John M. Sedivy - US grants

A large body of evidence indicates that cancer has a genetic basis, and that tumor progression is a multi-step, complicated set of events. The analysis of the transforming oncogenes has contributed significantly to our understanding of neoplasia, but many of the crucial causative steps remain elusive. Progress is hampered by our poor understanding of how proliferation signals are transmitted form the cell surface to the nucleus, and how they are executed. Two basic questions are of great importance. What are the normal functions of the cellular proto-oncogenes in cellular physiology? What are the links in the chain of command downstream from the oncogenes? Three proto-oncogenes, c-src, c-raf, and c-myc were chosen for the present study because each appears to act at a distinct and different point in the signal transduction process. The primary aim of this proposal is the elucidation of the role of these proto-oncogenes in normal cellular physiology. This goal will be approached using targeted gene modification. Since many biological phenomena can be profitably approached in tissue culture, all experiments will be conducted in appropriate in vitro model systems. The aim will be to use the phenotypes of the resultant null mutants as clues to unravel the molecular functions of the genes in question. In addition, it is hoped that gene targeting will provide an entry point for future genetic studies of the proto-oncogenes. Progress in the field of gene targeting in mammalian cells has been very rapid during 1988. Four distinct genetic selection methods for the recovery of homologous recombination events at nonselected chromosomal loci have been independently developed (Doetschman et al., 1988; Jasin and Berg, 1988; Mansour et al., 1988; Sedivy and Sharp, 1989). Gene targeting in cultured cells necessitates the modification of multiple gene copies in diploid or even polyploid backgrounds. This will be accomplished by performing targeted homologous recombination several times in succession. To circumvent the possible lethality of homozygous null phenotypes, a conditionally expressed copy of the target gene will be introduced into the genome. This will allow the propagation of a cell line in which both endogenous copies of the target gene are modified. Then, under conditions non-permissive for expression of the introduced gene copy, the null phenotype will become apparent and will be subjected to experimental analysis. Another aim of this proposal is to compare, generalize, and further develop the methods of gene targeting.

DESCRIPTION (Adapted from Applicant's Abstract): An important part in unraveling the causes of cancer will be a detailed understanding of how mitogen-triggered signal transduction is integrated into the molecular mechanisms that govern the cell cycle. A large body of evidence indicates that the product of the c-myc gene is a transcription factor involved in these regulatory processes. Our knowledge of the role played by Myc is fragmentary in three important areas: 1) the mechanisms that regulate expression of the c- myc gene: 2) the downstream targets of Myc activity; and, 3) the mechanisms by which Myc regulates its targets. The goal of this proposal is a comprehensive investigation of signaling pathways both upstream and downstream of Myc. Emphasis is place on genetic experiments to understand how these regulatory processes operate in vivo. The first specific aim is to mutate promoter elements directly in a chromosomal c-myc gene. This will be accomplished using targeted homologous recombination, and will allow the in vivo study of specific regulatory circuits in the regulation of the c-myc gene. The second specific aim is to identify downstream Myc target genes, and to determine how Myc regulates their expression. This will be accomplished by testing the expression of specific putative targets in cells devoid of Myc activity (Myc- cells). A second approach will be to use a general screening method, differential display, to search for novel targets whose expression is altered in Myc- cells. Follow up experiments will identify the cis-acting elements in the targets genes that mediate the action of Myc. These experiments will include an investigating of how Myc regulates the expression of cyclins E and A, which have been implicated as downstream targets. The third specific aim is to disrupt the cyclin E gene, which will be accomplished by gene targeting. The comparison of Myc- and cyclin E- phenotypes will reveal which aspects of the growth regulatory activity of Myc are mediated by cyclin E. Experiments to further improve and generalize gene targeting methodologies are also a part of this specific aim. All the proposed experiments are founded on previous accomplishments of this laboratory: 1) the development of a new and efficient gene targeting strategy; 2) the isolation of Myc- cell lines; and, 3) the identification of cyclins E and A as Myc targets. Taken together, the experiments outlined in this proposal are aimed at providing a better understanding of the function of Myc in normal cellular physiology, and ultimately, its oncogenic action.

Two fundamentally different aging phenomena have been described at the cellular level: 1) the gradual decline of life processes in postmitotic cells, and, 2) the decline and eventual complete cessation of cell division observed in most replicating cell lineages. The former is measured in simple chronological time, and comes into play during the aging of postmitotic adult organisms, such as the nematode, or during the aging of largely postmitotic tissues such as the brain or muscle in more complex organisms. In contrast, finite replicative lifespan, often referred to as cellular replicative senescence , is measured in terms of cell divisions rather than chronological time. The current consensus is that both postmitotic and replicative aging processes are causally related to the aging of humans. The topic of this research proposal is the molecular mechanism of replicative aging processes, specifically, the molecular machine that actually executes and maintains senescence. The major tool will be targeted homologous recombination (gene targeting), which will be performed in normal (nonimmortalized) human cell strains of both fibroblastic and epithelial origin. The targets of gene targeting will be the following genes: the tumor suppressors p53 and retinoblastoma (Rb), and the Cdk kinase inhibitors p16INK4A, p19ARF, and p21CIP1/WAF1. The objective will be to ablate gene action and subsequently investigate the resultant senescence phenotypes on both the cellular and molecular levels. This direct interventive approach is expected to reveal the functionally relevant components of the molecular senescence machine. The proposal is built on a model which predicts that the molecular machine that establishes senescence is composed of components that also play roles in cell cycle control during the normal proliferative lifespan of the cell. The experiments in this proposal are designed to test the hypothesis that three events are required to convert a normal human cell into an immortal cell: 1) loss of p53-p21 pathway function, 2) loss of pl6-Rb pathway function, 3) gain of telomerase function. Experiments will be performed in normal human cells grown in in vitro cell culture. This is because a large body of evidence indicates that the regulation of replicative senescence mechanisms is significantly different in humans and in rodents. Therefore, due to the ethical unacceptability of experimentally altering the human germ line, and the limited utility of the rodent model to address the specific issues under investigation, the whole-organism transgenic route is not appropriate and the experimental model has been confined to human somatic cell culture.

