Phillip Allen Sharp - US grants

Regulation of transcription by RNA polymerase II in mammalian cells will be studied at the biochemical level. Advances in the analysis of this fundamental process will be used to elucidate the molecular nature of control of malignant growth. Activation of transcription by sequence- specific transcription factors alters the structure of a basal complex composed of at least the IIA IID factors. The unique aspect of this complex which specifies an increase rate of initiation will be determined. Transcription factors interact with basal factors through specific protein-protein contacts. The chemical nature of these protein signals will be elucidated and the surfaces of the basal factors contacted will be identified. Results from the analysis of yeast suggest that polymerase II associates with the IIA IID complex as a holoenzyme containing other basal factors and cellular components. Evidence for a similar holoenzyme will be sought in mammalian systems. The Oct-1 and Oct-2 transcription factors bind the same octamer sequence but have different essential roles during B cell development. Retroviral vectors will be used to introduce chimeras of Oct-1 and Oct-2 to determine the protein signals which determine their unique roles in B cells. A protocol for the rational design of a sequence-specific transcription factor which will regulate in a dominant fashion a specific endogenous gene has been proposed. This protocol depends upon covalent linkage of sequence-specific domains to maximize affinity and specificity. The technology will be extended and tested by variation of the linker sequences joining the sequence-specific domains, mutation of the binding specificity of the zinc finger domains and fusion of other DNA binding domains. This methodology will be tested by designing a dominant factor which will stimulate or suppress transcription of c-Myc and other genes. Many oncogenes and tumor suppressor genes encode proteins which bind DNA and regulate transcription. In vitro and in vivo assays will be used to study the transcriptional activities of c-Myc, Rb-E2F type complexes and the Wilms' tumor factor-type factors.

precursor mRNA; spliceosomes; nucleic acid sequence; messenger RNA; genetic regulation; RNA splicing; introns; small nuclear ribonucleoproteins; photoactivation; serine; cell type; crosslink; nucleic acid chemical synthesis; arginine; structural genes; antigens; nucleic acid repetitive sequence; fibronectins; nuclear matrix; tissue /cell culture; laboratory rabbit; oligonucleotides; monoclonal antibody;

This is a request for five years of core support beginning September 1, 1980 following the termination of NCI Grant CA 14051. The Cancer Center Support (Core) Grant is requested to provide funds for shared facilities and services, including research salaries for professional staff (the faculty members who as a group operate the program of the Center), centralized services, shared equipment, developmental funds, and administration. Research will be carried out in three main areas of Cancer Biology: 1) Cancer Virology - this group, led by five faculty members, concentrates primarily on the identification of cancer-inducing genes and their products. 2) Cancer Immunology - this program, led by three faculty members, places its major effort in the study of the mechanism by which killer T lymphocytes destroy foreign or cancer cells in vivo and in vitro. 3) Cancer Cell Biology, with four faculty members, explores the biochemical changes that occur on the cell surface and in the cytoskeleton as a result of mailignant transformation. This Core Grant will aid further development of the Center by providing partial support for initial research costs of two new faculty members: a replacement in the Immunology Group and an addition to the Cell Biology Group. Development funds are also requested for the support of exploratory projects in the initial phase, prior to application for peer-reviewed funding.

Polyadenylation of messenger RNA precursors is a critical step in the synthesis of mRNAs and an event that is regulated for some transcription units. Studies of expression of modular dihydrofolate reductase genes have suggested that inefficient polyadenylation can limit the synthesis of mRNA from a DNA segment and that the efficiency of polyadenylation at some sites is cell growth dependent. In the latter case, polyadenylation at some sites occurs more efficiently in growing cells as compared to resting cells. The possible growth dependent regulation of polyadenylation will be further studied by using recombinant DNA methods to construct tandem arrangements of a potential growth dependent site and a non-growth dependent site. The relative amount of mRNA polyadenylated at the two sites will be compared under different growth and physiological conditions. In addition, sequences specifying the polyadenylation reaction will be explored by constructing systematic deletions of sequences both 5' and 3' to the consensus AAUAAA sequence. The in vivo efficiency of these deletions will be examined by subcloning into the early region of a SV40 vector. The biochemical components responsible for cleavage and polyadenylation of mRNA precursors will be identified and purified by developing an in vitro system that processes exogenously added template RNA. Initially, this template RNA will encompass the L3 site in the late transcription unit of adenovirus 2. We have shown previously that nuclear precursor RNA can be cleaved and polyadenylated at the L3 site during incubation of isolated nuclei. This will be the starting point for development of a soluble system.

