Michael Rosbash - US grants

DESCRIPTION (provided by applicant): The long-term objectives of this application are to understand the basic mechanisms of nuclear RNA processing in the yeast Saccharomyces cerevisiae. RNA processing is largely conserved between yeast and humans and many steps occur predominantly co-transcriptionally, including splicing and RNP assembly. Whereas some of these events are well-understood, others are not. Further investigation is needed to understand the mechanisms by which RNP proteins and splicing factors are recruited co-transcriptionally as well as the relationship between chromatin and completed transcripts, i.e., after 3 end formation but before nuclear export. A first goal is to continue our work on the early steps of splicing as well as the relationship of splicing and spliceosome assembly to transcription. Proteins that impact more general aspects of RNP function as well as splicing will be also be studied. A second goal is to characterize chromatin by mass spectrometry, with an emphasis on proteins involved in post-transcriptional events. The strategy will take advantage of yeast genetics and look at interesting and well-studied mutants, which should give rise to missing chromatin proteins. New relationships should be uncovered, i.e., new proteins involved in these defined biochemical processes. A third goal is to visualize active genes by light and electron microscopy. Genes will be separated from the rest of the chromosomal DNA by in vivo endonuclease cleavage and their movement within nuclei examined by light microscopy. Visualization of individual pot II genes will also be done by Miller spreading, with an emphasis on co-transcriptional spliceosome assembly and splicing. A fourth goal is to identify the function(s) of individual yeast RNA binding proteins, with an emphasis on nuclear proteins. We will also further characterize the nature of dots that contain RNAs retained near the site of transcription. Understanding these processes is critical for human health. It is estimated that at least 60% of the human genome undergoes alternative splicing, most of which probably occurs co-transcriptionally. This research therefore has relevance to public health, as splicing errors also generate aberrant proteins and are associated with important human genetic diseases, including cancer, muscular dystrophy, and Alzheimer's. In addition, the exosome - involved in RNA turnover and dot formation - is implicated in autoimmune disorders.

The long term objectives of this proposal are to understand the mechanisms which regulate gene expression and the ways in which gene expression affects development and behavior. We propose to study this problem in one experimental organism, Drosophila melanogaster, because of the wealth of genetic information, procedures, and existing or readily obtainable variants connected with this organism. Also, the recent advent of DNA-mediated transformation makes it now possible to examine the relationship between specific DNA sequence and gene expression or phenotype, in a manner unparalleled in any other complex metazoan. (i) We will clone the per gene, involved in the determination or maintenance of biological rhythms. This gene will be characterized from molecular, phenotypic, and evolutionary perspectives in order to understand how a single gene has such a complex effect on different biorhythms. (ii) We will utilize cloned genes to improve current procedures to generate genetic mosaics in Drosophila. Successful completion of this project will allow the undertaking of experiments that are almost impossible with existing techniques, (iii) We will analyze the way in which Drosophila controls the expression of its four alpha-tubulin genes. This will be pursued by exchanging regions and sub-regions of alpha-tubulin genes and examining the pattern of expression of these altered genes subsequent to DNA-mediated transformation into the Drosophila germ line. (iv) We will study the genetics plus molecular biology of the vnd and kp loci and their control of neurogenesis in Drosopila embryos. (v) We will pursue genetic and molecular experiments to characterise further the structure and possible function of the complex embryonic transcripts, which are coded for by chromosome region 99D. We will also undertake a study to define the number, location, and properties of open reading frame DNA segments in Drosophila. These five projects will be performed in an interactive fashion so that each of the laboratories will be able benefit from the individual project investigations, in proper program project fashion.

The long-term objectives of this application are to understand the molecular mechanisms which control differential gene expression in oogenesis and early development in Xenopus laevis. Our interests, therefore, are related to basic eukaryotic molecular biology as well as developmental biology. The methodological approach includes standard recombinant DNA technology. In addition, a significant fraction of the research plan consists of the completion and exploitation of a novel method to clone open ready frames (orfs) directly from genomic DNA. This will allow us to score the presence and behavior of "average" mRNAS during oogenesis and embryogenesis. We will utilize these orf clones, more standard cDNA clones (for known and unknown gene products), and in vitro translation to monitor a number of events in oogenesis and early embryogenesis. Specifically, we will measure what fraction of Xenopus genes are transcribed in oocytes, tadpoles, and liver and what fraction of these mRNA are common to all these tissues. We will characterize further the structure of oocyte RNA to search for RNA molecuses stored in non-mRNA (precursor?) form. We will characterize oocyte nuclear RNA to find out which sequences are present in oocyte germination vesicles and in what form. We will also continue to search for any oocyte sequences which accumulate during oogenesis. We will also pursue recent observations that we have made on the molecular events of oocyte maturation and early embryogenesis. These include the adenylation and deadenylation of specific mRNAs as well as the degradation of certain mRNAs. We will also define the relationship of these events to changes in in vivo protein synthesis at these times. An attempt will be made to define and characterize the mRNAs associated with the nuclei of early embryos. Finally, the actin genes of Xenopus will be characterized. Two of these genes will be used in oocyte injections experiments to constitute a homologous Xenopus oocyte injection system which might shed some light on transcriptional control in oocytes. These experiments are of significance to the biology of early development because amphibians are among the only vertebrate systems in which oogenesis and early embryogenesis can be studied biochemically.