The goal of this proposal is to answer the question: How does the c-myc protooncogene regulate cellular proliferation? The c-Myc protein is a transcription factor, and a number of genes have been proposed to be targets of its regulation. However, the misregulation of these genes cannot explain the diverse biological effects of c-Myc, implying that additional target genes remain to be discovered. c-Myc has been implicated in the control of cellular proliferation, differentiation, and programmed cell death, but the mechanisms by which it exerts its activity on the cellular machinery are complex and not well understood. Our fascination with c-Myc stems not only from our interest in understanding basic molecular regulatory mechanisms, but also because the deregulation of c-Myc activity is associated with a wide range of human cancers. A genetic analysis can often lead to profound insights into molecular mechanisms, and the construction of knockout mice can be especially powerful. Unfortunately, the c-myc knockout mouse, because of its early embryonic lethality, did not yield insights into either cellular or molecular phenotypes. All attempts to recover c-myc -/- cells from homozygous embryos have been frustrated by the outgrowth of cells that express one of the other Myc family members. To overcome this problem, we used gene targeting to eliminate c-myc expression in a fibroblast cell line shown not to express the other family members. These knockout cells are the first experimental system in which c-myc loss-of-function phenotypes can be thoroughly investigated. The c-myc -/- cells are viable but display profound cell cycle defects. We have initiated a systematic molecular analysis of known cell cycle regulators in the c-myc -/- cells. Our new insights to date are: 1) loss of c-myc affects progression through both the G1 and G2 phases of the cell cycle; 2) the activity of all cyclin-Cdk complexes is affected; 3) the earliest and most prominent defect is an impairment in the activation of cyclin D-Cdk4/6 complexes; 4) c-Myc affects multiple points in the cell cycle; 5) Cdk7 has been identified as a new putative c-Myc target gene; and 6) the expression of virtually all previously proposed c-Myc target genes is unchanged in c-myc -/- cells. Our objective is to build on these results to fully elucidate how c-Myc regulates transition through the cell cycle. We propose to: construct homozygous c-myc knockouts in human cells, isolate new c-Myc target genes by differential cDNA screening, define the biochemical lesions in G1 and G2 cell cycle mechanisms that result from loss of c-Myc activity, and genetically test the physiological relevance of the observed biochemical lesions.

A Strategic Plan developed for Brown University in 1996 identified as a top priority the establishment of an integrated and interdisciplinary program in genetics. In response to this mandate the Division of Biology and Medicine in 1999 initiated the creation of an interdepartmental Center for Genetics and Genomics, whose mission will be to spearhead research in contemporary molecular genetics and to coordinate multi-disciplinary approaches in molecular genetics with programs in clinical and human genetics at affiliated hospitals. These very real needs at Brown University coincide perfectly with the programmatic goals of the Centers of Biomedical Research Excellence (COBRE) Program. The cornerstone of this application is therefore to use COBRE funding as the seed for the nascent Center for Genetics and Genomics. An Executive Committee charged with the development of the Center identified two areas of critical need: the establishment of infrastructure in the form of core facilities, and the support of junior faculty research. It is therefore proposed to establish two new cores in mouse transgenics and flow cytometry and to upgrade an existing confocal imaging core, and to establish five interdisciplinary research projects to disperse seed money to junior investigators. The cores will provide access to major new technologies and equipment currently not available in the state of Rhode Island. The research projects, in addition to providing direct funding for individual investigators, will be configured specifically to foster mentoring of junior faculty and to provide a stimulating and interdisciplinary research environment. The administrative core will oversee a competitive postdoctoral fellowship program, an invitational seminar series, a bi-weekly intramural research forum, and an annual retreat.

Genetic studies both at the organismal and cellular levels demand a sophisticated downstream analysis of the phenotypes that result from genetic manipulations. The creation of the transgenic and knockout animals proposed in this application, as well as the increasing capabilities of investigators using cell culture models provided by modern transient transfection technologies, will greatly increase the need for evaluating patterns of gene expression. Modern flow cytometry is the method of choice to answer these needs, especially because it provides unsurpassed capability to evaluate changes at the single cell level in large populations of cells. To accommodate the needs of this diverse group of investigators it is therefore proposed to establish a state of the art preparative flow cytometry core facility. In addition to all analytical procedures, this facility will have the ability to sort transiently transfected cells in a sterile fashion and provide investigators with pools of productively transfected viable cells that can be returned to culture for further observations or manipulation. This capability can in many cases obviate the need for stable transfection and thus result in a significant time saving. In addition, this method can be especially effective in establishing antisense ablation. The core will be supervised by Dr. John Sedivy, Professor, an expert in the field of cell cycle regulation, who has a long track record of publications using the proposed technologies. On a day to day basis the core will be run by a highly trained technician who will aid users in the preparation and analysis of their samples. Users of this core facility will have access not only to a state of the art flow cytometer, but also to guidance, expertise and training towards applying this technology to their own research needs.

mitogen activated protein kinase; nuclear factor kappa beta; molecular genetics; biological signal transduction; genetically modified animals; laboratory mouse;

It is proposed to establish a single, integrated, stand alone mouse transgenic and knockout core facility that will serve the needs of investigators at Brown, affiliated teaching hospitals, and throughout the region. This core will be the first mouse transgenic facility in the state of Rhode Island. The core will be dedicated to serve investigators in many disciplines, and aid them in the development and implementation of transgenic mouse model systems in their area of study. The core will perform pro-nuclear microinjections as well as blastocyst injections. In addition, protocols, genotype ES cell clones as well as founder animals by PCR, and maintain a mouse colony to support these efforts. The core will also support the research efforts of a magnet investigator, a nationally recognized scientist in the field of mouse transgenics, who will be hired and who will become the Director of this core. On a day to day basis the core will be run by a highly trained, Ph.D. level Associate Director who will be appointed at the rank of Assistant professor (Research).