Previous support from the NSF has allowed the following objectives to be largely accomplished. Development of mutant tRNA genes that would suppress termination at amber codons 2) Isolation of mammalian cell lines or cell conditions that allow efficient suppression of termination at nonsense codons 3) solation of mutants of animal viruses with nonsense lesions in essential genes, and 4) the use of these reagents to explore the genetics and molecular biology of both animal viruses and mammalian cells. Further and continuing objectives are to 1) develop a series of cell lines with opal and ocher suppressor tRNA's 2) develop an inducible suppressor system that does not involve gene amplification 3) the isolation of nonsense mutants of adenovirus and 4) to continue to develop methods for homologous recombination in mammalian cells. Much of the power of genetic systems such as yeast and Drosophila is a result of the sophisticated technology available to investigators working with those systems. The general theme of this research is to develop such methods for use with mammalian cells. %%% The value of genetic systems often correlates with the methodology available to those working with those systems. The use of suppressor tRNA's in prokaryotic and yeast systems allowed data to be accumulated which could not have been otherwise obtained. The ease with which genes can be inserted at the proper position in the yeast genome has made yeast one of the most useful of experimental genetic systems. The work projected in this proposal is designed to make some of those tools and methods available to investigators working with mammalian cells and mammalian viruses. New suppressor tRNA's will be constructed, new nonsense mutants of adenovirus will be sought and methods for homologous recombination in mammalian cells will be devised.

virus genetics; genetic regulation; oncogenes; gene expression;

Polyadenylation of messenger RNA precursors is a critical step in the synthesis of mRNAs in eucaryotic organisms and an event that is regulated for some transcription units. We have recently developed a soluble reaction from extracts of Hela cells that accurately polyadenylates exogenous substrate RNAs. Processing of the L3 site of Adenovirus 5 has been partially characterized. We propose to extend this study by identification of the precise phosphodiester bonds cleaved and newly synthesized during the polyadenylation reaction. Processing at the L3 site involves endonucleolytic cleavage and thus generation of both an upstream cleavage product and a downstream cleavage product. Under normal conditions the upstream cleavage product is polyadenylation. Under specific reaction conditions, synthesis of the poly A tract can be inhibited and the upstream cleavage product generated with little or no added adenosing residues. We will isolate and characterize the termini of both the downstream and upstream cleavage RNAs. This should reveal whether the precise site of poly A addition is produced by endonucleolytic cleavage at that position or by endonucleolytic cleavage downstream and exonucleolytic processing to the poly A addition site. These results will also demonstrate that the processing reaction at the L3 site can be mechanistically separated from the poly A synthesis reaction. Site specific mutagenesis will be used to identify the sequences and structure essential for processing at the L3 site. Polyadenylation in the soluble reaction is probably dependent upon the activity of small nuclear ribonucleoprotein (snRNP) particles since a monoclonal antibody (Y12) which reacts with sn-RNP inhibits the reaction. We will identify the specific snRNPs required and test whether processing at different poly A sites requires different snRNPs. Although not definitely proven it is likely that the regulated synthesis of the two forms of mRNAs for the heavy chain immunoglobulin genes is due to regulation of the efficiency of polyadenylation at the two sites. Polyadenylation at the upstream site in mature B cells generates mRNA encoding the secreted antibody. Polyadenylation at the downstream site in immature B cells generates mRNA for membrane bound antibody., We will develop RNA substrates for these two polyadenylation sites and characterize the in vitro processing in both Hela and B cell extracts.

The National Science Foundation established the Cell Culture Center in 1974 and has continued to support it for the purpose of providing large quantities of animal cells and their products to scientists lacking facilities to produce their own cells. As planned from the beginning, NSF funding is now being phased out. In the past nine years, the Center has completed more than 300 projects involving large-scale animal cell production. Much of the material has been supplied to researchers outside the New England area, almost all of whom have NIH support. Since NSF funding will not continue after 1986, we are seeking partial support for the Center from NIH. We feel that this support will be cost effective since large-scale cell and virus preparation can be handled much more efficiently by the Center than by individual investigators. Over the next five years, the Center plans to meet or exceed its current annual production rate of a trillion cells. In addition, extensive efforts will be made to improve the services provided by conducting research in the following areas: (1) Reduction in serum requirements for cell production; (2) Development and application of microcarriers and other cell production systems; (3) The use of computer technology and instrumentation to optimize conditions for cell production.

