This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

It is clear that dendritic cells (DCs) are essential for priming of T cell responses against tumors. However, the distinct roles DC subsets play in regulation of T cell responses in vivo are largely undefined. In this study, we investigated the capacity of ovalbumin (OVA)-presenting CD48, CD4+8, or CD48+ DCs (OVA-pulsed DC (DCOVA)) from mouse spleen in stimulation of OVA-specific T cell responses. Our data show that each DC subset stimulated proliferation of allogeneic and autologous OVA-specific CD4+ and CD8+ T cells in vitro, but that the CD48 DCs did so only weakly. Both CD4+8 and CD48+ DCOVA induced strong tumor-specific CD4+ Th1 responses and fully protective CD8+ cytotoxic T lymphocyte (CTL)-mediated antitumor immunity, whereas CD48 DCOVA, which were less mature and secreted substantial transforming growth factor (TGF- ) upon coculture with T cell receptor (TCR)-transgenic OT II CD4+ T cells, induced the development of interleukin-10 (IL-10)-secreting CD4+ T regulatory 1 (Tr1) cells. Transfer of these Tr1 cells, but not T cells from cocultures of CD48 DCOVA and IL-10/ OT II CD4+ T cells, into CD48+ DCOVA-immunized animals abrogated otherwise inevitable development of antitumor immunity. Taken together, CD48 DCs stimulate development of IL-10-secreting CD4+ Tr1 cells that mediated immune suppression, whereas both CD4+8 and CD48+ DCs effectively primed animals for protective CD8+ CTL-mediated antitumor immunity. <p> Different DC subsets play distinct roles in immune responses. CD4-8- DCs secreting TGF-â stimulate CD4+ regulatory T type 1 (Trl) cell responses leading to inhibition of CD8 CTL responses and antitumor immunity. In this study, we explored the potential effect of three stimuli CpG, lipopolysaccharide (LPS) and anti-CD40 antibody in conversion of CD4-8- DC-induced tolerance. We demonstrated that when CD4-8- DCs were isolated from overnight culture and cultured for another 8 hrs in AIM-V plus recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) (15-20 ng/ml) and OVA (0.1 mg/ml) with CpG (5 ug/ml), LPS (2 ug/ml) and anti-CD40 antibody (10 ug/ml), their phenotype became more mature compared with the freshly isolated ones. CpG is the only agent that stimulates the DCs to secrete significant level of interleukin-6 (IL-6) and interleukin-15 (IL-15); DNA array analyses also indicate that CpG stimulates higher expression of IL-6 and IL-15 mRNA. CpG treatment most efficiently converts the tolerogenic DCs into immunogenic ones which stimulated the OTII CD4+ T cell to become T helper type 1 (Th1) and T helper type 17 (Th17) rather Tr1, while the other two stimulator-treated DCs could not induce Th17 response. Their vaccination also induced the strongest antitumor CTL responses and protective immunity against tumor cell challenge. When CD4-8- DCs were isolated from IL-6 knock out (IL-6-/-) mice, CpG-treated DCOVA vaccination almost completely lost their animal protection capacity. Wild type B6 DCOVA-vaccinated IL-15 receptor knock out (IL-15R-/-) mice can only provide up to 30% protection against tumor challenge. Those results indicate that IL-6/ IL-l5-induced Th17 plays a critical role in their conversion. Taken together, our findings indicate that CpG treatment is the most efficient agent that can convert tolerogenic DCs into immunogenic ones and induce long-lasting antitumor immunity. We previously demonstrated that the nonspecific CD4+ T cells can acquire antigen-specific DC-released exosomes (EXO) and these CD4+ T cells with acquired exosomal MHC I peptide complex (pMHC I) can stimulate antigen-specific CD8+ CTL responses. In my project we have found that CD4-8-DCs could induce regulatory T cell type 1(Tr1) response, thus it would be very necessary to know whether regulatory T cells would change their antigen specificity if they got the membrane complex from DC through coculture or DC-derived exosome pulsing. During the beginning of my regulatory T cell project, we found that CD8+CD25+ Tr were much more easily expanded, while CD4+CD25+ Tr usually began to die just after 3 days in vitro culture and its very hard to get enough cells for further research. Therefore, CD8+CD25+ were used as a model Tr cells in the following project. To assess whether the nonspecific CD8+CD25+ Tr cells can acquire antigen-specificity via acquired exosomal pMHC I, we purified CD8+CD25+ Tr cells from wild-type C57BL/6 mice and OVA-pulsed DCOVA-released EXOOVA expressing pMHC I complexes. We demonstrated that the nonspecific CD8+CD25+ Tr cells expressing forkhead box P3 (Foxp3), cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), perforin and granzyme B inhibited in vitro T cell proliferation and in vivo OVA-specific CD4+ T cell-dependent and independent CD8+ CTL responses and antitumor immunity. CD8+CD25+ Tr cells suppressive effect is possibly mediated through its inhibition of DC maturation, down-regulation of secretion of Th1 polarization cytokines by DCs and its induction of T cell anergy via cell-to-cell contact. The nonspecific CD8+CD25+ Tr cells acquired antigen specificity by uptake of DCOVA-released EXOOVA expressing pMHC I and enhanced its effect on inhibition of OVA-specific CD8+ T cell responses and antitumor immunity by 10-folds. The principles elucidated in this study may have significant implications not only in antitumor immunity, but also in other sectors of immunology (e.g, autoimmunity and transplantation).

