This thesis details my examination of several mechanisms for modulation of N-type calcium channels in neonatal rat superior cervical ganglion (SCG) neurons. The first part of this work characterizes cross-talk between two distinct mechanisms of modulation: readily-reversible inhibition induced by activation of heterotrimeric G-proteins (termed G-protein-mediated inhibition), and phosphorylation of the channel by protein kinase C (PKC). Data previously presented by other groups suggested that one effect of activating PKC is to prevent G-protein-mediated inhibition. The goal of this project was to confirm this hypothesis by testing functional competition between these two pathways. My findings show that G-protein-mediated inhibition blocks the effects of activating PKC, and that phosphorylation by PKC blocks G-protein-mediated inhibition, confirming that these two mechanisms are mutually exclusive. In addition, I investigated the effect of activating PKC on whole-cell barium currents in the absence of G-protein-mediated inhibition. When endogenous G-proteins were inactivated by dialyzing the cell with GDP-β-S, a guanine nucleotide that prevents activation of the G-protein's α subunit, activation of PKC with phorbol esters was without obvious effect on whole-cell current amplitude, fast and holding potential-dependent inactivation, and voltage-dependent activation, suggesting that PKC's principal role in modulating these currents is to prevent G-protein-mediated inhibition. From these results, I advanced Bean's 1989 model of reluctant and willing gating (induced by G-protein-mediated inhibition and relief of that inhibition, respectively). In this expanded model, reluctant channels, inhibited by G-proteins, are resistant to phosphylation by PKC (reluctant/P-resistant). Unmodulated channels are called willing/available, as they exhibit willing gating, and are available for either binding to a G-protein or phosphorylation by PKC. Finally, phosphorylation of a willing/available channel by PKC drives the channel into the willing/G-resistant state, in which the channel gates willingly, and is resistant to G-protein-mediated inhibition. These results are published in the Journal of General Physiology(2000; 115:277-286), and are presented in this thesis as Chapter II. In addition to membrane-delimited inhibition, N-type calcium channels are also subject to inhibition via a diffusible second-messenger pathway. In SCG neurons, this inhibition can be observed following stimulation of M1 muscarinic receptors by the agonist oxotremorine-M. Our lab previously hypothesized that the diffusible messenger involved might be the polyunsaturated fatty acid arachidonic acid (AA). To test this hypothesis, our lab examined the effect of bath-applied AA on whole-cell SCG calcium currents, and demonstrated that AA induces inhibition with similar properties as M1 muscarinic inhibition. An analysis of AA's effects on unitary N-type calcium currents, published by Liu and Rittenhouse in Journal of Physiology(2000; 525:391-404), revealed that this inhibition is mediated, at least in part, by both a significant increase in the occurrence of null-activity sweeps and a significant decrease in mean closed dwell time. Based on these results, our lab conducted an examination of AA's effects on whole-cell currents in SCG neurons, and found that AA-induced inhibition is mediated by an increase in holding potential-dependent inactivation and appears independent of AA metabolism. When I examined AA's effects in greater detail, I discovered that, in addition to inhibition, AA also appeared to cause significant enhancement of whole-cell currents. The results characterizing AA's general effects on whole-cell calcium currents in SCG neurons have been published in American Journal of Physiology - Cell Physiology(2001; 280:C1293-C1305). Because my finding that AA enhances whole-cell neuronal calcium currents revealed a novel pathway through which this current can be modulated, I proceeded to characterize this effect. My results showed that enhancement develops significantly faster than inhibition, suggesting different mechanisms or pathways. In addition, dialyzing the cell with BSA, a protein that binds fatty acids, blocked the majority of AA-induced inhibition, but did not reduce enhancement, suggesting that enhancement is independent of inhibition and might be mediated at