This is a request for five years of core support beginning September 1, 1980 following the termination of NCI Grant CA 14051. The Cancer Center Support (Core) Grant is requested to provide funds for shared facilities and services, including research salaries for professional staff (the faculty members who as a group operate the program of the Center), centralized services, shared equipment, developmental funds, and administration. Research will be carried out in three main areas of Cancer Biology: 1) Cancer Virology - this group, led by five faculty members, concentrates primarily on the identification of cancer-inducing genes and their products. 2) Cancer Immunology - this program, led by three faculty members, places its major effort in the study of the mechanism by which killer T lymphocytes destroy foreign or cancer cells in vivo and in vitro. 3) Cancer Cell Biology, with four faculty members, explores the biochemical changes that occur on the cell surface and in the cytoskeleton as a result of mailignant transformation. This Core Grant will aid further development of the Center by providing partial support for initial research costs of two new faculty members: a replacement in the Immunology Group and an addition to the Cell Biology Group. Development funds are also requested for the support of exploratory projects in the initial phase, prior to application for peer-reviewed funding.

The ultimate goal of the program is to elucidate the molecular nature of processes causing human cancer so that methods to treat or control this disease can be devised. The properties which distinguish a cancer cell from a normal cell reflect genetic changes that activate dominant oncogenes or inactivate anti-oncogenes. Both types of genetic alterations commonly result in changes of gene regulation. The central theme of this grant is the study of the malignant state by analysis of regulation of gene expression, particularly at the stage of initiation of transcription. We will address this theme by (a) study of the role of the Wilm's anti- oncogene in suppression transformation, (b) analysis of the nuclear events regulating transcription from the fos promoter caused by activation of a cell surface receptor by the oncogene protein PDGF, (c) use of a retroviral promoter trap vector to identify cellular genes critical for cell transformation by oncogenes and anti-oncogenes, (d) analysis of cell division and differentiation by studying the role of promoter specific and basal factors in regulating transcription by RNA polymerase II. The four programs constituting the grant have a conceptual and methodological unity which insure their synergistic interactions in accomplishing these objectives. This unity is reflected in the recent discovery that the Wilm's anti-oncogene protein contains a structural motif common of many sequence-specific DNA binding-proteins. The research will help reveal the cellular and biochemical mechanisms responsible for alterations in control of cell division and differentiation that cause malignant transformation. Understanding the cancer cell at the level of biochemical processes will generate new opportunities for intervening in the progression of the disease.

The goal of the proposed research is to elucidate the mechanisms of gene regulation by the human immunodeficiency virus (HIV-1) encoded proteins tat and rev. A particular emphasis will be the establishment, characterization and analysis of reactions in vitro which duplicate the respective in vivo processes. Tat stimulates transcription of the proviral DNA by recognition of the transactivation response (TAR) element as RNA. Recent results suggest that tat action increases the efficiency of elongation of the polymerase. A reaction in vitro has been developed which responds to addition of tat protein p68 by increased transcription of the HIV-1 promoter. The role of a cellular protein 68 which specifically binds the TAR element will be studied in this system following purification of the protein. The mechanism of tat activation of transcription will be analyzed by: kinetic studies of transcription in vitro, definition of the activation domain of the protein, characterization of tat-TAR interactions under reaction conditions, and reconstitution of reactions using partially purified factors which duplicate the in vivo process. A possible relationship between the process inhibited by the drug 5,6-dichloro-1-beta- ribofuranosylbenzimidazole (DRB) and the process simulated by tat will be studied. The HIV-1 promoter may be uniquely configured to be responsive to tat stimulation. This possibility will be investigated by construction of mutant promoters. Rev stimulates the cytoplasmic appearance of unspliced or singly spliced viral RNA. In the absence of rev only multiply spliced RNA is transported to the cytoplasm while unspliced or singly spliced RNA is retained in the nucleus. We have proposed that rev functions by regulating the splicing of nuclear RNA containing the rev-responsive element (RRE). Potential regulation of splicing in vitro by purified rev protein will be investigated. A short peptide containing the basic region of rev will specifically repress the splicing of an RNA substrate containing RRE but not a control substrate in vitro. This reaction will be characterized and conditions will be sought to obtain regulation by the complete rev protein. The step in assembly of splicing complexes inhibited by rev protein will be determined. Experiments analyzing the possible role of rev in transport of RNA from nucleus to cytoplasm will also be tested. The two primary benefits of this research will be establishment of (1) a knowledge base for the future development of agents that inhibit tat and rev mediated process, and (2) the relationship between regulation of viral transcription and RNA processing by these viral proteins and regulation of cellular genes by cellular factors.