This program project grant revolves around the development and function of the nervous system. The two experimental organisms, Drosophila and C. elegans, underscore an additional common theme of the application: the molecular basis of neural function and behavior in genetic systems. The links between the five projects/laboratories are therefore practical as well as intellectual. In the first and second project, the Drosophila circadian clock will be investigated. There is arguably more known about the molecular basis of circadian rhythms in Drosophila than in any other eukaryotic organism. Yet there are only two identified Drosophila clock components (PER and TIM), and neural substrate features of this system are primitive. The first Project proposes to identify biochemically additional clock components, and the second Project will largely focus on neural and physiological issues that address the Drosophila pacemaker mechanism. The third Project will focus on two Drosophila genes, elav and appl. ELAV provides a vital function essential for proper formation of the nervous system, and it has recently been shown that it functions as a regulator of neuron-specific alternate splicing. The application proposes to investigate with biochemical and genetic procedures how ELAV regulates splicing, with significant input from the Rosbash laboratory. Proposed experiments on APPL will test the hypothesis that it acts as a receptor that influences neuronal arbors and synapses. The fourth Project proposes to continue and extend studies on the contribution of CaM kinase to plastic behaviors, including learning and memory. Specific aims includes the identification of neurons requiring CaM kinase, the characterization of CaM kinase- dependent biochemical pathways, and characterization of the larval neuromuscular junction phenotype of animals with alterations in these biochemical pathway. Significant input for the Hall lab on courtship conditioning as well as from White lab on tyrosine beta hydroxylase mutant is anticipated. The fifth Project proposes to study olfaction in C. elegans. Specifically, the focus is on the regulatory gene odr-7 and its role in the determination of the AWA subtype of olfactory neurons and its sensory properties. Odr-7 encodes a unique member of the nuclear receptor family of transcription factors. Specific aims include the identification and characterization of targets of odr-7 regulation, analysis of the effects of spatial and temporal control of odr-7 expression, and the identification of additional genes required for the determination of sensory specificity of the AWA neurons.

This is an application to provide partial support for U.S. participants at the Arolla International Meeting on Mechanisms of Gene Regulation and Development, to be held in Arolla, Switzerland, August 14-21, 1988.

"The Conference on Mechanisms of Gene Regulation and Development" will be held in Arolla, Switzerland, August 14 through August 21, 1988. This meeting will bring together scientists working with different approaches in an environment conducive to discussions between scientists at all levels of their professional careers.

This Program Project grant revolves around the development and function of the nervous system in Drosophila. Many of the approaches involve genetics and molecular biology. In Project I (Rosbash) and II (Hall), circadian rhythms will be investigated. Several such studies will apply existing "clock" mutations and cloned DNA defined by mutant alleles at a locus called period (per). In addition (Project I), it is proposed to isolate several new circadian rhythm mutants, using chemical and transposon-mobilization mutageneses. It is also proposed to initiate molecular analyses of certain emerging rhythm mutants. In Project II, the neural substrates of Drosophila's circadian rhythms will be delved into. Cyclings of per product levels, known to fluctuate with circadian periodicities, will be dissected molecularly, immunohistochemically, and behaviorally. Searches for interspecific relatives of the per gene will be made. In project III (White), the influence of two genes on Drosophila's embryonic development will be analyzed. One is embryonic-lethal/abnormal-visual-system (elav), whose encoded. protein will be studied with regard to its presumed role in the control of neuronal-specific mRNA processing. Putative targets of ELAV's action will be looked for in genetic and biochemically based screens. Aberrant expression of elav will be analyzed, by molecularly characterizing conditional mutants, and eliciting ectopic expression of the gene's product(s). The other gene, erect-wing (ewg), which is expressed in the nuclei of developing neurons and muscle precursors, will be investigated with regard to possible involvement in transcriptional regulation. Project IV (Lisman) involves the function of Drosophila photoreceptors. The two reactions involved in the first stage of transducing visual stimuli [activation of G-protein by light-activated rhodopsin (M*), and the reaction by which M* is deactivated] will be quantitated. Molecular manipulations of a cloned opsin gene-- to eliminate most of the putative phosphorylation sites-- will be effected in order to: measure the rate of M * deactivation and gain and determine if the reliability of this deactivation is impinged upon. A separate photoreceptor-based subproject will connect with Projects I and II, whose previous studies showed that per expression is prominent in these visual-system cells. Project V (Griffith) will study the role of the multifunctional calcium/calmodulin protein kinase II (CaM kinase) and its relatives in experience-dependent behavior. Gene cloning, biochemical characterization, spatial expression studies, and the identification of other biochemical components that contribute to behavioral plasticity are the goals of this project.