an extracellular site. Using fatty acid analogs that cannot cross the cell membrane, I confirmed that enhancement occurs extracellularly. My data also indicate that AA-induced enhancement of whole-cell currents does not require metabolism of AA, consistent with enhancement being mediated directly by AA. I also examined the biophysical characteristics of enhancement, and found that both an increase in the voltage sensitivity of activation and an increase in activation kinetics underlie this effect. Finally, using both pharmacological agents and a recombinant cell line, I presented the first demonstration that AA enhances N-type calcium current. These findings are described in detail in a paper recently published in American Journal of Physiology - Cell Physiology(2001; 280:C1306-C1318), and are presented in this thesis as Chapter III. In our investigation of AA's effects on whole-cell calcium currents, we utilized a voltage protocol, in conjunction with pharmacology, to enhance the level of L-type current in these cells. Since whole-cell calcium currents in SCG neurons are comprised of mostly (80-85%) N-type current, with the remaining current comprised of mostly L-type current, this approach allowed us to examine both N- and L-type currents. When currents are recorded in the presence of 1 μM FPL 64174 (FPL), a benzoyl pyrrole L-type calcium channel agonist first described in 1989, stepping the membrane potential to -40 mV following a test pulse to +10 mV generates a slowly-deactivating ("tail") current. This tail current is made up entirely of L-type current, and allows us to readily investigate the effect of various modulatory mechanisms on this current type. Although FPL has been used for almost a decade to study L-type calcium currents, activity of FPL on N-type calcium currents has not been investigated. Because our lab routinely uses micromolar concentrations of FPL to measure whole-cell and unitary calcium currents in neuronal cells, I tested whether FPL has any effects on N-type calcium current. Therefore, I examined the effect of FPL on whole-cell calcium currents in an HEK 293 cell line that expresses recombinant N-type calcium channels. Application of 1 and 10 μM FPL caused significant, voltage-independent inhibition of currents, demonstrating that FPL inhibits N-type calcium current. Thus, at micromolar concentrations, FPL is not selective for L-type calcium current, and any examination of its effects on whole-cell calcium currents should take this into account. The results describing FPL's effects on L- and N-type calcium currents are included in a manuscript currently in preparation, and are presented as Chapter IV.

Coordination of mitosis and cytokinesis is crucial to generate healthy daughter cells with equal amounts of genetic and cytoplasmic materials. In the fission yeast Schizosaccharomyces pombe, an evolutionarily conserved Cdc14-like phosphatase (Clp1) functions to couple mitosis and cytokinesis by antagonizing CDK activity. The activity of Clp1 is thought to be regulated in part by its subcellular localization. It is sequestered in the nucleolus and the spindle pole body (SPB) during interphase. Upon mitotic entry, it is released into the cytoplasm and localized to the kinetochores, the actomyosin ring, and the mitotic spindle to carry out distinct functions. It is not clear how Clp1 is released from the nucleolus, however, once released, a conserved signaling pathway termed Septation Initiation Network (SIN) functions to retain Clp1 in the cytoplasm until completion of cytokinesis. The SIN and Clp1 function together in a positive feedback loop to promote each other’s activity. That is, the SIN promotes cytoplasmic retention of Clp1, and cytoplasmic Clp1 antagonizes CDK activity and reverses CDK inhibition on the SIN pathway to promote its function and activity. However, at the start of this thesis, the mechanism by which the SIN regulated Clp1 was unknown. The SIN pathway is also required to promote constriction of the actomyosin ring, and the septum formation. However, its downstream targets were still uncharacterized. In two separate studies, we studied how Clp1 is released from the nucleolus at mitotic entry and how the SIN kinase Sid2 acts to retain Clp1 in the cytoplasm. We identified several Sid2 candidate substrates, and revealed other functions of the SIN pathway in coordinating mitotic events.

