Giovanni Bosco - US grants

[unreadable] DESCRIPTION (provided by applicant): The retinoblastoma tumor suppressor protein (RB) is a critical regulator of cell proliferation. RB binds to the E2F transcription factor and together repress transcription of S-phase genes, thereby controlling the G1-S cell cycle transition. It has become increasingly clear that RB also has a direct function in regulating DNA replication at sites of replication initiation. The evidence for this direct role comes from five observations: (1) RB localizes to replication foci in primary human cells and regulates progression through S-phase as well as the G1-S transition. (2) RB interacts with the MCM7 replication factor and can repress replication in a Xenopus cell-free and transcription-free system. (3) RB can be recruited to mammalian replication origins after DNA damage and prevents endoreduplication. (4) Drosophila RB (RBF) interacts with the replication initiation factor, the Origin Recognition Complex (ORC). (5) Mutants in Drosophila E2F and RB fail to limit replication initiation and mis-localize ORC in ovarian follicle cells. The mechanisms by which RB functions to control replication directly are not known. This proposal builds on our previous observations that Drosophila RB associates with ORC and that RB mutants mislocalize ORC and fail to limit [unreadable] DNA replication. We now have shown that the N-terminus of RBF (a.a. 151-330), that is conserved [unreadable] throughout RB family proteins, is critical for its interaction with ORC. This and recent reports that human RB localizes to replication initiation sites strongly suggests that RB control of replication initiation is an evolutionarily conserved function. Our central hypothesis is that RB regulates DNA replication by two distinct mechanisms: First, it represses transcription of S-phase specific genes by responding to antiproliferative and developmental cues; Second, RB associates with ORC at origins to inhibit replication initiation. There are two big picture questions we are asking here. (1) What are the mechanisms by which RB directly represses replication initiation at origins? (2) How are endo cycles and chorion amplification regulated by developmental signals thus coordinating development with RB regulated cell cycle events? Using Drosophila oogenesis as a model system, our proposed studies will give us new insights as to how the RB tumor suppressor protein in response to developmental cues regulates DNA replication. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Drug addiction, long-term memory and other lasting behavioral changes derive from plasticity of underlying neural circuits, driven by activity-regulated gene expression. Recent behavioral analyses demonstrate critical roles for two transcription factors, CREB and AP1 (usually a dimer of Fos and Jun) in regulating cocaine addiction. Induction of FosB, a dominant-negative Fos isoform, in rodent nucleus accumbens causes long-term behavioral sensitization and craving for cocaine; in contrast, induction of CREB reduces drug-reward by inducing adaptation to higher levels of cocaine. Despite the obvious importance of AP1 and CREB for behavioral plasticity, little is known about the mechanism of their action. Our recent observations are consistent with the unexpected, important hypothesis that AP1 acts upstream of CREB at the top of the hierarchy of known plasticity-associated transcription factors. While testing this hypotheses, this proposal aims to: a) more completely elaborate cellular functions of AP1; and b) identify molecular mechanisms that operate upstream and downstream of AP1. In the short term, the work will provide a description of cellular biological consequences of AP1 manipulations, and outline the hierarchies and relationships among regulatory proteins, like AP1, CREB and MAP kinases that initiate plasticity processes. In the longer term, effector molecules that mediate the processes of synaptic change will be identified and their functions analyzed. Drosophila melanogaster is an excellent model organism for these analyses. The commonality of underlying mechanisms involved in plasticity regulation in mammals and insects is indicated by the functional conservation of almost all known regulators of plasticity in both phyla. However, the rate of progress of the proposed analyses in Drosophila is much faster, facilitated not only by its short generation time and facility for genetics, but also by novel resources from Drosophila genome projects, microarray technologies and newly developed procedures for gene disruption, perturbation and replacement in vivo. The proposed experiments address an area of fundamental importance in synaptic remodeling events that underlie behavioral change. The work is particularly significant because it addresses the function of Fos and Jun, two critical regulators of drug addiction. In addition, by identifying activity-regulated neuronal proteins the program may contribute new, molecular markers of plasticity processes that underlie addiction. Finally, results from these experiments may identify and validate new molecules to target for pharmacological therapy.

DESCRIPTION (provided by applicant): Drug addiction, long-term memory and other lasting behavioral changes derive from plasticity of underlying neural circuits, driven by activity-regulated gene expression. Recent behavioral analyses demonstrate critical roles for two transcription factors, CREB and AP1 (usually a dimer of Fos and Jun) in regulating cocaine addiction. Induction of FosB, a dominant-negative Fos isoform, in rodent nucleus accumbens causes long-term behavioral sensitization and craving for cocaine; in contrast, induction of CREB reduces drug-reward by inducing adaptation to higher levels of cocaine. Despite the obvious importance of AP1 and CREB for behavioral plasticity, little is known about the mechanism of their action. Our recent observations are consistent with the unexpected, important hypothesis that AP1 acts upstream of CREB at the top of the hierarchy of known plasticity-associated transcription factors. While testing this hypotheses, this proposal aims to: a) more completely elaborate cellular functions of AP1; and b) identify molecular mechanisms that operate upstream and downstream of AP1. In the short term, the work will provide a description of cellular biological consequences of AP1 manipulations, and outline the hierarchies and relationships among regulatory proteins, like AP1, CREB and MAP kinases that initiate plasticity processes. In the longer term, effector molecules that mediate the processes of synaptic change will be identified and their functions analyzed. Drosophila melanogaster is an excellent model organism for these analyses. The commonality of underlying mechanisms involved in plasticity regulation in mammals and insects is indicated by the functional conservation of almost all known regulators of plasticity in both phyla. However, the rate of progress of the proposed analyses in Drosophila is much faster, facilitated not only by its short generation time and facility for genetics, but also by novel resources from Drosophila genome projects, microarray technologies and newly developed procedures for gene disruption, perturbation and replacement in vivo. The proposed experiments address an area of fundamental importance in synaptic remodeling events that underlie behavioral change. The work is particularly significant because it addresses the function of Fos and Jun, two critical regulators of drug addiction. In addition, by identifying activity-regulated neuronal proteins the program may contribute new, molecular markers of plasticity processes that underlie addiction. Finally, results from these experiments may identify and validate new molecules to target for pharmacological therapy.

DESCRIPTION (provided by applicant): Cognitive and memory loss disorders affect a large part of the US population, and as life expectancy of humans continues to increase, the occurrence of late onset disorders leading to behavioral deficits and memory loss is on the rise. The molecular basis of long-term memory of learned behaviors is poorly understood, and thus any molecular genetic insights into the mechanisms of learning and memory will have broad and possibly profound implications for the prevention, treatment and reversal of memory loss in humans. We propose to test the hypothesis that epigenetic factors play critical functions in maintenance of long- term memory. By using Drosophila we will explore the possible function of epigenetic factors and will investigate models that will address the interactions among learned behaviors and epigenetic processes during maintenance of long- term memory over time. PUBLIC HEALTH RELEVANCE: Cognitive and memory loss disorders affect a large part of the US population, and as life expectancy of humans continues to increase, the occurrence of late onset disorders leading to behavioral deficits and memory loss is on the rise. We will explore the possible function of epigenetic factors and will investigate models that will address the interactions among learned behaviors and epigenetic processes during maintenance of long-term memory.