This Program Project grant revolves around the development and function of the nervous system in Drosophila. Many of the approaches involve genetics and molecular biology. In Project I (Rosbash) and II (Hall), circadian rhythms will be investigated. Several such studies will apply existing "clock" mutations and cloned DNA defined by mutant alleles at a locus called period (per). In addition (Project I), it is proposed to isolate several new circadian rhythm mutants, using chemical and transposon-mobilization mutageneses. It is also proposed to initiate molecular analyses of certain emerging rhythm mutants. In Project II, the neural substrates of Drosophila's circadian rhythms will be delved into. Cyclings of per product levels, known to fluctuate with circadian periodicities, will be dissected molecularly, immunohistochemically, and behaviorally. Searches for interspecific relatives of the per gene will be made. In project III (White), the influence of two genes on Drosophila's embryonic development will be analyzed. One is embryonic-lethal/abnormal-visual-system (elav), whose encoded. protein will be studied with regard to its presumed role in the control of neuronal-specific mRNA processing. Putative targets of ELAV's action will be looked for in genetic and biochemically based screens. Aberrant expression of elav will be analyzed, by molecularly characterizing conditional mutants, and eliciting ectopic expression of the gene's product(s). The other gene, erect-wing (ewg), which is expressed in the nuclei of developing neurons and muscle precursors, will be investigated with regard to possible involvement in transcriptional regulation. Project IV (Lisman) involves the function of Drosophila photoreceptors. The two reactions involved in the first stage of transducing visual stimuli [activation of G-protein by light-activated rhodopsin (M*), and the reaction by which M* is deactivated] will be quantitated. Molecular manipulations of a cloned opsin gene-- to eliminate most of the putative phosphorylation sites-- will be effected in order to: measure the rate of M * deactivation and gain and determine if the reliability of this deactivation is impinged upon. A separate photoreceptor-based subproject will connect with Projects I and II, whose previous studies showed that per expression is prominent in these visual-system cells. Project V (Griffith) will study the role of the multifunctional calcium/calmodulin protein kinase II (CaM kinase) and its relatives in experience-dependent behavior. Gene cloning, biochemical characterization, spatial expression studies, and the identification of other biochemical components that contribute to behavioral plasticity are the goals of this project.

CORE ABSTRACT NOT AVAILABLE

DESCRIPTION (provided by applicant): This renewal of a program project application focuses on the molecular basis of behavior and neuronal function in two genetically tractable model organisms: Drosophila melanogaster and C. elegans. A subtheme is the behavioral and neuronal control of gene expression, which also relies on state-of-the-art microarray technology. In Project 1 (Rosbash and Hall), the Drosophila circadian clock will be further investigated. The goal is to identify new rhythm genes and new clock-controlled genes, by genetic and biochemical means. The possibility of circadian clocks in C. elegans and yeast will also be addressed. Project 2 (White and Rosbash) will focus on the Drosophila gene ELAV. Its protein product ELAV has effects on 3' end formation as well as neuronal splicing. The goals include the elucidation of the biochemical mechanisms that underlie ELAV-mediated pre-mRNA processing in neurons. They also include the identification of direct and indirect targets of ELAV. Project 3 (Griffith) will analyze the contribution of CaMKII to courtship conditioning in flies. The full extent of the adult circuit will be determined, and the contribution of CamKII modulation of Eag potassium channel excitability will be tested. The developmental role of CaMKII in the assembly of the neuronal circuitry will be analyzed, and the sensitive cell groups defined. To determine the temporal pattern of kinase activation, genetically based sensors will measure the real time-activation of CaMKII and PKA in neurons of intact behaving animals. Project 4 (Welte and White) will study the role of the cytoplasmic dynein intermediate chain (Cdic) in specific neuronal functions. It will also determine the extent to which nuclear migration and axonal transport rely on the same components of the dynein transport machinery. Finally, microarrays will be used to determine how nuclear positioning affects gene expression? Project 5 (Sengupta) will address the contribution of signal transduction