DESCRIPTION (provided by applicant): BU applied for COBRE funding in the first round of applications in the Spring of 2000 (awarded in September of 2000). COBRE funding was used to establish an Administrative Core; the following three Research Cores: 1) Transgenic Mouse and Knockout Core (TMKC), Genomics and Bioinformatics Core (GBC), and Microscopy and Bioimaging Core (MBC); and five Research Projects (Events Regulating the Balance Between Resistance and Infection, Genetic Models of Human Dementias, Molecular Genetics of Ion Channels, Regulation of MAPK and NF-kB Activity and Downstream Signaling, and Mechanisms of Cellular Injury and Transformation). Each research project is directed by a senior Faculty Leader-Mentor, and comprised of three to five Sub-Projects headed by Junior Faculty Investigators. The Core Facilities are typically headed by a tenure-track Faculty Leader and directed on a day-to-day basis by a research-track Faculty Director. With the exception of some staffing to be completed in the near future, all Core Facilities have been fully implemented in the first one and a half years of the COBRE award. The supplemental funding application is being submitted for the purpose of purchasing additional large equipment items for the Core Facilities. Specifically, it is proposed to: 1) enhance the functionality of the TMKC by significantly upgrading an existing Histopathology Facility; 2) expand the operation of the GBC by incorporating the technology of expression profiling using spotted cDNA arrays; and 3) to upgrade the MBC through the acquisition of versatile light microscopy and digital electron microscopy capabilities. These upgrades are necessitated by the rapidly expanding use of the COBRE Core Facilities.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] In subproject C, "Molecular Genetics of Ion Channels," we are examining the nicotinic acetylcholine receptor, L-type calcium channels, and kainate receptors as well as the scaffolding proteins that couple to them. To obtain a detailed understanding of the molecular mechanism of the physiological consequences of the transgenic knock-in experiments, a series of biochemical and biophysical assays are carried out to characterize the modified protein. The targeted proteins are structurally characterized, and mutations aimed to abate the biological function without altering the protein structure are designed and then tested. The results provide a unique insight into the interpretation of the in viva findings from our knock-in experiments. Currently, the NMR instrumentation utilized for the structural studies is at a point of saturation and the research projects covered by this COBRE award (RR 15578) as well as other NIH-sponsored research efforts at Brown University cannot be aggressively pursued. Here we request funds for the acquisition of a cryogenic probe for the 600 MHz high resolution NMR which serves as the sole high-field instrument at Brown. Resulting in a 16-fold reduction in the time to collect data, the cryogenic probe is a cost-effective manner to address the critical demand for NMR measurement time that is currently limiting the scientific problems that can be undertaken. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The myc proto-oncogenes encode transcriptional regulators whose inappropriate expression is correlated with a wide array of malignancies. At the cellular level Myc activity has been linked with cell division, accumulation of mass, differentiation, and programmed cell death. The molecular mechanisms by which these end points are achieved have been show to emerge. It is now clear that Myc can directly influence the expression of hundreds, perhaps thousands, of genes with diverse functions. A significant challenge for the future will be to integrate this wealth of information into mechanistic models that explain the biological functions of Myc. This proposal is centered on a genetic analysis of cellular functions controlled by Myc. Three c-myc loss-of-function model systems will be used: an already existing c-myc knockout in the Rat-1 fibroblast cell line, mouse embryo fibroblasts (MEF) derived from conditionally targeted c-myc mice, and human c-myc knockout fibroblasts, whose construction and analysis constitutes aim 1 of this proposal. In all three models conditional Myc expression will be engineered on the knockout background. Aim 2 will analyze mutations in c-Myc effector domains that will be knocked into one copy of the endogenous c-myc gene. After turning off the wild-type c-myc gene copy, cells will be examined for known Myc phenotypes. Expression of individual target genes will be studied in detail, biochemical functions known to be affected by Myc will be assayed, and interaction of the mutant Myc proteins with known partners will be determined. Aim 3 will reconstitute functional expression of selected Myc target genes in the knockout cell lines. Myc-activated genes will be overexpressed using retrovirus vectors, and Myc-repressed genes will be ablated using RNAi. Phenotypes of these interventions will be studied, and multiple interventions will be attempted in combination. Aim 4 will determine the mechanisms by which Myc promotes cell cycle entry. D-type cyclin expression and activation of Cdk4/6 will be examined in c-myc/- MEF, rescue with a constitutively active Cdk4 will be attempted, the presence or absence of rescue will be analyzed in molecular terms, and posttranslational mechanisms leading to thr activation of cyclin D-Cdk4/6 complexes will be examined. These studies are expected to shed light on how specific outputs, such as the promotion of cell growth or cell Cycle progression, are causally associated with specific cellular targets and molecular mechanisms. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The vast majority of normal cell types from all vertebrate species display this response. It is becoming increasingly evident that what has classically been described as cellular senescence is a collection of interrelated states that can be triggered by distinct intrinsic and extrinsic stimuli. We have found that when normal human diploid fibroblasts are subcultured into replicative exhaustion, three [unreadable] essentially independent processes can take place, each of which is sufficient to establish a senescent growth arrest state. The first pathway is initiated by telomere shortening. The second pathway is initiated by an unknown, spontaneous and stochastic process that leads to the upregulation of the cyclin-dependent kinase inhibitor p16 n K4A.The third pathway is initiated by mitochondrial damage leading to the production of sufficient oxidative stress to activate the p53 tumor suppressor protein. We have developed an in vitro cell based model system that allows us to selectively track and study senescent cells in unperturbed cultures, and to prepare homogeneous populations of cells that have activated a single senescence pathway. Aim 1 will examine the role of p53 and upstream effectors in telomere-initiated senescence. Posttranslational modifications of p53 and the activation status of signaling pathways upstream of p53 will be studied. Aim 2 will seek to discover what causes the spontaneous, age-dependent upregulation of pl6. Effectors implicated in regulating the p 16 gene will be examined, pharmacological methods will be used to probe cytoplasmic kinase cascades, and the physiological state elicited by the p16 pathway will be explored by microarray expression profiling. Aim 3 will investigate the causes and consequences of spontaneous upregulation of reactive oxygen species (ROS). The functional relationships between the ROS, p 16 and telomere pathways will be examined, and the effect of the IGF signaling pathway implicated in organismal aging on the pathways of cellular senescence will be studied. In all cases, reverse-genetic interventions utilizing dominant and constitutively active proteins as well as siRNA-mediated ablation will be used to probe the transmission of the senescence signals. Interventions will be sought to elicit senescence responses in naive cells, to prevent a natural senescence response caused by replicative exhaustion, and to reverse an established senescent state. Studies described in this proposal will give us a better understanding of cellular senescence processes, and will begin to shed light on the roles of cellular senescence in organismal aging. [unreadable] [unreadable]