The ultimate goal of the program is to elucidate the molecular nature of processes causing human cancer so that methods to treat or control this disease can be devised. The properties which distinguish a cancer cell from a normal cell reflect genetic changes that activate dominant oncogenes or inactivate anti-oncogenes. Both types of genetic alterations commonly result in changes of gene regulation. The central theme of this grant is the study of the malignant state by analysis of regulation of gene expression, particularly at the stage of initiation of transcription. We will address this theme by (a) study of the role of the Wilm's anti- oncogene in suppression transformation, (b) analysis of the nuclear events regulating transcription from the fos promoter caused by activation of a cell surface receptor by the oncogene protein PDGF, (c) use of a retroviral promoter trap vector to identify cellular genes critical for cell transformation by oncogenes and anti-oncogenes, (d) analysis of cell division and differentiation by studying the role of promoter specific and basal factors in regulating transcription by RNA polymerase II. The four programs constituting the grant have a conceptual and methodological unity which insure their synergistic interactions in accomplishing these objectives. This unity is reflected in the recent discovery that the Wilm's anti-oncogene protein contains a structural motif common of many sequence-specific DNA binding-proteins. The research will help reveal the cellular and biochemical mechanisms responsible for alterations in control of cell division and differentiation that cause malignant transformation. Understanding the cancer cell at the level of biochemical processes will generate new opportunities for intervening in the progression of the disease.

The goal of the proposed research is to elucidate the mechanisms of gene regulation by the human immunodeficiency virus (HIV-1) encoded proteins tat and rev. A particular emphasis will be the establishment, characterization and analysis of reactions in vitro which duplicate the respective in vivo processes. Tat stimulates transcription of the proviral DNA by recognition of the transactivation response (TAR) element as RNA. Recent results suggest that tat action increases the efficiency of elongation of the polymerase. A reaction in vitro has been developed which responds to addition of tat protein p68 by increased transcription of the HIV-1 promoter. The role of a cellular protein 68 which specifically binds the TAR element will be studied in this system following purification of the protein. The mechanism of tat activation of transcription will be analyzed by: kinetic studies of transcription in vitro, definition of the activation domain of the protein, characterization of tat-TAR interactions under reaction conditions, and reconstitution of reactions using partially purified factors which duplicate the in vivo process. A possible relationship between the process inhibited by the drug 5,6-dichloro-1-beta- ribofuranosylbenzimidazole (DRB) and the process simulated by tat will be studied. The HIV-1 promoter may be uniquely configured to be responsive to tat stimulation. This possibility will be investigated by construction of mutant promoters. Rev stimulates the cytoplasmic appearance of unspliced or singly spliced viral RNA. In the absence of rev only multiply spliced RNA is transported to the cytoplasm while unspliced or singly spliced RNA is retained in the nucleus. We have proposed that rev functions by regulating the splicing of nuclear RNA containing the rev-responsive element (RRE). Potential regulation of splicing in vitro by purified rev protein will be investigated. A short peptide containing the basic region of rev will specifically repress the splicing of an RNA substrate containing RRE but not a control substrate in vitro. This reaction will be characterized and conditions will be sought to obtain regulation by the complete rev protein. The step in assembly of splicing complexes inhibited by rev protein will be determined. Experiments analyzing the possible role of rev in transport of RNA from nucleus to cytoplasm will also be tested. The two primary benefits of this research will be establishment of (1) a knowledge base for the future development of agents that inhibit tat and rev mediated process, and (2) the relationship between regulation of viral transcription and RNA processing by these viral proteins and regulation of cellular genes by cellular factors.