DESCRIPTION (provided by applicant): This is a proposal to continue NINDS funding of a Core Facility at Brandeis University. This will allow us to fully exploit the three subcomponents that are now firmly established: a microarray and FACS facility, an imaging facility and a mouse/transgenic facility. They are the underpinnings of a large number of NINDS- funded and other neuroscience-relevant research projects on campus. We also propose major expansions of all three existing cores and the establishment of a fourth core. The microarray/FACS facility will be expanded to encompass proteomics thereby becoming the Genomics/Proteomics Core Facility. The imaging facility will be expanded to include Correlated Light and Electron Microscopy (CLEM) and ultra high- resolution cryo-fluorescence imaging thereby become the Imaging/CLEM Core Facility. The mouse/transgenic facility has recently added the capacity to produce Lentiviral vectors for transfection and transgenesis, and is now referred to as the Transgenic Mouse and Viral Transfection Core Facility. Finally, we will establish a new Computational Core Facility to support large scale neural simulations and computational biology projects as part of a large high-performance computing cluster. These additions will allow the Brandeis community to remain at the cutting edge technologically, and ensure that we can continue to generate exciting and ground-breaking new science. The projects supported by the cores are joined together through the shared interests of multiple neuroscience faculty members in basic as well disease- related aspects of brain and neuron function: cell identity, synaptic transmission and circuits, plasticity, behavior and its modulation. The proposed studies will exploit vertebrate and invertebrate model systems, with a strong emphasis on transgenic animals. They address basic and applied problems that are pertinent to a wide range of neurological and psychiatric diseases including disturbances of excitability, such as epilepsy, disturbances of sleep, waking and mood, neurodevelopmental disorders such as Autism and Rett Syndrome, neurodegenerative disorders, like Amyotrophic Lateral Sclerosis, and disturbances of long and short -term memory such as those that accompany Alzheimer's Disease and Schizophrenia.

[unreadable] DESCRIPTION (provided by applicant): Animal behavior is both stable and plastic. To these ends, molecules and neural circuits respond to environmental changes, both short-term and long-term pertubations. In some cases, the responses maintain a fixed behavior despite a radically altered external environment, whereas in others the responses underlie an adaptive change. Learning and memory, for example, are an important class of adaptive changes to altered environmental stimuli. This Program Project group is principally focused on temperature as the environmental stimulus of interest, and all five laboratories work on poikliotherm invertebrates. There are three Drosophila groups, one C. elegans group and one group working on the crab stomatogastric ganglion (STG). All are interested in the molecules, neurons, networks and mechansms that underlie stability, sensory responsiveness, and change (learning) in the context of altered environmental stimuli. Although each of the five projects is self-contained, there are strong and important connections between groups: In Project 1, Rosbash is collaborating with Sengupta and asking, are there circadian rhythms in C. elegans and what are the relevant molecules? In this same project, Rosbash is collaborating with Garrity and asking, what are the temperature sensors that govern circadian entrainment by temperature in Drosop/7/7a? Even projects that are not directly collaborative are well-connected, as the STG group (Marder, 4) is asking a question of profound interest to a circadian laboratory (Rosbash, 1): how does network output remain constant despite substantial changes in temperature? This is a classic circadian problem but has never been addressed in the context of non-circadian molecules, cells and circuits. Project 1 also addresses the same overarching question in the traditional circadian context. Both Project 2 and Project 5 are addressing thermotaxis and thermosensory responses, in Drosophila and C. elegans, respectively. Project 5 (S [unreadable] [unreadable]