laboratory mouse; mitogen activated protein kinase

bioinformatics; functional /structural genomics; molecular biology information system; genome; biomedical facility;

functional /structural genomics

[unreadable] DESCRIPTION (provided by applicant): [unreadable] It is proposed to establish a COBRE comprised of 5 research projects headed by junior and early career faculty and 3 research core facilities. The scientific theme of the Center will be cancer signaling networks, namely, the molecular mechanisms by which signaling networks relevant to carcinogenesis are regulated: transcription, protein phosphorylation and protein degradation. Some of the focal points of the proposed research will be DNA repair, Wnt and IGF-1 signaling, proteome-wide analysis of protein phosphorylation, ubiquitin-mediated proteolysis, global RNA polymerase II machinery, transgenic mouse models of carcinogenesis, and bioinformatic inference of network systems. Cancers under investigation will include hepatocellular carcinoma and ovarian follicular cancer. Two of the investigators will continue from the current COBRE (Drs. Singer, Zhitkovich), and 3 are newly hired junior faculty. The proposed research core are Mouse Transgenics, Genomics, and Bioinformatics. The first two carry over from the current Center, while the latter will be established as a new core facility. Each project leader and core director has an assigned senior faculty mentor. The mentors comprise the IAC which is chaired by the PI The EAC evaluates all aspects of the Center's operation and advises the PI and the IAC. The PI is also advised by the Steering Committee of the Brown Center for Genomics and Proteomics, and reports to the Dean of the Medical School and the senior administration. The of the program is to establish both a thematic focus and the caliber of science required for a transition to a PPG funded by the NCI. The following Specific Aims are proposed to achieve these goals: Aim 1: To investigate the role of the gonad-specific transcriptional coactivator TAF4b in the development of ovarian granulosa cell cancer; Aim 2: To examine the function of Wnt signaling networks in the development of hepatocellular carcinoma and to address whether targets in this pathway may be useful for cancer therapy; Aim 3: To describe and functionally test, on a proteome-wide basis, cascades of tyrosine protein phosphorylation triggered by receptor crosslinking in mast cells and by IGF-1 stimulation in hepatocellular carcinoma cells; Aim 4: To elucidate the mechanisms by which Cullin 3 complexes recognize regulatory proteins causally connected to cell cycle aberrations found in cancer and target them for ubiquitin-mediated proteolysis; Aim 5: To discover novel mechanisms by which normal levels of oxidative stress found in all cells participate in generating DNA damage that ultimately leads to the development of cancer. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The vast majority of normal cell types from all vertebrate species display this response. It is becoming increasingly evident that what has classically been described as cellular senescence is a collection of interrelated states that can be triggered by distinct intrinsic and extrinsic stimuli. We have found that when normal human diploid fibroblasts are subcultured into replicative exhaustion, three [unreadable] essentially independent processes can take place, each of which is sufficient to establish a senescent growth arrest state. The first pathway is initiated by telomere shortening. The second pathway is initiated by an unknown, spontaneous and stochastic process that leads to the upregulation of the cyclin-dependent kinase inhibitor p16 n K4A.The third pathway is initiated by mitochondrial damage leading to the production of sufficient oxidative stress to activate the p53 tumor suppressor protein. We have developed an in vitro cell based model system that allows us to selectively track and study senescent cells in unperturbed cultures, and to prepare homogeneous populations of cells that have activated a single senescence pathway. Aim 1 will examine the role of p53 and upstream effectors in telomere-initiated senescence. Posttranslational modifications of p53 and the activation status of signaling pathways upstream of p53 will be studied. Aim 2 will seek to discover what causes the spontaneous, age-dependent upregulation of pl6. Effectors implicated in regulating the p 16 gene will be examined, pharmacological methods will be used to probe cytoplasmic kinase cascades, and the physiological state elicited by the p16 pathway will be explored by microarray expression profiling. Aim 3 will investigate the causes and consequences of spontaneous upregulation of reactive oxygen species (ROS). The functional relationships between the ROS, p 16 and telomere pathways will be examined, and the effect of the IGF signaling pathway implicated in organismal aging on the pathways of cellular senescence will be studied. In all cases, reverse-genetic interventions utilizing dominant and constitutively active proteins as well as siRNA-mediated ablation will be used to probe the transmission of the senescence signals. Interventions will be sought to elicit senescence responses in naive cells, to prevent a natural senescence response caused by replicative exhaustion, and to reverse an established senescent state. Studies described in this proposal will give us a better understanding of cellular senescence processes, and will begin to shed light on the roles of cellular senescence in organismal aging. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The survival of populations in developed nations into extreme old age is a remarkable phenomenon in human history. This change has come about largely through improved public hygiene, decreased child mortality and a decrease in infectious diseases. However, the steady increases in the average age of the population have resulted in an ever increasing burden of the degenerative diseases of aging, such as osteoporosis, Alzheimer's disease, diabetes and cancer. Currently, there are several mutually non-exclusive models to explain the mechanisms behind these degenerative aging processes. One theory is centered on the idea that replicative senescence of cells limits their proliferative capacity and hence tissue renewal. Other models suggest that accumulation of genetic and/or epigenetic (chromatin) damage with age eventually impairs cell and tissue function. In this application, we test a hypothesis that links two of these ideas - cellular senescence and epigenetics - to a pathway that has not previously been considered to be a major regulator of aging, namely, Wnt signaling. Although not widely viewed as a regulator of aging, the Wnt signaling pathway is well documented to be an evolutionarily conserved determinant of tissue and organismal development, and later in life, adult tissue homeostasis. The research proposed here is based on recent and exciting discoveries in the two collaborating laboratories. First, the work of Peter D. Adams (PDA) has implicated Wnt signaling in the regulation of cellular senescence by showing that in vitro - human fibroblast cell culture - repression of Wnt signaling triggers extensive heterochromatinization and cellular senescence. Second, the in vivo studies of John M. Sedivy (JMS) have found that a marked expansion of facultative heterochromatin occurs in association with cellular senescence and aging in mouse and primate tissues. Based on these results we propose that a Wnt-Chromatin-Senescence signaling axis is an important determinant of organismal aging. We propose here a series of experiments to initiate the investigation of this novel signaling axis, the mechanisms of its operation, and its role in organismal aging. Aim 1 will perform high resolution, genome-wide mapping of senescence-associated changes in chromatin structure that are triggered by reduced Wnt signaling in vitro. Aim 2 will investigate the links between Wnt signaling, heterochromatinization, cellular senescence and aging in vivo, using mouse, primate and human models. Aim 3 will assess cellular senescence and genome- wide chromatin changes in mice harboring hypomorphic Wnt pathway mutations. Our goal is to use a discovery-based approach to reveal epigenome-wide characteristics of aging processes, functionally connect these with Wnt signaling pathways, and ultimately open the road to new pharmaceutical targets. PUBLIC HEALTH RELEVANCE: Aging is a fundamental biological process with a profound impact on society. An important aspect of aging is believed to be biological structures that are inherently difficult to maintain. Our genomes, which are compacted into a complex network of DNA and protein referred to as chromatin, are likely to be one such structure. This proposal will perform a global analysis of age-associated chromatin modifications and remodelling in mammalian organisms. These discoveries will advance our understanding of the basic processes of aging, as well as potentially uncover targets for pharmaceutical intervention to ameliorate age-related disorders.

