion bis heute nur unzureichend verstanden, wobei besonders die Rolle der lernabhängigen Genexpression unklar ist. Wiederholungen im Lernprozess fördern die Bildung von stabilen Gedächtnisinhalten. Die Lerneffiktivität kann hierbei durch lernfreie Zeitintervalle, insbesondere durch eingeschobene Schalfperioden, zusätzlich gesteigert werden. Entsprechend kann man den mehrtägigen Morris Water Maze (MWM)-Test mit einer verborgenen Plattform als einen mehrstufigen räumlichen Lernprozess bezeichnen. Dieser Test ist Hippokampus-abhängig und produziert Langzeitgedächtnisspuren in Nagern. Für diese Studie wurden FVB/NxC57Bl/6-Mäuse der F1-Generation über vier Tage in der MWM trainiert, das Erlernte in einem Probe Trial an Tag 5 überprüft und die Tiere gemäß ihrer Lernleistung in die beiden Gruppen „gute“ und „schlechte Lerner“ eingeteilt. Eine Analyse der hippokampalen Expression von Kandidatengenen per Microarray und Real-Time PCR erfolgte eine, sechs beziehungsweise 24 Stunden nach dem jeweils letzten Trainingslauf eines Tages. Durch den Vergleich von Schwimmkontrollen mit Test-naiven Mäusen wurde eine gleichgeschaltete, mit dem impliziten Lernen der MWM-Erfahrung der Tiere assoziierte unspezifische Genexpression festgestellt. Beim Vergleich der Schwimmkontrollen (ohne Plattform) mit den trainierten Tieren (verborgene Plattform mit konstanter Lokalisation) wurde in guten Lernern zu bestimmten Zeitpunkten eine Hochregulation von Genen, die mit Lernen und Gedächtnis (PP1, Kibra), neuronaler Aktivität (mt-CO1), Epigenetik (Dnmt3a, Dnmt3b) und neurodegenerativen Erkrankungen (Mapt, Sorl1) assoziiert sind, gefunden. Im Hippokampus der schlechten Lerner wurde eine im Vergleich zu den guten Lernern gesteigerte Synthese der mRNA von Genen festgestellt, die mit Lernen und Gedächtnis (Reelin, PP1, Kibra), Epigenetik (Dnmt1, Dnmt3a, Dnmt3b) und neurodegenerativen Erkrankungen (Mapt, Sorl1, APP) in Zusammenhang stehen. Diese Studie liefert somit den bisher ersten Hinweis, dass während eines mehrtägigen MWM-Lernprozesses eine abnormal erhöhte de novo-mRNA-Synthese mit verminderter Lernleistung in Zusammenhang steht.

Comparative DNA sequence and methylation analyses of orthologous genes in humans and non-human primates: Genetic and epigenetic footprints of evolution