FACILITIES AVAILABLE Our five laboratories are adequate to carry out the specific five projects as requested. There are in addition a number of collective facilities, which we share. The labs have a media kitchen with approximately 600 sq. ft. of space dedicated to preparation of Drosophila media and worm plates. Artificial seawater is also produced by this facility. The kitchen currently contains its own autoclave, office, and storage facilities. In addition, the labs share a special space in the basement of the Bassine building ("the pit": approximately 1000 sq. ft.) in which several fly incubators are housed that are used to monitor circadian and thermal activity. The light tight and constant temperature nature of this space makes it optimal for entrainment in constant dark as well as controlled light-dark and temperature conditions. This is where much of the behavioral monitoring takes place. In addition to this there is a 60 sq ft. temperature and humidity controlled Harris room in the Garrity/Griffith space that is used for cold behavior and learning. All of the labs have access to available departmental resources including a phosphorimager facility, confocal facility, microarray/FACS facility, etc. The Confocal laboratory is directed by and used heavily by all laboratories. In addition to this there is also a shared Imaging Facility that has a two-photon microscope and two real-time epifluorescence microscopes. The Fly labs share an injection facility (320 sq. ft.) with two microinjection setups for the generation of transgenic flies. The Microarray/FACS Facility are shared resources 2 buildings away. These facilities contain Affymetrix microarray processing instrumentation (hybridization oven, fluidics station, and scanner), an Agilent 2100 Bioanalyzer system, and a BD FACSAria Flow Cytometer. We have a NMR facility, and a new MALDI and peptide analysis center. The structural biology group at Brandeis owns and operates a superb state-of-the art electron microscope facility. A machine shop and electronics shop are available to the Biology Department and the Science library is located in the adjacent building. ADMINISTRATIVE ARRANGEMENTS The PI of this grant is and has been M. Rosbash for the past twenty-three years. Hall and White who played important leadership roles during the past five years of the grant are passing the torch to Marder and Griffith. Rosbash is principally in charge of grant management and both Rosbash and Marder are responsible for overall scientific leadership and quality control. Griffith is in charge of the media kitchen and other aspects of the Core, including oversight of the microscope facility and its technician. Rosbash's assistant/lab manager Heather Felton, who holds a doctorate in Molecular Biology, as well as the Biology Department administrator Barbara Wrightson provide administrative support for grant-related matters. Felton is an HHMI employee and Wrightson is 100% paid by Brandeis University. All five Pis are members of the Brandeis Biology Department, which is their primary affiliation from an administrative standpoint, and of the Volen Center for Complex Systems. Rosbash has received 100% salary from HHMI since 1989 eliminating any salary from this grant at that time. Most students and post-docs in the group attend the ample seminars and journal clubs hosted by the Biology Department, the Biochemistry Department and the Neuroscience community. There are also Program Project monthly seminars to foster closer contact between the five laboratories. The fly groups hold a monthly "FlyFest" at which both inside and outside speakers present their work.

We recently received funding to replace an obsolete BioRad 600 confocai microscope with a Leica TCS SP Spectral confocal microscope (S10 RR16708). The day-to-day running of the new confocal is under the direction of Mr. Edward Dougherty, an experienced microscopist. Dougherty's salary is currently paid in part by the Drosophila program project grant (N $44232), partly by Marder's N S 17813, and partly by Biology Department funds. The confocal is available on a fee-for-usage basis for all investigators at Brandeis. The confocal is presently heavily used (60-70 hours/week) as several laboratories (Marder, White, Hall, Griffith) have projects that intensively depend on the confocal, for the study of fixed and processed tissue. There is an urgent need for additional instrumentation and personnel to meet the needs of our user group. A subset of our user group (Turrigiano [PI], Marder, Griffith, Lisman and Nelson) applied for a Shared Equipment Grant to purchase a Leica Spectral Confocal/Multiphoton microscope, to allow for the study of live tissue combhled with electrophysiology (S10 RR017938). This grant did very well and stands an excellent chance of being funded. The University has committed funds for the installation of this instrument and renovations to accommodate it, but there are currently no institutional funds available for support personnel. Mr. Ed Dougherty will provide oversight for the new confocal and the multiphoton instrument, but the day-to-day running of these new pieces of equipment will require an additional technician. We also lack funds to purchase the electrophysiology equipment needed to fully utilize the potential of the multiphoton set-up. The availability of combined electrophysiology and multiphoton imaging to the user group would allow us to extend our research programs in important new directions. A second gap in our imaging arsenal is the lack of instrumentation and software for time-lapse imaging. A number of the projects of our user group require time-lapse measurements of multiple fluorophores. The set-up costs for this imaging rig (manipulators, stimulators, culture chamber stage) have been prohibitive for any individual, since it would not receive full-time use in a single lab. The addition of equipment and software for this type of analysis would extend existing projects and allow users to investigate these new, highly relevant avenues of research.