DESCRIPTION (provided by applicant): Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The great majority of normal cell types from all vertebrate species examined display this response. It is becoming increasingly evident that what has classically been described as cellular senescence is a collection of interrelated states that can be triggered by distinct intrinsic and extrinsic stimuli. The underlying cause of senescence due to replicative exhaustion is telomere shortening. In addition, it is now apparent that many types of stress, reactive oxygen species, pharmacological agents, and even nutrient imbalances can trigger a senescence response. Activation of some oncogenes also induces senescence in normal cells, and recent data have implicated cellular senescence as an important in vivo tumor suppression mechanism. In contrast, the connections between cellular senescence and the aging of organisms are significantly more tenuous. The necessary first step is to distinguish senescent cells from the majority of healthy but quiescent cells found in normal tissues. We, and others, have recently developed a method based on the microscopic detection of DNA damage markers localized to telomeres, designated the `TIF'assay (for `telomere dysfunction-induced foci'). TIFs are a robust biomarker of telomere-initiated senescence, which we used to demonstrate a marked age-associated accumulation of senescent cells in normal primate tissues. This proposal is aimed to give us a better understanding of multiple cellular senescence processes, focusing on their roles in organismal aging. Aim 1 will examine the in vivo occurrence of telomere-induced senescence in mouse, primate and human models, and probe the links between cellular senescence and pathways that functionally influence aging. Aim 2 will extend recent studies linking genome-wide changes in chromatin structure with cellular senescence by developing new assays to assess in vivo states of heterochromatin in cells and tissues. These new biomarkers of cellular senescence will then be applied to the models developed in Aim 1. Aim 3 will seek to discover what causes the age-dependent upregulation of the cyclin-dependent kinase inhibitor p16, an important effector implicated in regulating multiple senescent states. PUBLIC HEALTH RELEVANCE: Replicative cellular senescence was discovered and first described as an irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. These findings generated two hypotheses regarding the significance of cellular senescence: that it contributes to aging, and that it suppresses cancer. Recent data have implicated cellular senescence as an important in vivo tumor suppression mechanism in a variety of human and mouse tissues. In contrast to tumor suppression, the connections between cellular senescence and the aging of organisms are significantly more tenuous. The necessary first step is to distinguish senescent cells from the majority of healthy but quiescent cells found in normal tissues. This proposal will develop new biomarkers of cellular senescence that will be applied in vivo investigate the occurrence of senescent cells in rodents, primates and humans. Mechanisms that lead to the generation of senescent cells will also be investigated, as well as the persistence of senescent cells.