The Smad proteins transduce the signal of transforming growth factor-β (TGF-β) and related factors from the cell surface to the nucleus. Following C-terminal phosphorylation by a corresponding receptor kinase, the R-Smad proteins form heteromeric complexes with Smad4. These complexes translocate into the nucleus, bind specific transcriptional activators and DNA, ultimately modulating gene expression. Though studied through a variety of means, the stoichiometry of the R-Smad/Smad4 complex is unclear. We investigated the stoichiometry of the phosphorylation-induced R-Smad/Smad4 complex by using acidic amino acid substitutions to simulate phosphorylation. Size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry analysis revealed that the R-Smad/Smad4 complex is a heterotrimer consisting of two R-Smad subunits and one Smad4 subunit. In addition, a specific mechanism for phosphorylation-induced R-Smad/Smad4 complex formation was studied. Although it had been previously established that part of the mechanism through which phosphorylation induces Smad oligomerization is through relieving MH1-domain mediated autoinhibition of the MH2 (oligomerization) domain, it is also evident that phosphorylation serves to energetically drive Smad complex formation. Through mutational and size exclusion chromatography analysis, we established that phosphorylation induces oligomerization of the Smads by creating an electrostatic interaction between the phosphorylated C-terminal tail of one R-Smad subunit in a Smad trimer with a basic surface on an adjacent R-Smad or Smad4 subunit. The basic surface is defined largely by the L3 loop, a region that had previously been implicated in R-Smad interaction with the receptor kinase. Furthermore, the Smad MH2 domain shares a similar protein fold with the phosphoserine and phosphothreonine-binding FHA domains from proteins like Rad53 and Chk2. Taken together, these results suggest that the Smad MH2 domain may be a distinct phospho serine-binding domain, which utilizes a common basic surface to bind the receptor kinase and other Smads, and takes advantage of phosphorylation-induced allosteric changes dissociate from the receptor kinase and oligomerize with other Smads. Finally, the structural basis for the preferential formation of the R-Smad/Smad4 heterotrimeric complex over the R-Smad homotrimeric complex was explored through X-ray crystallography and isothermal titration calorimetry. Crystal structures of the Smad2/Smad4 and Smad3/Smad4 complexes revealed that specific residue differences in Smad4 compared to R-Smads resulted in highly favorable electrostatic interactions that explain the preference for the interaction with Smad4.

The study of DNA templated events is not complete without considering the chromatin environment. Histone modifications help to regulate gene expression, chromatin compaction and DNA replication. Because DNA damage repair must occur within the context of chromatin, many remodeling enzymes and histone modifications work in concert to enable access to the DNA and aid in restoration of chromatin after repair is complete. CK2 has recently been identified as a histone modifying enzyme. In this study we identify CK2 as a histone H3 tail kinase in vitro, identify the phospho-acceptor site in vitro, and characterize the modification in vivo in S. cerevisiae. We also characterize the DNA damage phenotype of a strain lacking a single catalytic subunit of CK2. We further characterize the CK2- dependent phosphorylation of serine 1 of histone H4 in vivo. We find that it is recruited directly to the site of a DSB and this recruitment requires the SIN3/RPD3 histone deacetylase complex. We also characterize the contribution of H4 serine 1 phosphorylation in chromatin compaction by using reconstituted nucleosomal arrays to study folding in the analytical ultracentrifuge.

Oxidative stress is a cellular condition where cells are challenged by elevated levels of reactive oxygen species (ROS) that are produced endogenously or exogenously. ROS can damage vital cellular components, including lipid, protein, DNA and RNA. Oxidative damage to DNA often leads to cell death or mutagenesis, the underlying cause of various human disease states. Previously our laboratory discovered that human PC4 gene can prevent oxidative mutagenesis in the bacterium Escherichia coli and that the yeast homolog SUB1 has a conserved function in oxidation protection. In this thesis I examined the underlying mechanisms of PC4’s oxidation protection function. My initial efforts to examine the predicted role of SUB1 in transcription-coupled DNA repair essentially negated this hypothesis. Instead, results from our experiments suggest that PC4 and yeast SUB1 can directly protect genomic DNA from oxidative damage. While testing SUB1’s role in double strand DNA break (DSB) repair, I found the sub1Δ mutant resects DSB ends rapidly but still ligates chromosomal breaks effectively, suggesting that DSB resection is not inhibitory to nonhomologous end-joining, an important DSB repair pathway. Finally, in the course of studying transcription recovery after UV damage, I found UV induces a longer form of RPB2 mRNA and demonstrated that this is caused by alternative polyadenylation of the RPB2 mRNA and that alternative polyadenylation contributes to UV resistance. Based on results of preliminary experiments, I propose that UV activates an alternative RNA polymerase to transcribe RNA POL II mRNA, a novel mechanism to facilitate recovery from inhibition of transcription resulting from UV damage. The hypothetical polymerase switch may account for the UV-induced alternative polyadenylation of the RPB2 mRNA.