Brandeis University has a long history in computational neuroscience. This work has been characterized by the close and intimate relationships between theoretical and computational work, and many of the experimentalists on this grant (Marder, Lisman, Turrigiano, Nelson) have published numerous computational and modeling papers that have illuminated and/or driven many of their experimental findings. At present, we have a strong computational journal club that meets every Monday with attendance of about 20 faculty, postdocs, and graduate students, many of whom work in NINDS-funded labs. For example, Marder now has 4 postdocs and 3 graduate students who are doing exclusively or partially computational work, one of whom is now building models of Birren's neurons Lisman has longstanding computational projects and Paul Miller, a theorist and new hire, is already in collaborative work with Lisman, Fiser, Katz and Turrigiano. We believe that our ability to build and analyze computational models is becoming increasingly important in generating new hypotheses relevant to experimental work. We are now in an era in which reductionist approaches to biology are being supplemented by efforts to account for system behavior in terms of their underlying components. Whether we call this "systems biology" or another name, it remains the case that if one wants to understand the interaction of multiple, non-linear processes, it becomes useful, even necessary, to build models and determine which properties of the system depend on which component interactions. For example, the Birren/Marder computational collaboration arose in response of data generated in the Birren laboratory that were "begging" for a model to help understand these data and make further predictions from them. Therefore, we envision that more and more of our experimental colleagues will find it useful to have both computational expertise on campus, as well as a Core facility that will be available to run simulations, large and small. The addition of a Computational Core will make tangible our firm belief that computational and modeling work will become increasingly important to all of our experimental laboratories, and that these facilities be easily accessible and supported. Initially our computational work was done with lab-based computers, and/or small individual clusters of processors. This has two disadvantages: the relatively small number of processors available to any given investigator, and too much time being spent in maintaining too many small clusters. Consequently, a number of faculty around campus decided, with the advice and help of Dr. Steven Karel, our Biology Computational Expert, and LTS, the university's technology department, to consolidate the various small clusters found in neuroscience, biochemistry, and physics, into one large cluster. We are in the process of doing so, by taking all of our existing machines and then adding to them new hardware;we expect this process to be complete by the end of 2007. This new computational cluster will need oversight and maintenance for most effective management, and it will also need periodic replacements and upgrades. Because computational work is so integral to the experimental components of so many of us, we feel that this cluster will provide a significant asset to the NINDS-funded research we are now doing, and want to add this newly centralized facility to this Core grant. This is expecially important to maintaining consistency in the cluster maintenance and operation.

Brandeis has a long and rich history of neurogenetics in invertebrate model organisms, but only in the last two years, have we begun to begin to take full advantage of existing transgenic mouse strains and develop new ones ourselves. Prior mammalian work in the Nelson, Turrigiano, Birren and Lisman laboratories has been primarily conducted in rats. Thanks to establishment of the Mouse/Virus Core facility powerful new knockout and transgenic approaches in mice are now part of the research program in each of these laboratories, and these state-of-the-art approaches have engendered new collaborations between Brandeis faculty. For example, Birren and Sengupta are studying signaling associated with the transcription factor Arx in worms and mice, and they have begun work on a conditional knockout of Arx in mice. The Nelson lab has generated new transgenic lines which allow labeling and manipulation of specific forebrain cell types. The first characterized line labels layer 4 star-pyramid neurons in primary sensory cortices. It will enhance a collaborative project with Turrigiano on the molecular basis of changing forms of synaptic plasticity in layer 4 of visual cortex. The new mice have been made using lentiviral transgenesis and reflect the facilities newly developed capability to creat and harvest lentiviral vectors for brain transfection and transgenesis. In order to optimize delivery of lentivirus to delicate tissues we proposed to purchase a Burliegh Piezo Impact Drill system. Newer additions to the Brandeis faculty will also make use of the facility. Katz is adapting his rat multielectrode recording techniques to mice and will collaborate in multiple projects with Birren, Nelson and Turrigiano on in vivo analyses of transgenic mouse models. Paradis will join the faculty in January 2008;she will use the facility to make knockout mice important for analyzing molecules that are critical regulators of glutamatergic and GABAergic synapse development in the mammalian CNS. These include class 4 Semaphorins, which she identified in a largescale RNAi screen 1. Continued support of the facility would permit these investigators to obtain sufficient preliminary results to seek additional NINDS funding.

Brandeis has a long and rich history of neurogenetics in invertebrate model organisms, but only in the last two years, have we begun to begin to take full advantage of existing transgenic mouse strains and develop new ones ourselves. Prior mammalian work in the Nelson, Turrigiano, Birren and Lisman laboratories has been primarily conducted in rats. Thanks to establishment of the Mouse/Virus Core facility powerful new knockout and transgenic approaches in mice are now part of the research program in each of these laboratories, and these state-of-the-art approaches have engendered new collaborations between Brandeis faculty. For example, Birren and Sengupta are studying signaling associated with the transcription factor Arx in worms and mice, and they have begun work on a conditional knockout of Arx in mice. The Nelson lab has generated new transgenic lines which allow labeling and manipulation of specific forebrain cell types. The first characterized line labels layer 4 star-pyramid neurons in primary sensory cortices. It will enhance a collaborative project with Turrigiano on the molecular basis of changing forms of synaptic plasticity in layer 4 of visual cortex. The new mice have been made using lentiviral transgenesis and reflect the facilities newly developed capability to creat and harvest lentiviral vectors for brain transfection and transgenesis. In order to optimize delivery of lentivirus to delicate tissues we proposed to purchase a Burliegh Piezo Impact Drill system. Newer additions to the Brandeis faculty will also make use of the facility. Katz is adapting his rat multielectrode recording techniques to mice and will collaborate in multiple projects with Birren, Nelson and Turrigiano on in vivo analyses of transgenic mouse models. Paradis will join the faculty in January 2008;she will use the facility to make knockout mice important for analyzing molecules that are critical regulators of glutamatergic and GABAergic synapse development in the mammalian CNS. These include class 4 Semaphorins, which she identified in a largescale RNAi screen 1. Continued support of the facility would permit these investigators to obtain sufficient preliminary results to seek additional NINDS funding.