The ultimate goal of this program is to elucidate the molecular nature of processes controlling human cancer in order to further its prevention, diagnosis and treatment. The function of tumor suppressor genes Rb and related genes p107 and p130, p53, and Wilms' WTI in normal physiology and in suppressing the development of tumors will be analyzed using gene targeting methods to generate mutant mice. The Rb protein interacts with the E2F family of transcription factors in controlling cell division. These types of interactions will be studied both by generating mouse strains deficient in the E2F-2 and E2F-3 proteins and by analyzing the biochemistry of cell cycle control using cell lines established from these and other strains. The WT1 protein binds DNA in a sequence-specific fashion and transcription of genes critical for normal kidney cell growth and development. Proteins which interact directly with the WT1 protein in mediating its effects on suppressing tumor growth will be sought. In addition, genes regulated by WT1 will be identified. The WT2 locus also suppresses development of kidney tumors and the gene responsible for this activity will be identified. Loss of p53 activity from a tumor cell renders it multidrug resistant. The relationship between p53 status and tumor cell phenotype will be further investigated. The tumor suppressor protein Rb, p53 and WT1 as well as the oncogene Myc all either bind DNA in a sequence-specific fashion or are part of a complex that has this property. The mechanisms by which these proteins as well as the Oct-1 and Oct-2 transcription factors regulate initiation of transcription will be investigated, both in vitro and in vivo. The unique function of Oct-1 and Oct-2 proteins in development of B cells will be analyzed to identify the nature of protein-protein interactions which determine this specificity. Finally, a method to design novel transcription factors to regulate specific endogenous genes in a dominant fashion will be tested.

Science is a communal activity which is best advanced with collaborations both intellectual and experimental. The overlapping research interests and collaborations of laboratories in the program is strongly reinforced by sharing of several essential core facilities. All investigators in this grant have offices and laboratories in contiguous space on the 5th floor of the Center for Cancer Research. To both promote interactions between scientists in the lab and to more cost-effectively use large equipment, the four groups share several common facilities. These include: -200 degrees cell line storage devices (some liquid nitrogen cooled), -70 degrees freezers, ultracentrifuges and rotors, intermediate speed centrifuges (Sorvall), gel dryers and vacuum pumps, bacterial fermentation facilities, spectrophotometer, phosphorimager, dark room with automatic developer, photographic device for gels, scintillation counter and computers. This equipment is located in three communal rooms which are accessible from public halls. The four groups share the work of a lab aide who is responsible for handling glassware. The four groups also share a common dry ice chest which is located with direct access from the hall. The sharing of these common facilities/equipment is important for building a feeling of shared responsibility, goals and ideas among the four laboratories. It also increases the intensity of use which reduces costs. Finally, as students and fellows compete for use of this equipment, they exchange ideas and begin collaborations. The sharing of common equipment breaks down the isolation of the four groups.

DESCRIPTION: (Adapted from Investigator's Abstract) The objective of the application is to investigate the molecular mechanisms that underlie Tat and Rev regulation of HIV expression. The Sharp laboratory has recently identified a cellular factor, Tat-SF, that is required for Tat function. It was purified biochemically, using its intrinsic activity to monitor purifications. It has been microsequenced and the corresponding cDNA cloned. Tat-SF appears to assist in processivity of elongation by RNAP II in the presence of Tat, and it appears to interact with both Tat and TAR. The application proposes to further characterize Tat-SF-Tat interaction, and the role of these proteins in the processiveness of transcription in vitro and in vivo. The kinase that phosphorylates Tat-SF, and the components that further increase Tat-responsiveness in vitro will be further purified. The laboratory has also shown that the pol II elongation factor SIII increases polymerase processivity, and reduces Tat activation, suggesting that Tat may function through facilitation in the activity of SIII. The role of this factor in Tat activation will be further examined. Finally, the application also proposes to further study Rev regulation of RNA splicing, and the interaction of Rev with the nucleoporin protein Rab.

DESCRIPTION (provided by Applicant) The goals of this program are elucidation of the genetic changes that underlie tumorigenesis and determination of the molecular mechanisms by which these genetic changes cause cancer. A current focus of the program is the mechanisms by which the retinoblastoma (Rb/E2F) pathway suppresses cancer. Mutant mice deficient in individual and combinations of the Rb family of genes, pRb, p107, and p130, will be studied for tumorigenesis, overlapping functions in development, and interactions with other genes in the Rb/E2F pathway. In addition, the roles of the E2F proteins E2F-1 and E2F-3, which are primarily responsible for activation of transcription in the pathway, in tumorigenesis and development will be studied by combining mutations in these genes with those of the Rb family. Deregulation of either E2F-1 or E2F-3 is proapoptotic and their relative contributions to cell growth and cell death will be determined. The p53 dependence of their induction of cell death will be further characterized by crossing in mutations in p53 and/or pl9ARF. Both transcriptional profiling and SNP genomic analysis will be used to characterize the nature of tumors in the p16INK4a/tyrRas melanoma model. The frequency of melanomas in this model varies dramatically between genetic backgrounds. This offers the potential to identify modifier genes by genetic analysis. A new core facility is proposed which will provide both microarray for gene expression profiles and high throughput genetic analysis using SNPs that vary between inbred mouse strains. Loss of heterozygosity when combined with transcription profiles and tumor properties will define at a new and fundamental level tumor-host biology. A similar analysis will be done on tumors arising from mutations in the Rb/E2F pathway whose malignant potential varies dependent upon genetic backgrounds. The profiling technology will also be used to study new possibilities for dominant inhibition of gene expression based on RNA interference (RNAi) in mammalian cells and the relationship between activation of transcription by Oct-1, Oct-2 and OCA-B/Bob-1 and development in B cells.