Histone lysine methylation is an important epigenetic mark for regulation of gene expression and chromatin organization. Setdb1 (Set domain, bifurcate 1), one of the histone lysine methyltransferases, specifically methylates histone H3 at lysine 9 (H3K9) and participates in transcriptional repression and heterochromatin formation. The major task of my thesis work was to investigate the epigenetic roles of Setdb1 in regulating brain functions. I started my thesis work by examining Setdb1 expression pattern during mouse brain development. The most robust signal of Setdb1 was detected in the fetal brains at embryonic day 12.5, with a ubiquitous distribution in all the proliferative zones, as well as the cortical plate and other regions comprised of postmitotic neurons. The expression of Setdb1 decreased as the brain developed, and this down-regulation profile was correlated to neuronal maturation as examined in a primary culture model of mouse cortical neurons. I then generated CK-Setdb1 transgenic mice, in which a myc-tagged full length mouse Setdb1 was constantly expressed in postmitotic neurons under the control of the CaMK II alpha promoter (CK). The expression of mycSetdb1 was detected in NeuN positive cells throughout most forebrain regions including cerebral cortex, striatum and hippocampus. A sustained increase of Setdb1 in CK-Setdb1 transgenics was verified at both mRNA and protein levels. Furthermore, an increase of H3K9 trimethylation was detected at major satellite DNA repeats in CK-Setdb1forebrains, which indicated that transgene-expressed mycSetdb1 was functionally active in adult brains. The behavioral phenotype of CK-Setdb1 transgenics was examined by using two separate founder lines. Gross neurological functions including body weight, locomotion activity, motor coordination, and breeding behavior were generally normal in CK-Setdb1 mice. CK-Setdb1 mice were further subjected to behavioral paradigms related to mood and cognitive functions. Intriguingly, as compared to the littermate controls, CK-Setdb1 mice represent a lower level of depression as indicated by decreased total immobility in two different behavioral despair tests. Moreover, CK-Setdb1 mice showed an accelerated extinction in the learned helplessness paradigm after a delayed interval (7 days), indicating a faster recovery from an established status of despair. The potential confounding factors, like memory deficits, were ruled out as CK-Setdb1 mice showed normal or even improved performances in different memory-related paradigms. Anxiety scores and stimulant drug response were normal in CK-Setdb1mice. Taken together, these findings suggested that a specific antidepressant-like phenotype was elicited by the over-expression of Setdb1 in adult mice forebrains. To further study the molecular mechanism underlying Setdb1-associated antidepressant-like behavioral changes, I screened for Setdb1-binding sites in a genome-scale by ChIP-on-chip using a tiling microarray from Affymetrix. Unexpectedly, Setdb1 showed a very restricted binding profile with a high specificity towards ionotropic glutamate receptor genes including the NMDA receptor 2B subunit gene Grin2b, which is a new target for the treatment for major depression. An increase of H3K9 dimethylation at Setdb1-binding site on Grin2b locus was detected in CK-Setdb1 hippocampus, which was correlated to a decrease of Grin2b expression as well as an accelerated desensitization of NMDA receptor. Furthermore, Chromosome Conformation Capture (3C) on Grin2b locus revealed a repressive chromatin loop structure, which tethered the distal Setdb1-binding site (~ 32 Kb downstream of transcriptional start site (TSS)) to a proximal intronic region (~12 Kb downstream of TSS) that is enriched for the binding of KAP1, a well-studied Setdb1-interacting transcriptional corepressor. Taken together, our data indicated that Setdb1 repressed Grin2b expression via H3K9 hypermethylation and higher-order chromatin loop formation, which may contribute to the antidepressant-like phenotype we observed in CK-Setdb1mice. The second part of my thesis work was to investigate the role of Setdb1 in the animal model of a neurodevelopmental disorder - Rett syndrome (RTT). Loss-of-function mutations of the gene encoding methyl-CpG binding protein 2 (MECP2) is the primary cause of RTT. There is an overlap between Setdb1- and Mecp2-associated repressive chromatin machineries, which both include histone deacetylase complex, H3K9 methyltransferase, DNA methyltransferase and heterochromatin protein 1 (HP1). Moreover, in contrast to Setdb1, which is downregulated during the cortical neuronal differentiation, Mecp2 is upregulated and the expression level is positively correlated to neuronal maturation. Therefore, we hypothesized that there is a functional redundancy between Setdb1 and Mecp2, and the up-regulation of Setdb1 in mature neurons will compensate for brain deficiency due to the loss of Mecp2. To test this hypothesis, I crossed CK-Setdb1 transgenic mice