Welche genetische Unterschiede machen uns verschieden von unseren nächsten Verwandten, den Schimpansen, und andererseits so ähnlich zu den Schimpansen? Was wir untersuchen und auch verstehen wollen, ist die komplexe Beziehung zwischen den multiplen genetischen und epigenetischen Unterschieden, deren Interaktion mit diversen Umwelt- und Kulturfaktoren in den beobachteten phänotypischen Unterschieden resultieren. Um aufzuklären, ob chromosomale Rearrangements zur Divergenz zwischen Mensch und Schimpanse beigetragen haben und welche selektiven Kräfte ihre Evolution geprägt haben, habe ich die kodierenden Sequenzen von 2 Mb umfassenden, die perizentrischen Inversionsbruchpunkte flankierenden Regionen auf den Chromosomen 1, 4, 5, 9, 12, 17 und 18 untersucht. Als Kontrolle dienten dabei 4 Mb umfassende kollineare Regionen auf den rearrangierten Chromosomen, welche mindestens 10 Mb von den Bruchpunktregionen entfernt lagen. Dabei konnte ich in den Bruchpunkten flankierenden Regionen im Vergleich zu den Kontrollregionen keine höhere Proteinevolutionsrate feststellen. Meine Ergebnisse unterstützen nicht die chromosomale Speziationshypothese für Mensch und Schimpanse, da der Anteil der positiv selektierten Gene (5,1% in den Bruchpunkten flankierenden Regionen und 7% in den Kontrollregionen) in beiden Regionen ähnlich war. Durch den Vergleich der Anzahl der positiv und negativ selektierten Gene per Chromosom konnte ich feststellen, dass Chromosom 9 die meisten und Chromosom 5 die wenigsten positiv selektierten Gene in den Bruchpunkt flankierenden Regionen und Kontrollregionen enthalten. Die Anzahl der negativ selektierten Gene (68) war dabei viel höher als die Anzahl der positiv selektierten Gene (17). Eine bioinformatische Analyse von publizierten Microarray-Expressionsdaten (Affymetrix Chip U95 und U133v2) ergab 31 Gene, die zwischen Mensch und Schimpanse differentiell exprimiert sind. Durch Untersuchung des dN/dS-Verhältnisses dieser 31 Gene konnte ich 7 Gene als negativ selektiert und nur 1 Gen als positiv selektiert identifizieren. Dieser Befund steht im Einklang mit dem Konzept, dass Genexpressionslevel unter stabilisierender Selektion evolvieren. Die meisten positiv selektierten Gene spielen überdies eine Rolle bei der Fortpflanzung. Viele dieser Speziesunterschiede resultieren eher aus Änderungen in der Genregulation als aus strukturellen Änderungen der Genprodukte. Man nimmt an, dass die meisten Unterschiede in der Genregulation sich auf transkriptioneller Ebene manifestieren. Im Rahmen dieser Arbeit wurden die Unterschiede in der DNA-Methylierung zwischen Mensch und Schimpanse untersucht. Dazu wurden die Methylierungsmuster der Promotor-CpG-Inseln von 12 Genen im Cortex von Menschen und Schimpansen mittels klassischer Bisulfit-Sequenzierung und Bisulfit-Pyrosequenzierung analysiert. Die Kandidatengene wurden wegen ihrer differentiellen Expressionsmuster zwischen Mensch und Schimpanse sowie wegen Ihrer Assoziation mit menschlichen Krankheiten oder dem genomischen Imprinting ausgewählt. Mit Ausnahme einiger individueller Positionen zeigte die Mehrzahl der analysierten Gene keine hohe intra- oder interspezifische Variation der DNA-Methylierung zwischen den beiden Spezies. Nur bei einem Gen, CCRK, waren deutliche intraspezifische und interspezifische Unterschiede im Grad der DNA-Methylierung festzustellen. Die differentiell methylierten CpG-Positionen lagen innerhalb eines repetitiven Alu-Sg1-Elements. Die Untersuchung des CCRK-Gens liefert eine umfassende Analyse der intra- und interspezifischen Variabilität der DNA-Methylierung einer Alu-Insertion in eine regulatorische Region. Die beobachteten Speziesunterschiede deuten darauf hin, dass die Methylierungsmuster des CCRK-Gens wahrscheinlich in Adaption an spezifische Anforderungen zur Feinabstimmung der CCRK-Regulation unter positiver Selektion evolvieren. Der Promotor des CCRK-Gens ist anfällig für epigenetische Modifikationen durch DNA-Methylierung, welche zu komplexen Transkriptionsmustern führen können. Durch ihre genomische Mobilität, ihren hohen CpG-Anteil und ihren Einfluss auf die Genexpression sind Alu-Insertionen exzellente Kandidaten für die Förderung von Veränderungen während der Entwicklungsregulation von Primatengenen. Der Vergleich der intra- und interspezifischen Methylierung von spezifischen Alu-Insertionen in anderen Genen und Geweben stellt eine erfolgversprechende Strategie dar.