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) accounts for 80-90% of primary liver tumors, and is one of the most common and devastating malignant diseases worldwide. Insulin-like growth factors (IGF-I and II) have been shown to play a key role in HCC by activating intracellular signaling cascades. Chief among these is the mitogen-activated protein kinase (MAPK) signaling pathway. Raf-1, the apical kinase, couples the MAPK pathway to extracellular tyrosine kinase receptors. Raf Kinase Inhibitory Protein (RKIP) is the prototype of a highly conserved family of proteins that bind directly to both Raf-1 and the next kinase in the pathway, MEK, disrupting their interaction, and antagonizing the activation of the entire signaling cascade. RKIP expression has been found to be reduced in breast, colon, liver and prostate cancers, among others. What is especially striking is the frequency of this event in HCC: almost 90% of human HCC specimens display reduced RKIP protein expression. Even more provocative are indications that RKIP may play a role in metastatic processes, implicating it as one of only a handful of known metastasis suppressor genes. This raises the possibility that modulation of RKIP protein expression or activity may have therapeutic value. These hopes are further fueled by observations that while RKIP protein expression is clearly reduced in many advanced stage tumors, it is seldom completely absent. The broad objective of this proposal is to initiate the development of mouse models to investigate in vivo the HCC tumor suppressor activity of RKIP. In pursuit of these goals two Specific Aims are proposed. The first will investigate the effect of downregulating RKIP. An already existing RKIP knockout will be used to determine the functional consequences of RKIP loss-of-function during the development and progression of HCC. The RKIP knockout will be combined with an exciting new mouse HCC model: a double transgenic with liver-specific expression of the hepatitis virus Bx transcriptional regulator (HBx) and insulin receptor substrate-1 (IRS-1). HBx/IRS-1 animals develop hepatocellular dysplasia that progresses to HCC. This model is of great interest because it recapitulates in the mouse many of the histological and molecular hallmarks of the human disease. The second aim will investigate the effect of restoring RKIP expression. A new transgenic model will be established to evaluate the functional consequences of upregulating RKIP during the development and progression of HCC. The approach will be to make a mouse with a liver-specific Tet- regulated RKIP transgene. These studies will provide critical in vivo functional evidence whether RKIP is a target with therapeutic potential. If affirmative, the in vivo data will justify future studies to unravel in detail the mechanisms that regulate RKIP expression and dysregulation in HCC as well as other cancers. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinoma accounts for 80-90% of liver cancers, one of the most common malignancies worldwide. Intensive research has discovered the importance of a key cellular mechanism, the MAPK signaling pathway, in the development of this devastating disease. The main control point of the MAPK pathway, regulated by a protein kinase called Raf, has been targeted by the newly developed drug Nexavar, which has shown extraordinary promise in stage III clinical trials. Raf kinase is known to be influenced by other cellular regulators, Raf Kinase Inhibitory Protein (RKIP) being an especially important one. Epidemiological data has linked RKIP to cancer progression, raising the prospect that it may also be a valuable therapeutic target. The objective of this proposal is to develop mouse models of hepatocellular carcinoma in which this hypothesis can be directly tested.

DESCRIPTION (provided by applicant) Aging is a fundamental biological process, and age-related diseases are the leading causes of death in modern societies. The objective of the proposed Molecular Biology of Aging Predoctoral Graduate Training Program (MBoA) is simple: to train the next generation of scientists to attack the immensely challenging yet important task of understanding and eventually manipulating human aging. The MBoA will bring together molecular biologists, computational and population biologists, and geriatricians to provide instruction and training to graduate students in the molecular mechanisms of aging. The aim will be to provide PhD candidates with a strong academic and experimental foundation in the current landscape of molecular aging research, and to equip them with the skills to pursue a research career in this field. Our understanding of aging has reached a watershed in the past 10 years that was enabled by the increasing use of forward genetics in simple model systems. Thirteen faculty from 6 different departments have come together to launch the MBoA Training Program. Their research interests span from insulin/IGF signaling, chromatin structure, cellular senescence, mitochondrial function and protein quality control to degenerative disorders of the nervous system, heart and cartilage. The experimental systems span from yeast through nematode and Drosophila to mammalian models including the mouse and a variety of cell culture models. Support is requested for 2 trainees in year 1 and for a total of 4 trainees in year 2 through 5. Trainees will be supported for a period of 1 to 2 years. Candidates for support will be drawn from talented students in the program, either in their first year based on academic excellence, or in subsequent years based on the caliber of their research. The MBoA Training Program will operate as a track under the auspices of 2 existing and well-established programs at Brown University, the Molecular Biology, Cell Biology and Biochemistry Graduate Program (MCB), and the Graduate Program in Pathobiology. These successful programs have different, but with respect to the biology of aging, very complementary and synergistic areas of activity: MCB is the seat of molecular expertise and model organisms, whereas Pathobiology holds the keys to mammalian and human physiology and pathology. The combination and integration of these approaches is the cornerstone of the philosophy of the MBoA. While research in invertebrate model systems will provide leads to the underlying molecular mechanisms of aging, these principles will have to be interpreted in terms of mammalian physiology and ultimately integrated with human pathology. Only a truly interdisciplinary approach can hope to eventually implement therapies to alleviate the suffering caused by age-associated degenerative processes.

? DESCRIPTION (provided by applicant): Aging is associated with more than a ten-fold increase in the incidence of sudden cardiac death (SCD). This multi-PI proposal brings together two investigators with very different but complementary areas of expertise to investigate the role of fibroblast senescence in the increased fibrosis and abnormal electrical activity found in SCD. The Koren laboratory has developed a minimally invasive myocardial infarction (MI) model in aged rabbits that exhibits increased incidence of SCD and closely recapitulates human pathophysiology. The Sedivy laboratory has developed biomarkers and genetically manipulated mice to study cellular senescence in vitro and in vivo. We propose that the interplay between aging cardiomyocytes (CMs) that have a lower threshold for arrhythmogenic activity, and the increased fibrosis in the infarct border zone (IBZ) that slows the conduction of electrical impulses, are the two main elements that promote the initiation of triggered activity and its spread to the rest of the heart, leading to SCD. We hypothesize that cellular senescence of cardiac fibroblasts (CFs) plays a key role in post-myocardial infarction (MI) wound healing processes. We postulate that aging results in changes that modify the senescence response of CFs, leading to excess fibrosis after MI. In Aim 1 the Koren laboratory will create MI by implanting a coil in the circumflex coronary artery and monitor the rabbits for spontaneous and inducible arrhythmias. The hearts will be optically mapped ex vivo to study action potential dispersion, conduction velocities and blocks in the IBZ, triggered activity, and heterogeneities in electrical and mechanical restitution at the IBZ and remote zones (RZ). CMs cultured from the IBZ and RZ will be studied for changes in action potential durations (APDs), depolarizing and repolarizing currents, calcium dynamics and triggered activity. In Aim 2 the Sedivy laboratory will use mouse models to specifically focus on fibrosis, and its regulation by the senescence of CFs. CFs will be cultured from the IBZ and RZ after MI and investigated in vitro for a variety of senescence markers, regulation of the senescence response, and phenotypes elicited in senescent cells. Corresponding tissue specimens will be investigated for evidence of these processes in vivo. Young and old animals of both species will be studied. Senescence will be manipulated in vivo using genetic (transgenic mice) and pharmacological (drugs that promote senescence) approaches. Recent work on liver, cutaneous and post-MI wound healing suggests that senescence normally limits fibrosis. Hence, we postulate that accelerating senescence will reduce fibrosis, which should have a beneficial effect by alleviating SCD. In summary, we envision that by understanding and controlling the cellular senescence mechanisms we will be able to reduce the aging fibrotic response and alleviate the propensity for arrhythmogenesis.