with nestincre-Mecp2 conditional knockout mice (Mecp2-/y). The behavior changes of CK-Setdb1/Mecp2-/y mice, including body weight, locomotion, motor coordination, and life span, were then compared to Mecp2-/y mice. No significant improvements in behaviors or survival were observed from CK-Setdb1/Mecp2-/y mice. Because the activation of CK promoter is limited to defined population of postmitotic neurons in forebrain, I tested our hypothesis by generating another strain of Setdb1 overexpression mice – tauSetdb1, in which the expression of mycSetdb1 is under the control of an endogenous pan-neuronal active promoter Tau. However, the introduction of tauSetdb1 also failed to rescue Mecp2 deficiency. The life span of tauSetdb1/ Mecp2-/y was even shorter as compared to Mecp2-/y mice (Kaplan-Meier, p=0.07). In conclusion, up-regulation of Setdb1 in adult brain was not sufficient to rescue Mecp2deficiency in the mouse model of RTT. One of the most challenges to study neuronal dysfunctions in brain diseases is the cellular heterogeneity of central nervous system. Current techniques for chromatin studies, including chromatin immunoprecipitation (ChIP) assays, usually lack of single cell resolution and are unable to examine the neurobiological changes in defined cell populations. In the third part of my thesis work, I developed a modified protocol to isolate neuronal nuclei from brain homogenates via Fluorescence-Activated Cell Sorting (FACS). In general, total nuclei was extracted from frozen brains, neuronal nuclei were then immuno-tagged with NeuN and sorted via FACS. Besides the NeuN labeling-FACS protocol, I also generated CK-H2BeGFP transgenic mice, in which a histone H2B-eGFP (enhanced green fluorescent protein) fusion protein was expressed in the nuclei of postmitotic neurons in mouse forebrain. Nuclei extracted from CKH2BeGFP brain were directly applied for FACS sorting. By using this protocol, we routinely got around 6-8 x106neuronal nuclei from one adult mouse forebrain, which was sufficient for ChIP applications followed by single gene PCR and microarray studies. In conclusion, our protocol permits large-scale studies of chromatin modifications or any other nuclei events in defined cell populations from distinct brain regions. Taken together, my dissertation work will lead to a better understanding of the epigenetic roles of histone H3K9 methyltransferase Setdb1 in brain functions, and may provide new targets for the therapeutic treatment of major depression.

Cancer is the second leading cause of death among men and women after heart disease. Though our knowledge associated with the complexities of the cancer network has significantly improved over the past several decades, we have only recently started to get a more complete molecular understanding of the disease. To better comprehend signaling pathways that prevent disease development, we focused our efforts on investigating endogenous tumor suppression networks in controlling effectors of cancer cell survival and proliferation. Survivin is one such effector molecule that controls both cell proliferation and survival. In order to identify how this protein is overexpressed in cancer cells as opposed to normal cells, we looked at signaling molecules that negatively regulate this inhibitor of apoptosis protein. PTEN and caspase 2 are two of the identified proteins that utilize their enzymatic activity to suppress tumor growth by inhibiting downstream cell survival effectors, namely survivin. PTEN uses its phosphatase activity to suppress the PI3K/AKT pathway and maintain cellular homeostasis. In the absence of AKT activity, FOXO transcription factors are able to target downstream gene expression and regulate cell proliferation and survival. Here we have identified survivin as a novel gene target of FOXO, which binds to a specific promoter region of survivin and suppresses its transcription. Alternatively, caspase 2 uses its catalytic activity to suppress survivin gene expression by targeting the NFκB pathway. Caspase 2 acts by cleaving a novel substrate known as RIP1 that prevents NFκB from entering the nucleus, thus inhibiting target gene transcription. Interestingly, survivin is known to be a direct gene target of NFκB that controls cancer cell survival. In our investigation, we found that survivin is downregulated upon caspase 2 activation via the NFκB pathway, resulting in decreased cell cycle kinetics, increased apoptotic threshold and suppressed tumor growth in mice. These studies conclude that survivin is a common effector molecule that is regulated by tumor suppressors to maintain cellular homeostasis. However, upon deactivation of the tumor suppressor pathway, survivin is deregulated and contributes significantly to disease progression. These observations may lead to potential therapeutic implications and novel targeting strategies that will help eradicate harmful cancer cells and spare surrounding healthy cells; often the most persistent problem of most conventional chemotherapy.