Transkriptom des Meristemgewebes des Kolumnarapfels

Kolumnare Apfelbäume (Malus x domestica) stellen aufgrund ihres auffälligen Phänotyps eine ökonomisch interessante Wuchsform dar. Diese extreme Form des Kurztriebwuchses zeichnet sich durch einen insgesamt sehr schlanken, säulenförmigen Habitus aus, welcher eine dichte Pflanzung und damit einhergehend Ertragssteigerungen im Vergleich zu normalwüchsigen Bäumen ermöglicht. Verursacht wird der Phänotyp durch die Anwesenheit eines einzelnen, dominanten Allels des Columnar (Co)-Gens. Bis auf die approximative Lokalisation des Gens auf Chromosom 10 ist über mögliche Identität und Funktion bislang nichts bekannt.rnIn der vorliegenden Arbeit wurde ein erster Versuch unternommen, mit Hilfe von Next Generation Sequencing (NGS) Technologien und RNA-Seq Einblicke in das Transkriptom des Sprossapikalmeristems (SAM) kolumnarer Apfelbäume zu gewinnen. So konnte gezeigt werden, dass unabhängig vom Zeitpunkt der Entnahme des Materials mehrere hundert Gene differentiell reguliert werden. Diese lassen sich funktional in mehrere überrepräsentierte Kategorien gruppieren, von denen sich einige wiederum mit dem kolumnaren Phänotyp assoziieren lassen. Durch den Einsatz weiterer Expressionsstudien (Microarrays, qRT-PCR) konnten frühere Ergebnisse bezüglich des Hormonhaushalts auf Genebene bestätigt und neue Erkenntnisse gewonnen werden, die eine mögliche Erklärung für den Phänotyp darstellen. Weiterhin ergab der Vergleich aller durchgeführten Expressionsstudien eine Anreicherung signifikant differentiell regulierter Gene auf Chromosom 10, was auf einen „selective sweep“ hindeutet. Eine potentielle epigenetische Regulation dieser Gene durch das Genprodukt von Co könnte daher möglich sein. Mehr als die Hälfte dieser Gene lassen sich darüber hinaus aufgrund ihrer Funktion direkt mit dem kolumnaren Phänotyp assoziieren.rnDiese Ergebnisse zeigen, dass die Anwesenheit des Co-Allels massive Veränderungen in der Genregulation des SAMs mit sich bringt, wobei einige dieser differentiell regulierten Gene mit großer Wahrscheinlichkeit an der Etablierung des kolumnaren Phänotyps beteiligt sind. Auch wenn die Funktion des Co-Genproduktes nicht abschließend geklärt werden konnte, sind doch anhand der Resultate schlüssige Hypothesen diesbezüglich möglich.rn

Aureobasidium pullulans as biological control agent: modes of action

The postharvest phase has been considered an environment very suitable for successful application of biological control agents (BCAs). However, the tri-interaction between fungal pathogen, host (fruit) and antagonist is influenced by several parameters such as temperature, oxidative stresses, oxygen composition, water activity, etc. that could be determining for the success of biocontrol. Knowledge of the modes of action of BCAs is essential in order to enhance their viability and increase their potentialities in disease control. The thesis focused on the possibility to explain the modes of action of a biological control agent (BCA): Aureobasidium pullulans, in particular the strains L1 and L8, control effective against fruit postharvest fungal pathogen. In particular in this work were studied the different modes of action of BCA, such as: i) the ability to produce volatile organic compounds (VOCs), identified by SPME- gas chromatography-mass spectrometry (GC-MS) and tested by in vitro and in vivo assays against Penicillium spp., Botrytis cinerea, Colletotrichum acutatum; ii) the ability to produce lytic enzymes (exo and endo chitinase and β-1,3-glucanase) tested against Monilinia laxa, causal agent of brown rot of stone fruits. L1 and L8 lytic enzymes were also evaluated through their relative genes by molecular tools; iii) the competition for space and nutrients, such as sugars (sucrose, glucose and fructose) and iron; the latter induced the production of siderophores, molecules with high affinity for iron chelation. A molecular investigation was carried out to better understand the gene regulation strictly correlated to the production of these chelating molucules. The competition for space against M. laxa was verified by electron microscopy techniques; iv) a depth bibliographical analysis on BCAs mechanisms of action and their possible combination with physical and chemical treatments was conducted.

Response of gram-positive bacteria to copper stress

The Gram-positive bacteria Enterococcus hirae, Lactococcus lactis, and Bacillus subtilis have received wide attention in the study of copper homeostasis. Consequently, copper extrusion by ATPases, gene regulation by copper, and intracellular copper chaperoning are understood in some detail. This has provided profound insight into basic principles of how organisms handle copper. It also emerged that many bacterial species may not require copper for life, making copper homeostatic systems pure defense mechanisms. Structural work on copper homeostatic proteins has given insight into copper coordination and bonding and has started to give molecular insight into copper handling in biological systems. Finally, recent biochemical work has shed new light on the mechanism of copper toxicity, which may not primarily be mediated by reactive oxygen radicals.

Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. Results We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. Conclusions Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Alternative splicing of pre-mRNA in cancer: focus on G protein-coupled peptide hormone receptors

Through alternative splicing, multiple different transcripts can be generated from a single gene. Alternative splicing represents an important molecular mechanism of gene regulation in physiological processes such as developmental programming as well as in disease. In cancer, splicing is significantly altered. Tumors express a different collection of alternative spliceoforms than normal tissues. Many tumor-associated splice variants arise from genes with an established role in carcinogenesis or tumor progression, and their functions can be oncogenic. This raises the possibility that products of alternative splicing play a pathogenic role in cancer. Moreover, cancer-associated spliceoforms represent potential diagnostic biomarkers and therapeutic targets. G protein-coupled peptide hormone receptors provide a good illustration of alternative splicing in cancer. The wild-type forms of these receptors have long been known to be expressed in cancer and to modulate tumor cell functions. They are also recognized as attractive clinical targets. Recently, splice variants of these receptors have been increasingly identified in various types of cancer. In particular, alternative cholecystokinin type 2, secretin, and growth hormone-releasing hormone receptor spliceoforms are expressed in tumors. Peptide hormone receptor splice variants can fundamentally differ from their wild-type receptor counterparts in pharmacological and functional characteristics, in their distribution in normal and malignant tissues, and in their potential use for clinical applications.

Mechanistic and therapeutic implications of angiogenesis in endometriosis

Like tumor metastases, endometriotic implants require neovascularization to proliferate and invade into ectopic sites within the host. Endometrial tissue, with its robust stem cell populations and remarkable regenerative capabilities, is a rich source of proangiogenic factors. Among the most potent and extensively studied of these proteins, vascular endothelial growth factor has emerged as a critical vasculogenic regulator in endometriosis. Accordingly, angiogenesis of the nascent endometriotic lesion has become an attractive target for novel medical therapeutics and strategies to inhibit vascular endothelial growth factor action. Vascular endothelial growth factor gene regulation in endometrial and endometriosis cells by nuclear receptors, other transcription factors, and also by infiltrating immune cells is emphasized. New data showing that oxidative and endoplasmic reticulum stress increase vascular endothelial growth factor expression are provided. Finally, we review the clinical implications of angiogenesis in this condition and propose potential antiangiogenic therapies that may become useful in the control or eradication of endometriotic lesions.

Differential expression and activity of 11beta-hydroxysteroid dehydrogenase in human placenta and fetal membranes from pregnancies with intrauterine growth restriction

OBJECTIVES: To study the expression and the function of the 11beta-hydroxysteroid dehydrogenase enzyme 1 (11beta-HSD1) and 2 (11beta-HSD2) in placenta and the fetal membranes from pregnancies with intrauterine growth restriction (IUGR) and from controls. METHODS: Amnion, chorion, decidua and cotyledon were separated from placenta; mRNA was analyzed by TaqMan real-time technology and proteins by Western blot; enzyme activities were measured by the conversion of 3H-cortisol to 3H-cortisone and vice versa. RESULTS: Predominant mRNA expression (p < 0.001) was found for 11beta-HSD1 in chorion and for 11beta-HSD2 in decidua and cotyledon. In pregnancies with IUGR, 11beta-HSD1 was upregulated in chorion (mean DeltaCt 11beta-HSD:18S mRNA 193.5 vs. 103.0 in controls respectively, p < 0.05) and 11beta-HSD2 was downregulated in decidua (mean DeltaCt 11beta-HSD2:18S mRNA 0.18 vs. 15.88 in controls respectively, p < 0.05). 11beta-HSD1 protein levels were reduced in amnion and 11beta-HSD1 and 11beta-HSD2 oxidase activity in decidua and cotyledon were reduced from pregnancies with IUGR. CONCLUSION: Reduced synthesis or activity of 11beta-HSD1 or 2 in cases of IUGR is shown in some but not in all tissues. The local mRNA expression of 11beta-HSD1 in chorion may reflect a mechanism on the post-transcriptional gene regulation to stimulate the formation of cortisone in IUGR. To provoke increasing activity with oxidase stimulators could be a future therapy in cases of IUGR.