Project Summary Retrotransposable elements (RTEs) comprise approximately 45% of the human genome. RTEs are mobile DNA elements that can insert into new genomic positions using a copy and paste mechanism. This process, termed retrotransposition, can be deleterious at multiple levels by causing mutagenesis and genome structural instability, triggering epigenetic changes, and disrupting normal patterns of gene regulation. Numerous single- gene mutations in humans have been documented to result from germline retrotransposition. Organisms have evolved multiple transcriptional and post-transcriptional silencing mechanisms to protect their genomes against RTEs. Until recently RTEs were thought to be silent in the soma, however, new evidence points to activity in the brain and in cancer cells. Indeed, initial indications are that somatic retrotransposition is much more frequent than previously anticipated. We have reported that retrotransposition is activated during aging and cellular senescence, and hypothesized that it may represent a hitherto unappreciated molecular aging process. The long-term goal of our research is to determine the impact of retrotransposition on our genomes and transcriptomes during aging. As a first step, our objective in this proposal is to study, using high-throughput DNA and RNA sequencing methods, the mobilization of RTEs during cellular senescence and aging, and their impact on the transcriptome. In Aim 1 we will perform genome-wide high-throughput DNA sequencing of in senescent cells to determine where new insertions occur and to evaluate whether these events have the potential to exert deleterious effects by disrupting regions of the genome important for cell function. We have evidence that many new insertions likely occur in individual post-mitotic cells after they have ceased dividing. To comprehensively profile the spectrum and frequency of these events we will use single-cell whole genome DNA sequencing. In Aim 2 we will perform these same studies in the mouse to investigate to what degree different tissues are affected by increased retrotransposition with age. In Aim 3 we will investigate the effects of RTEs derepression and transposition on the transcriptome of senescent cells and in the aging mouse. Throughout our research plan we will apply interventive methods to modulate RTE activity to investigate the consequences on the landscape of somatic transposition as well as on the physiological function of the cells. Our research will employ innovative state-of-the-art high-throughput sequencing strategies, and we will develop new bioinformatic tools to integrate and validate the output from multiple algorithms. The information obtained will address the question: To what extent is retrotransposition damaging to the genomes of somatic cells in our bodies, and is this a plausible mechanism of aging? The experience gained and tools developed will be highly informative for future studies aimed at examining these processes directly in aging human tissues. Our efforts will also inform us whether proof-of-principle studies using interventions that inhibit retrotransposition should be investigated as potential therapeutics for age-associated diseases.

PROJECT SUMMARY (PROJECT 1) Retrotransposable elements (RTEs) comprise ~45% of the human genome. RTEs are mobile DNA elements that can insert into new genomic positions using a copy and paste mechanism. This process, termed retro- transposition, can be deleterious at multiple levels by causing mutagenesis and genome structural instability, triggering epigenetic changes, and disrupting normal patterns of gene regulation. Organisms have evolved multiple transcriptional and post-transcriptional silencing mechanisms to protect their genomes. Until recently RTEs were thought to be silent in the soma, however, new evidence points to activity in the brain and in cancer cells. Indeed, initial indications are that somatic retrotransposition is much more frequent than previously anticipated. Our group has reported that retrotransposition is activated during aging and cellular senescence, and hypothesized that it may represent a hitherto unappreciated molecular aging process. These findings however raise important new questions: what is the magnitude of the 'retrotransposition problem', and to what extent is this process damaging to our somatic cells? What are the mechanisms that hold RTEs in check in somatic tissues, and how do they fail with age? Our lack of knowledge in these areas is a key barrier to under- standing the role of RTEs in aging and disease. To break new ground in these efforts our research seeks to elucidate what we broadly refer to as 'the landscape of somatic retrotransposition'. In Aim 1 we will perform a genome-wide quantitative analysis of novel transpositions. The frequency of these events in the genomes of senescent or aged cells, their structures, and their locations are completely unknown. With Jef Boeke and Core B we will apply high-throughput DNA sequencing, including TIP-seq and single-cell whole genome sequencing to comprehensively profile novel transposition events. As an independent method we will use retrotransposon reporters. Aim 2 is based on our preliminary data that occupancy of pRb on the L1 promoter decreases in senescent human cells and aging mouse tissues, that overexpression of pRb antagonizes the activation of L1 in senescent cells, and knockdown in normal cells promotes L1 activation. We hypothesize that pRb, a known regulator heterochromatin, represses L1 in normal cells and that this mechanism fails during senescence and aging. We will therefore examine the composition of pRb-containing complexes and their interaction with L1s, observe their behavior during cellular senescence and mouse aging, and perturb specific components to test effects on L1 activity. In Aim 3 we will modulate RTE activity using genetic and pharmaceutical interventions and investigate the consequences on cellular function. We will use engineered regulatable L1 elements to drive increased retrotransposition activity, develop shRNA or CRISPR interventions to block the transcription of endogenous L1s, and treat cells and mice with reverse transcriptase inhibitors to determine whether age- associated phenotypes can be ameliorated. Our long-term goal is to assess the extent to which RTEs contribute to aging or age-associated disorders, and hence are potential targets for therapeutic interventions.