This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.

Galectins are a family of secreted animal lectins with a high affinity to betagalactosides commonly involved in cellular functions such as apoptosis, adhesion and migration. Galectin 15 (LGALS15), a newest member of the galectin superfamily, has a unique C-terminal RGD sequence and participates in integrin-mediated ovine trophectoderm cell attachment and migration. In the ovine uterus, LGALS15 is expressed only by the endometrial luminal (LE) and superficial glandular (sGE) epithelia, induced by progesterone between Days 10 and 12 of the cycle and pregnancy, and then stimulated by interferon tau (IFNT) from the conceptus after Day 14 of pregnancy. During early pregnancy, the canonical janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is not active in the endometrial LE/sGE. Therefore, IFNT may utilizes a non-canonical signaling pathway to increase transcription of genes, including CST3, CTSL, HIF2A, LGALS15, and WNT7A, specifically in the endometrial LE/sGE. Alternatively, IFNT and progesterone could indirectly affect epithelial gene expression by influencing gene expression in the stroma, which then communicates with the epithelium. Although the LGALS15 gene is present in ovine, caprine and bovine species, it is only expressed in uteri of sheep and goats. Available data shows a tissue- and speciesspecific expression pattern for LGALS15, likely involving multiple layers of transcription regulation in the ruminant endometrium. Further analysis of the LGALS15 5? promoter/enhancer region revealed similar predicted transcription factor binding sites in all three species, including; PU.1, Ets-1, AP1, Sp1, and GRE or PRE sites. Interestingly, the proximal promoter region of the LGALS15 gene in all three species exhibited a conserved Sp1 binding site upstream of an AP1 binding site on both sense and antisense strands, and with similar spacing between binding sites. Sequence analysis revealed key differences in LGALS15 gene structure between ruminant species including the proximity of repetitive DNA sequences to the transcription start site (+1). Bovine LGALS15 has repetitive DNA sequences start at - 145 whereas in ovine or caprine LGALS15 it starts at about -300. The length of the repetitive DNA sequence is similar (~1.2 kb) in the 5' promoter/enhancer region of LGALS15 in all three species. Transient transfection analyses found that repetitive DNA sequences reduced basal promoter activity and responsiveness to treatments. None of the promoter construct showed responsiveness to interferon tau (IFNT). The bovine LGALS15 gene promoter showed no activity under any experimental conditions. The current studies indicate that uterine LGALS15 is expressed in ovine and caprine but not bovine species. Additionally, repetitive DNA sequences found in the promoter region may contribute to modulating the LGALS15 gene expression. Therefore, the ruminant LGALS15 gene, like other galectins, is under tight transcriptional control involving hormones, requisite transcription factors and potentially chromatin remodeling complexes working synergistically for LGALS15 promoter transactivation.

Recently, the prediction of promoters has attracted many researchers' attention. Unfortunately, most previous prediction algorithms did not provide high enough sensitivity and specificity. The goal of this thesis is to develop an efficient prediction algorithm that can increase the detection power (power = 1 - false negative). We do not try to find more distinct features in promoters one by one, such as transcriptional elements. Our main idea is to use the computer power to calculate all possible patterns which are the possible features of promoters. Accordingly, we shall define some scoring methods for training a given set of sequences, which involve promoter sequences and non-promoter sequences. Then, we can obtain a threshold value for determining whether a testing sequence is a promoter or not. By the experimental results, our prediction has higher correct rate than other previous methods.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-144).