DESCRIPTION (provided by applicant): We request partial support for the 17th Chronobiology Gordon Research Conference, to be held in Barga, Italy from June 12 - 17 in 2011. The conference will focus on topics that represent key research areas in chronobiology, which include molecular biology, genetics, behavior (including sleep), neurobiology, metabolism, cancer and (healthy) aging. The subtheme of the conference will thus be 'Integration: from molecule to organism'. All of the speakers and discussion leaders are recognized leaders in their fields and were chosen in consultation with scientists who represent the breadth of the field of chronobiology. Special attention has been given to cultural diversity and the tentative program reflects this in terms of gender balance. We have been successful in building an extremely high-quality program that features over 40% female speakers and discussion leaders. We aim to improve diversity further through registrations;preference will be given to members of under-represented groups. Training aspects have traditionally been formally incorporated into the program in the form of Hot Topic talks, which are reserved for students and post doctoral fellows. The specific objectives of the conference are: 1) to provide a forum for discussion of the latest (unpublished) chronobiology research. 2) to bring together scientists working primarily with molecular aspects of chronobiology with those who focus on higher levels of organization, as well as selected representatives of industry, to integrate across all levels - from fundamental to applied research. 3) to identify priority research themes for the coming years (e.g. aging, metabolism, pathologies) by identifying gaps in knowledge and improving conceptual models. 4) to continue - as chronobiology tradition dictates - to provide a high quality and accessible arena for interaction between trainees and senior researchers. PUBLIC HEALTH RELEVANCE: Chronobiology GRC 2011 Project narrative: The clock regulates a myriad of processes in organisms from all phyla. Recent discoveries showing robust circadian rhythms in virtually all cells emphasize the pervasive impact of the circadian clock on physiology and behavior. Accordingly, epidemiological and genetic studies on humans show that a disrupted circadian clock - whether due to genetic predisposition or to socially influenced behavior - leads to pathologies that are associated with aging in our society: sleep disorders, metabolic disease and obesity, and cancer, for example. This conference will bring together experts and trainees who are in a position to integrate and translate basic research into applied science and policy, renewing the focus of the chronobiology field to keep pace with the latest research results and the interests of society.

DESCRIPTION (provided by applicant): A new tool for the cell-specific identification of RNA binding protein targets. This proposal has its origins in my interest in post-transcriptional regulation in the Drosophila circadian system. Although historically the focus has been on transcriptional regulation, there is now substantial interest in all of the post-transcriptional regulation that occurs within the central brain neurons that govern circadian locomotor activity rhythms. Of particular interest are 8-9 neurons on each side of the adult brain, which express the neuropeptide PDF and include key pacemaker cells. We have genetic and biochemical evidence that the RNA binding protein (RBP) Hrp48 makes a substantial contribution to circadian timekeeping within these cells. The overarching question then becomes, what are the RNA targets of Hrp48. However, the identification of in vivo mRNA targets of specific RBPs is challenging, especially within small numbers of discrete neurons. This challenge is by no means limited to Hrp48 and extends to many other important RNA-binding proteins like dFMRP (Fragile X Mental Retardation Protein). The gold standard for RNA-protein identification is arguably HITS-CLIP (High Throughput Sequencing- Cross Linking and Immunoprecipitation), i.e., in vivo cross-linking of protein to RNA with UV followed by immunoprecipitation, RNAse digestion, and deep-sequencing of RNA fragments cross-linked to that protein . However, the method is imperfect: for example, the efficiency of UV cross-linking is variable, and typically very low (1-5%). More importantly for our purposes, there is unlikely to be sufficient material of a sufficient purity from a tagged protein in discrete populations of brain neurons for successful identification of target mRNAs. This proposal is designed to circumvent these issues by developing an entirely different approach to the identification of RBP substrates. It involves creating a fusion protein between an RBP and the catalytic domain of a RNA editing enzyme like ADAR (adenosine deaminase). This enzyme deaminates adenosine to inosine, which is interpreted by the ribosomal machinery as a guanosine. ADAR edited substrates can be identified by sequencing RNA, i.e., the presence of a substantial percentage of G where there is normally only a genomically-encoded A. The fusion protein will have removed the RNA recognition features of ADAR, so the catalytic domain is delivered at high local concentration to RNA targets of the RBP. To improve sensitivity if necessary, RNA can be sequenced from the cells of interest, either after IP or after neuronal purification. Preliminary data from cell culture experiments indicate that the approach is promising and warrants further development, both in tissue culture and in fly neurons, as it will provide a novel and widely applicable new technique for the cell-specific identification of RBP targets.