Induction of tenascin-C by cyclic tensile strain versus growth factors: distinct contributions by Rho/ROCK and MAPK signaling pathways

Expression of the extracellular matrix (ECM) protein tenascin-C is induced in fibroblasts by growth factors as well as by tensile strain. Mechanical stress can act on gene regulation directly, or indirectly via the paracrine release of soluble factors by the stimulated cells. To distinguish between these possibilities for tenascin-C, we asked whether cyclic tensile strain and soluble factors, respectively, induced its mRNA via related or separate mechanisms. When cyclic strain was applied to chick embryo fibroblasts cultured on silicone membranes, tenascin-C mRNA and protein levels were increased twofold within 6 h compared to the resting control. Medium conditioned by strained cells did not stimulate tenascin-C mRNA in resting cells. Tenascin-C mRNA in resting cells was increased by serum; however, cyclic strain still caused an additional induction. Likewise, the effect of TGF-beta1 or PDGF-BB was additive to that of cyclic strain, whereas IL-4 or H2O2 (a reactive oxygen species, ROS) did not change tenascin-C mRNA levels. Antagonists for distinct mitogen-activated protein kinases (MAPK) inhibited tenascin-C induction by TGF-beta1 and PDGF-BB, but not by cyclic strain. Conversely, a specific inhibitor of Rho-dependent kinase strongly attenuated the response of tenascin-C mRNA to cyclic strain, but had limited effect on induction by growth factors. The data suggest that regulation of tenascin-C in fibroblasts by cyclic strain occurs independently from soluble mediators and MAPK pathways; however, it requires Rho/ROCK signaling.

The Eyes Have It: Regulatory and Structural Changes Both Underlie Cichlid Visual Pigment Diversity

A major goal of evolutionary biology is to unravel the molecular genetic mechanisms that underlie functional diversification and adaptation. We investigated how changes in gene regulation and coding sequence contribute to sensory diversification in two replicate radiations of cichlid fishes. In the clear waters of Lake Malawi, differential opsin expression generates diverse visual systems, with sensitivities extending from the ultraviolet to the red regions of the spectrum. These sensitivities fall into three distinct clusters and are correlated with foraging habits. In the turbid waters of Lake Victoria, visual sensitivity is constrained to longer wavelengths, and opsin expression is correlated with ambient light. In addition to regulatory changes, we found that the opsins coding for the shortest-and longest-wavelength visual pigments have elevated numbers of potentially functional substitutions. Thus, we present a model of sensory evolution in which both molecular genetic mechanisms work in concert. Changes in gene expression generate large shifts in visual pigment sensitivity across the collective opsin spectral range, but changes in coding sequence appear to fine-tune visual pigment sensitivity at the short-and long-wavelength ends of this range, where differential opsin expression can no longer extend visual pigment sensitivity.

Quantification of global DNA methylation with infrared fluorescence in liver and muscle tissues of differentially fed boars

Methylation of cytosine residues at CpG sites is involved in various biological processes to control gene regulation and gene expression. Global DNA methylation is changed in different tumors and in cloned animals. Global DNA methylation can be accurately quantified by dot blot analysis with infrared (IR) fluorophores. Methylated lambda DNA was used as model DNA to develop and validate an immunochemical assay with IR fluorescence detection. Two different IR fluorophores were used, one to detect 5-methylcytosine and another to account for DNA loading. A sensitive infrared detection method was established which is suitable for accurate and reproducible quantification of global DNA methylation across a wide dynamic range. This method was subsequently employed to quantify global DNA methylation in liver and in muscle tissues of boars which have received either a control diet or a methyl supplemented diet in an ongoing study. A significant difference in global DNA methylation is indicated in muscle but not in liver tissue between the two groups of boars.

The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis. Trypanosomes are early diverged protozoan parasites and show significant differences in their gene expression compared with higher eukaryotes. Due to a lack of individual gene promoters, large polycistronic transcripts are produced and individual mRNAs mature by trans-splicing and polyadenylation. In the absence of transcriptional control, regulation of gene expression occurs post-transcriptionally mainly by control of transcript stability and translation. Regulation of mRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about mRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved, only a few orthologues can be readily identified in the genome of T. brucei. Hence, biochemical approaches are needed to identify the export machinery of trypanosomes. Here, we report the functional characterization of the essential mRNA export factor TbMex67. TbMex67 contains a unique and essential N-terminal zinc finger motif. Furthermore, we could identify two interacting export factors namely TbMtr2 and the karyopherin TbIMP1. Our data show that the general heterodimeric export receptor Mex67-Mtr2 is conserved throughout the eukaryotic kingdom albeit exhibiting parasite-specific features.

FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.