Toll-like receptors (TLRs) recognize microbial components as danger signals and induce immune responses. TLR’s are expressed in many tissues of the host that are involved in immune responses including the intestines where they are abundantly expressed. This situation presents a challenge in the gastrointestinal tract which is constantly exposed to a wide variety of commensal organisms. Therefore, innate immune recognition in the intestine must be tightly regulated to prevent unwanted inflammation against harmless commensal micro-organisms and yet allow for the induction of protective immunity to invading pathogens. A dysregulation of this balance can result in intestinal inflammation. Peyer’s patches (PP) are the primary site for the induction of immune responses in the intestine and abundantly express TLRs. It is not known how PP regulate microbial signals from commensal bacteria and yet mount vigorous immune responses against dangerous pathogens. CpG DNA, an agonist for TLR9, can strongly activate immune cells in blood, lymph nodes and spleen. However, CpG very poorly activates immune cells from Peyer’s patches, although these cells express TLR9 [1, 2]. Understanding how TLR responses are regulated in PP cells will unveil important information on how immune responses are regulated in the intestine. Investigations from our laboratory have revealed a B cell population (CD5-CD11c-CD21+) in PP that spontaneously secrete high levels of IL-10 which in turn down regulates TLR9 induced IFN and IL-12 production. These IL-10-secreting PP B cells represent a novel subset of the recently proposed regulatory B cells (Bregs) in the intestine [1, 3]. Bregs may have a role in maintaining tolerance to commensal bacteria thereby achieving intestinal homeostasis. The overall goal of the work described in this thesis was to improve our understanding of the immunobiology of Bregs. We performed several experiments to achieve this goal. First, we studied the development of regulatory B cells in lambs of different ages. Jejunal PP were collected from 3-4 month old, neonatal and fetal lambs and the production of IL-10 (the immunoregulatory cytokine secreted by Bregs) was assayed. We found that IL-10 was secreted by CD21+ B cells from the PP in all the three age groups, confirming that Bregs develop prior to birth. We then wondered whether our CD21+ B cells might be contaminated with other cells or activated when using MACS to enrich B cells. To address this issue, we prepared very highly purified CD21+ B cell population using high speed cell sorting to negatively enrich for B cells. We also sorted DCs and assayed IL-10 production in both cell populations. Only the PP B cells spontaneously secreted IL-10. In contrast, dendritic cells, T cells, macrophages, neutrophils and NK cells did not secrete detectable IL-10. Since B cells exist as regulatory and effector populations in mice, we wondered whether an effector B cell population also existed in ovine PP that secreted the pro-inflammatory cytokines IFN-, IFN- and IL-12. Therefore, ovine PP B cells were fractionated into CD72+CD21+and CD72+CD21- subpopulations to assess their capacity to secrete pro-inflammatory cytokines. Interestingly, the CD72+CD21- B cell population secreted the cytokines IFN-, IFN- and IL-12 suggesting there was an effector population. We then surveyed for Bregs in different mucosal and peripheral tissues in sheep. We observed the Bregs frequency varied among the different lymphoid tissues. Finally, we investigated whether Bregs were present in PP of other ruminant species. We identified Bregs exist in PP of neonatal calves. In conclusion, our investigations reveal that ovine Bregs develop in utero prior to antigen exposure, and are present in a variety of mucosal and peripheral tissues. We also report the novel observation that two distinct B cell sub-populations are present in ovine jejunal PP’s: Regulatory and effector B cells.