? DESCRIPTION (provided by applicant): Retrotransposable elements (RTEs) comprise ~45% of the human genome. They are mobile DNA elements that can insert into new genomic sites using a copy and paste mechanism. This process, known as retrotransposition, is deleterious at multiple levels. RTEs inhabit the genomes of all life forms, from archaebacteria to humans. Not surprisingly, multiple defense mechanisms have evolved to protect genomes against RTEs. The first line of defense is to incorporate the genomic locations where the elements reside into repressive heterochromatin to prevent their expression. Combined with other posttranscriptional mechanisms these defenses are quite effective, and hence the great majority of RTEs have become passive genome passengers, accumulating mutations over evolutionary time. Most organisms, however, harbor a small number of elements that remain active; in humans, the long interspersed nuclear element-1 (LINE-1). New L1 insertions occur at a frequency of one per several hundred births, and numerous single-gene mutations have been documented to result from L1 activity in our germlines. What is the situation in our tissues? Historically, little attenion has been given to this, the prevailing opinion being that RTEs were largely dormant. However, derepression of L1 transcription and even de novo insertions are increasingly being found in a variety of somatic contexts, including embryogenesis, adult brain, and some stem cells. In cancer new L1 insertions occur in a variety of tumor types. Four members of our team (Sedivy, Gorbunova, Helfand, and Seluanov) have recently published evidence that RTEs become active during aging, in human cells, flies, and mice. In support, a rapidly accumulating literature shows that somatic retrotransposition is much more frequent than previously anticipated, and that its activation during aging is deeply conserved. With Jef Boeke, a retrotransposon expert, we have developed the hypothesis that the somatic activation of retrotransposition is a novel and hitherto unappreciated molecular aging process. The underlying mechanism, suggested by our work and that of others, is an aging-associated compromise of heterochromatin, leading to its decondensation and loss of repressive capacity. The goal of this Program Project Grant (PPG) proposal is to shed light on this new and fascinating aspect of RTE biology. We bring together three Projects in a highly integrated research plan that exploits diverse model systems (from Drosophila, through mammalian cell culture to the mouse) and methods of inquiry (from high-throughput genomics, through molecular biology to mouse physiology). The research performed by this PPG will: 1) Define the 'landscape of somatic retrotransposition' in aged tissues and senescent cells; 2) Investigate the mechanisms that lead to the failure of host defense systems with age; 3) Study the downstream consequences of RTE activation on cellular and tissue function; 4) Explore strategies for interventions to alleviate age-associated conditions that may arise from RTE activation. A Transposon Engineering and Genomics Core and a Mouse Interventions and Aging Core will provide critical and centralized services to support this research.

PROJECT SUMMARY (CORE A) The overall goals of the Administrative Core (Core A) are to provide scientific, programmatic and fiscal leader- ship, facilitate lines of communication between the different researchers involved in the PPG, maintain cohe- rence in the PPG's overall and long-range goals, and ensure that resources resulting from the PPG will benefit the scientific community. Core A will thus provide the mechanisms to manage, evaluate, and evolve the three Research Projects and two Research Cores in the program. In the context of the above, the task of Core A will be the effective coordination of the activities of all the components, such as the selection and design of jointly used models and resources (mouse lines, transposon reporters, sequencing strategies, etc.), the development of new technologies and tools to more effectively promote the research goals, providing statistical services to the members of the PPG, and enabling the scientific community access to the PPG's resources, technologies, and databases. The following activities will also contribute to achieving the overall goals: 1) Core A will ensure that the administrative and financial requirements of the NIH and the participating institutions are met; 2) Core A will organize monthly videoconferences and annual face-to-face retreats; 3) Core A will be responsible for communications between the PPG and the NIH; 4) Core A will facilitate the submission of joint publications and, in collaboration with Core B, the deposition of high-throughput datasets into public databases; 5) Core A will establish the External Advisory Board (EAB) and organize the annual reviews of the PPG.

? DESCRIPTION (provided by applicant): Retrotransposable elements (RTEs) comprise ~45% of the human genome. They are mobile DNA elements that can insert into new genomic sites using a copy and paste mechanism. This process, known as retrotransposition, is deleterious at multiple levels. RTEs inhabit the genomes of all life forms, from archaebacteria to humans. Not surprisingly, multiple defense mechanisms have evolved to protect genomes against RTEs. The first line of defense is to incorporate the genomic locations where the elements reside into repressive heterochromatin to prevent their expression. Combined with other posttranscriptional mechanisms these defenses are quite effective, and hence the great majority of RTEs have become passive genome passengers, accumulating mutations over evolutionary time. Most organisms, however, harbor a small number of elements that remain active; in humans, the long interspersed nuclear element-1 (LINE-1). New L1 insertions occur at a frequency of one per several hundred births, and numerous single-gene mutations have been documented to result from L1 activity in our germlines. What is the situation in our tissues? Historically, little attenion has been given to this, the prevailing opinion being that RTEs were largely dormant. However, derepression of L1 transcription and even de novo insertions are increasingly being found in a variety of somatic contexts, including embryogenesis, adult brain, and some stem cells. In cancer new L1 insertions occur in a variety of tumor types. Four members of our team (Sedivy, Gorbunova, Helfand, and Seluanov) have recently published evidence that RTEs become active during aging, in human cells, flies, and mice. In support, a rapidly accumulating literature shows that somatic retrotransposition is much more frequent than previously anticipated, and that its activation during aging is deeply conserved. With Jef Boeke, a retrotransposon expert, we have developed the hypothesis that the somatic activation of retrotransposition is a novel and hitherto unappreciated molecular aging process. The underlying mechanism, suggested by our work and that of others, is an aging-associated compromise of heterochromatin, leading to its decondensation and loss of repressive capacity. The goal of this Program Project Grant (PPG) proposal is to shed light on this new and fascinating aspect of RTE biology. We bring together three Projects in a highly integrated research plan that exploits diverse model systems (from Drosophila, through mammalian cell culture to the mouse) and methods of inquiry (from high-throughput genomics, through molecular biology to mouse physiology). The research performed by this PPG will: 1) Define the 'landscape of somatic retrotransposition' in aged tissues and senescent cells; 2) Investigate the mechanisms that lead to the failure of host defense systems with age; 3) Study the downstream consequences of RTE activation on cellular and tissue function; 4) Explore strategies for interventions to alleviate age-associated conditions that may arise from RTE activation. A Transposon Engineering and Genomics Core and a Mouse Interventions and Aging Core will provide critical and centralized services to support this research.