Werner syndrome (WS) is an autosomal recessive disorder associated with premature aging and cancer predisposition. WS cells show increased genomic instability and are hypersensitive to DNA-damaging agents. WS is caused by mutations of the WRN gene. WRN protein is a member of RecQ DNA helicase family. In addition to a conserved 3’–5’ helicase activity, the WRN protein contains unique 3’–5’ exonuclease activity. WRN recognizes specific DNA structures as substrates that are intermediates of DNA metabolism. WRN physically and functionally interacts with many other proteins that function in telomere maintenance, DNA replication, and DNA repair. The function of WRN is regulated by post–translational modifications that include phosphorylation, acetylation, and sumoylation. SIRT1 is a NAD-dependent histone deacetylase (HDAC) that deacetylates histones and a numbers of cellular proteins. SIRT1 regulates the functions of many proteins, which are important for apoptosis, cell proliferation, cellular metabolism, and DNA repair. SIRT1 is also regulated by other proteins or molecules from different levels to activate or inhibit its deacetylase activity. In this study, we found that SIRT1 interacts with and deacetylates WRN. We further identified the major acetylation sites at six lysine residues of the WRN protein and made a WRN acetylation mutant for functional analysis. We found that WRN acetylation increases its protein stability. Deacetylation of WRN by SIRT1 reverses this effect. CREB-binding protein (CBP) dramatically increased the half-life of wild-type WRN, while this increase was abrogated with the WRN acetylation mutant. We further found that WRN stability is regulated by the ubiquitination pathway, and that WRN acetylation by CBP dramatically reduces its ubiquitination level. We also found that acetylation of WRN decreases its helicase and exonuclease activities, and that SIRT1 reverses this effect. Acetylation of WRN alters its nuclear distribution. Down-regulation of SIRT1 increases WRN acetylation level and prevents WRN protein translocating back to nucleolus after DNA damage. Importantly, we found that WRN protein is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells that were stably expressing WRN acetylation mutant display significantly higher sensitivity to MMC than the cells expressing wild-type WRN. Taken together, these data demonstrated that acetylation pathway plays an important role in regulating WRN function in response to DNA damage. A model has been proposed based on our discoveries.

Pim (Provirus Integration site for Moloney murine leukemia virus) kinases are a family of three serine/threonine kinases involved in cell cycle, survival and metabolism. These kinases were first identified in malignant cells and are most often associated with their role in cancer. Their role in immunity and lymphocytes is less well known. To date, it has been shown that Pim 1 and/or Pim 2 are important for T lymphocyte survival and activation when the Akt signaling pathway is inhibited by rapamycin. In addition, our laboratory has shown that Pim 2 is critical for BLyS-mediated naive B lymphocyte survival in the presence of rapamycin. This thesis extends the role(s) for Pim 1 and/or 2 to include functions during B cell activation and the generation of immune responses. We found that during in vitro activation of purified resting splenic B cells from wild type mice with a variety of activators that use multiple signaling pathways, including the BCR, TLR and CD40 receptors, both Pim 1 and 2 kinases were induced by 48 hours post-activation, suggesting that they could play a role in B cell activation and differentiation to antibody secreting or memory B cells. Immunization of Pim 1-/-2-/- knockout mice with T cell dependent antigens showed impairment in antibody and antibody secreting cell generation as well as lack of germinal center formation clearly demonstrating an involvement of Pim 1 and/or 2 in the immune response. FACS examination of B cell populations from naive Pim 1-/-2-/- knockout mice revealed normal levels of splenic marginal zone and follicular B cells and T cells, however, decreased numbers of all peritoneal B cell populations and decreased B cells in Peyer's Patches was seen. An examination of serum antibody found in naive Pim 1-/-2-/- knockout mice showed decreased levels of natural antibody, which is likely due to loss of the peritoneal B1 cells but does not explain the significantly decreased TD immune response. To determine whether the defect was B cell intrinsic or a more complex interaction between B and T cells, we determined whether Pim 1-/-2-/- mice would respond to T cell independent, TI-1 and TI-2, antigens. Antibody production and antibody secreting cell formation were also significantly decreased in these mice supporting our notion of a B cell intrinsic defect. To further examine the B cell response problem, we attempted to establish chimeric mice using either bone marrow derived cells or fetal liver cells from WT or Pim 1-/-2-/- donors so that the B cells were derived from Pim 1-/-2-/- mice and the T cells would be WT. Unfortunately, we were not able to consistently engraft and develop mature Pim 1-/-2-/- B cells, which indicate that there is a stem cell defect in these knockout mice that requires further investigation. Because one of the major failures in activated Pim 1-/-2-/- B cells is the generation of antibody secreting cells, an analysis of the expression of transcription factors IRF-4 and BLIMP-1, known to play a role in this process was carried out. Although IRF-4 induction was not affected by the loss of Pim 1 and 2, the number of cells able to increase BLIMP-1 expression was significantly decreased, revealing a partial block in the generation of ASCs. Taken together the data presented in this thesis reveals a new and critical role for Pim 1 and 2 kinases in the humoral immune response.