In eukaryotic genomes ranging from plants to mammals, DNA methylation is a major epigenetic modification of DNA by adding a methyl group exclusively to cytosine residuals. In mammalian genomes such as humans, these cytosine bases are usually followed by guanine. Although it does not change the primary DNA sequence, this covalent modification plays critical roles in several regulatory processes and can impact gene activity in a heritable fashion. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant DNA methylation is implicated in several human diseases, in particular in neuro-developmental syndromes (such as the fragile X and Rett syndromes) and cancer. These biological significances disclose the importance of understanding genomic patterns and function role of DNA methylation in human, as a initial step to get to know the epigenotype and its manner in connecting the phenotype and genotype. Two key papers back in 1975 independently suggested that methylation of CpG dinucleotides in vertebrates could be established de novo and inherited through somatic cell divisions by protein machineries of DNA methyltransferases that recognizes hemi-methylated CpG palindromes. They also indicated that the methyl group could be recognized by DNA-binding proteins and that DNA methylation directly silences gene expression. After almost four decades, several key points in these foundation papers are proved to be true. Take the mammalian genome for example, there are several findings indicating the epigenetic repression of gene expression by DNA methylation. These include X-chromosome inactivation, gene imprinting and suppressing the proliferation of transposable elements and repeat elements of viral or retroviral origin. In addition to these, many novel roles of DNA methylation have also been revealed. For example, DNA methylation can regulate alternative splicing by preventing CTCF, an evolutionarily conserved zinc-finger protein, binding to DNA. By using the technique of fluorescence resonance energy transfer (FRET) and fluorescence polarization, DNA methylation has also been shown to increase nucleosome compaction through DNA-histone contacts. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant change of DNA methylation has been related to disease such as cancer. However, it is also notable there are several lines of evidence contradicting the relationship between DNA methylation and gene silencing. For example, comparison of DNA methylation levels in human genome on the active and inactive X chromosomes showed reduced methylation specifically over gene bodies on inactive X chromosomes. Not only in human, DNA methylation is found to be usually targeted to the transcription units of actively transcribed genes in invertebrate species. These results prove that the function of DNA methylation is challenging to be unravel. Besides, due to the development of sequencing technique, whole genome DNA methylation profiles have been detected in diverse species. Comparing genomic patterns of DNA methylation shows considerable variation among taxa, especially between vertebrates and invertebrates. However, even though extensive studies reveal the patterns and functions of DNA methylation in different species, in the mean time, they also highlight the limits to our understanding of this complex epigenetic system. During my Ph.D., in order to perform in-depth studies of DNA methylation in diverse animals as a way to understand the complexity of DNA methylation and its functions, I dedicated my efforts in investigating and analyzing the DNA methylation profiles in diverse species, ranging from insects to primates, including both model and non-model organisms. This dissertation, which constitutes an important part of my research, mainly focuses on the DNA methylation profile in primates including human and chimpanzee. In general, I will use three chapters to elucidate my work in generating and interpreting the whole genome DNA methylation data. Firstly, we generated nucleotide-resolution whole-genome methylation maps of the prefrontal cortex of multiple humans and chimpanzees, then comprehensive comparative studies for these DNA methylation maps have been performed, by integrating data on gene expression as well. This work demonstrates that differential DNA methylation might be an important molecular mechanism driving gene-expression divergence between human and chimpanzee brains and also potentially contribute to the human-specific traits, such as evolution of disease vulnerabilities. Secondly , we performed global analyses of CpG islands (CGIs) methylation across multiple methylomes of distinctive cellular origins in human. The results from this work show that the human CpG islands can be distinctly classified into different clusters solely based upon the DNA methylation profiles, and these CpG islands clusters reflect their distinctive nature at many biological levels, including both genomic characteristics and evolutionary features. Moreover, these CpG islands clusters are non-randomly associated with several important biological phenomena and processes such as diseases, aging, and gene imprinting. These new findings shed lights in deciphering the regulatory mechanisms of CpG islands in human health and diseases. At last, by utilizing the DNA methylome from human sperm and genetic map generated from the International HapMap Consortium project, we investigated the hypothesis suggesting a potential role of germ line DNA methylation in affecting meiotic recombination, which is essential for successful meiosis and various evolutionary processes. Even thought the results imply that DNA methylation is a important factor affecting regional recombination rate, the strength of correlation between these two is not as strong as the previous report. Besides, high-throughput analyses indicate that other epigenetic modifications, tri-methylation of histone 3 lysine 4 and histone 3 lysine 27 are also global features at the recombination hotspots, and may interact with methylation to affect the recombination pattern simultaneously. This work suggests epigenetic mechanisms as additional factors affecting recombination, which cannot be fully explained by the DNA sequence itself. In summary, I hope the results from these work can expand our knowledge regarding the common and variable patterns of DNA methylation in different taxa, and shed light about the function role and its major change during animal evolution.

The rates and patterns at which nucleotide substitutions occur vary significantly across the genome sequence of vertebrates. A prominent example is the difference in the rate of evolution of functional sequences versus nonfunctional (neutrally evolving) sequences, which is explained by the influence of natural selection on functional sequences. However, even within neutrally evolving sequences there is striking variation in the rates and patterns of nucleotide substitutions. Unraveling the underlying processes that induce this variation is necessary to understand the basic principles of variation in neutral substitution profiles, which in turn is crucial for the identification of regions in the genome where natural selection acts. This research question builds the main focus of the present thesis. I have studied the causes and consequences of variation in different patterns of nucleotide substitutions. In particular, I have investigated substitutional strand asymmetries in mammalian genes and could show that they result from the asymmetric nature of DNA replication and transcription. Comparative analysis of substitutional asymmetries then suggested that the organization of DNA replication and the level of transcription are conserved among mammals. Further, I have examined the variation in CpG mutation rate among human genes and could show that beside DNA methylation also GC content plays a decisive role in CpG mutability. In addition, I have studied the signatures of GC-biased gene conversion and its impact on the evolution of the GC isochore structure in chicken. By comparison of the results in chicken to previous results in human I found evidence that karyotype stability is critical for the evolution of GC isochores. Finally, beside the empirical studies, I have performed theoretical investigations of substitution rates in functional sequences. More precisely, I have explored the temporal dynamics of estimates of the ratio of non-synonymous to synonymous substitution rates dN/dS in a phylogentic-population genetic framework.