DESCRIPTION (provided by applicant): We request partial support for the 18th Chronobiology Gordon Research Conference, to be held in Newport, R.I. from July 14-19, 2013. Funds received from the NIH will be used to support registration fees and/or travel costs for participants, especially post-doctoral fellows and graduate students. The conference will focus on key research areas in chronobiology, which include molecular genetics, sleep, neuroscience, metabolism and related diseases, cancer, cardiovascular disease and aging. The subtheme of the conference will be, from molecule to man, which reflects the extent to which circadian rhythms affect virtually all features of physiology and disease. The session chairs are leaders in the field and have played a major role in recruiting the speakers. Special attention has been given to diversity, and the current program reflects this balance, for example gender and youth. To this end, several chairs have agreed to share a time slot to make room for additional younger speakers, and a portion of our requested budget will be to support a new pre-conference program for student and post-doc attendees (Gordon Research Seminar (GRS). In short, we will focus on youth and training to complement the stars of the field. Lastly, we will aim to improve diversity further through registrations; preference will be given to members of under- represented groups. The specific objectives of the conference are: 1. To bring together scientists working primarily on molecular aspects of chronobiology with those who focus on human disease. We expect representatives from industry to attend, which will help integrate across all levels of investigation, from fundamental to applied. 2. To provide a forum for discussion of the latest (unpublished) chronobiology research. More specifically, this conference has been designed to focus on gaps in our knowledge and areas of controversy. To this end, each session has two prestigious chairs, who are tasked with featuring and even debating interesting and unresolved issues in their opening presentations. This strategy should improve conceptual models and help to prioritize future research themes. 3. To continue - in the tradition of Chronobiology - to provide a high quality arena for interactions between trainees and senior researchers. Whereas section chairs will be leaders in the field, younger people (students, post-docs and untenured faculties) will have opportunities to present their work.

Project Summary/Abstract Recent publications indicate that post-transcriptional regulation makes an important contribution to circadian rhythms, in flies and in mammals. This evidence is now quite extensive and includes translational regulation, which impacts the synthesis of key clock proteins within the mammalian SCN as well as within the small number of central brain neurons that govern Drosophila circadian locomotor activity rhythms. These neurons pose a significant challenge ? and opportunity ? for circadian biology and for molecular biology more generally: how can one assess biochemical events within a few neurons? For almost all measurements, there is not enough signal and/or a problematic signal:noise ratio when an extract is made and assayed starting with only a few cells/brain. Even methods that tag brain neurons are plagued with this problem if the fraction of tagged neurons/brain is very low. To address this issue in the context of RNA binding proteins and their targets, my lab developed a new technique, which we call TRIBE (Targets of RNA Binding proteins Identified by Editing). It fuses the catalytic domain of the RNA editing enzyme ADAR to RNA binding proteins. TRIBE takes advantage of the fact that one can make clean RNA and sequence it even from a single cell despite the inability to work with an extract from the same source material. We have used TRIBE with three different RBPs and also showed that it can work within the ?small number of specific neurons? context. We have recently validated TRIBE and improved its efficacy, so that it has many fewer false negatives, i.e., it recognizes a much greater fraction of RBP- associated mRNAs. We will extend the method in two new translation-centric directions: to identify specific targets of the important translation factor eIF4E-BP and to identify ribosome-associated transcripts. We also propose to address some remaining issues, and extend TRIBE in new directions, with CRISPR-mediated knock-ins as well as with dimerization approaches. The latter will bring the editing moiety to the RNA on command, i.e., in response to a drug- or light-mediated dimerization signal. In all cases, assaying successfully small numbers of discrete neurons is the key biological focus. It is an important goal relevant to many human health problems like mental illness, neurodegeneration, stroke, substance abuse and addiction.