Faithful duplication and segregation of undamaged DNA is critical to the survival of all organisms and prevention of oncogenesis in multicellular organisms. To ensure inheritance of intact DNA, cells rely on checkpoints. Checkpoints alter cellular processes in the presence of DNA damage preventing cell cycle transitions until replication is completed or DNA damage is repaired. Several checkpoints are specific to S-phase. The S-M replication checkpoint prevents mitosis in the presence of unreplicated DNA. Rather than outright halting replication, the S-phase DNA damage checkpoint slows replication in response to DNA damage. This checkpoint utilizes two general mechanisms to slow replication. First, this checkpoint prevents origin firing thus limiting the number of replication forks traversing the genome in the presence of damaged DNA. Second, this checkpoint slows the progression of the replication forks. Inhibition of origin firing in response to DNA damage is well established, however when this thesis work began, slowing of replication fork progression was controversial. Fission yeast slow replication in response to DNA damage utilizing an evolutionarily conserved kinase cascade. Slowing requires the checkpoint kinases Rad3 (hATR) and Cds1 (hChk2) as well as additional checkpoint components, the Rad9-Rad1-Hus1 complex and the Mre11-Rad50-Nbs1 (MRN) recombinational repair complex. The exact role MRN serves to slow replication is obscure due to its many roles in DNA metabolism and checkpoint response to damage. However, fission yeast MRN mutants display defects in recombination in yeast and, upon beginning this project, were described in vertebrates to display S-phase DNA damage checkpoint defects independent of origin firing. Due to these observations, I initially hypothesized that recombination was required for replication slowing. However, two observations forced a paradigm shift in how I thought replication slowing to occur and how replication fork metabolism was altered in response to DNA damage. We found rhp51Δ mutants (mutant for the central mitotic recombinase similar to Rad51 and RecA) to slow well. We observed that the RecQ helicase Rqh1, implicated in negatively regulating recombination, was required for slowing. Therefore, deregulated recombination appeared to actually be responsible for slowing failures exhibited by the rqh1Δ recombination regulator mutant. Thereafter, I began a search for additional regulators required for slowing and developed the epistasis grouping described in Chapters II and V. We found a wide variety of mutants which either completely or partially failed to slow replication in response to DNA damage. The three members of the MRN complex, nbs1Δ, rad32Δ and rad50Δ displayed a partial defect in slowing, as did the helicase rqh1Δ and Rhp51-mediator sfr1Δ mutants. We found the mus81Δ and eme1Δ endonuclease complex and the smc6-xhypomorph to completely fail to slow. We were able to identify at least three epistasis groups due to genetic interaction between these mutants and recombinase mutants. Interestingly, not all mutants’ phenotypes were suppressed by abrogation of recombination. As introduced in Chapters II, III and IV checkpoint kinase cds1Δ, mus81Δ endonuclease, and smc6-x mutant slowing defects were not suppressed by abrogation of recombination, while the sfr1Δ, rqh1Δ, rad2Δ and nbs1Δ mutant slowing defects were. Additionally, data shows replication slowing in fission yeast is primarily due to proteins acting locally at sites of DNA damage. We show that replication slowing is lesion density-dependent, prevention of origin firing representing a global response to insult contributes little to slowing, and constitutive checkpoint activation is not sufficient to induce DNA damage-independent slowing. Collectively, our data strongly suggest that slowing of replication in response to DNA damage in fission yeast is due to the slowing of replication forks traversing damaged template. We show slowing must be primarily a local response to checkpoint activation and all mutants found to fail to slow are implicated in replication fork metabolism, and recombination is responsible for some mutant slowing defects.