EIA-0218506
Burge, Christopher B
MIT

CRCNS: Bioinformatics of Alternative Splicing in the Nervous System

Almost every human cell contains a huge instruction manual called the genome with many thousands of pages (the genes), each of which tells the cell how to make a particular building block (protein) that it needs to live or grow or to perform its assigned function in the body. The cell uses this manual in a complicated way, first copying (transcribing) each page that it needs to a piece of scratch paper (the pre-mRNA), and then cutting and pasting (splicing) pieces of the scratch paper (the exons) together to form the final recipe (mRNA) for the protein product. Interestingly, this cutting and pasting is often carried out in different ways in different types of cells or under different conditions in a process called alternative splicing (AS), generating many different varieties of a protein under different conditions. Alternative splicing is particularly common in neurons, helping to generate protein variants whose properties are optimized to the local environment of the neuron. For example, AS is used to tune the electrical properties of ion channels which help different sensory neurons in the inner ear respond to different frequencies of sound. In addition, mutations that affect AS are associated with a number of neurodegenerative diseases. The goal of the proposal is to gain a better understanding of the signals in a gene that determine how that gene will be spliced when it is expressed in a particular part of the brain, and of how alternative splicing is used to modulate brain function.

The three projects in this PO1 will continue to share a core set of facilities which has two favorable results. First, this sharing is highly effective in cost and management time. Second, students and fellows interact during use of this common equipment promoting collaboration and cross fertilization. All Project Leaders and PI in this Program have offices and laboratories within 100 feet of one another. Some of the facilities supported in part by the PO1 core include a shared darkroom, centrifuges and microscopes. In these cases, maintenance contracts and supplies are supported. A Core facility for shRNA-vector libraries technician for use of short RNA hairpin vectors will be shared by all three projects. This technician will have laboratory space on the same floor as the three projects. All of the projects propose experiments where either large or small libraries of retro viral shRNA vectors will be used. These libraries are currently available from three sources, the Broad Institute RNA/Consortium, a library developed at Cold Spring Harbor by Dr. Greg Hannon, and a library developed by Dr. Ren6 Bernards of the Netherlands Cancer Institute. The RNAi Consortium based at MIT's Broad Institute is led by Professor William Hahn, a consultant to the Core. The Consortium has developed approximately 150,000 shRNA-lentivirus vectors which are targeted to silence approximately 30,000 genes orisoforms of genes. There will be other such libraries developed in the future. The Core will acquire, maintain and develop methods for optimalscreens with these RNAi libraries. The technician will also interact with the Virus Production Core Facility in the Center for Cancer Research. This will be both cost effective and insure a high quality of experimentation. Many of the above viral stocks are arrayed in microtiter dishes and to fully implement their use, a robotic system must be used to automate their distribution.

Cancer is a major disease burden and one hope of changing this is to develop new treatments through a better understanding of the disease. Recent discoveries concerning the activities of short RNAs in mammals may provide both new insights and new treatments of cancer. A common goal of this Program is to investigate the roles of short RNAs, such as microRNAs, in regulation of genes in normal and cancer cells. There is strong and rapidly growing evidence suggesting that changes in miRNA regulation are related to malignant transformation and in fact could be a critical event in oncogenic transformation. The function of the mir-17-92-1 cluster which is frequently overexpressed/amplified in a subset of human cancers will be investigated by creation of specific mutations of these microRNAs in the context of mouse models of cancer. Changes in microRNA populations in normal cells and tumor cells of the same developmental state will be analyzed using both bead-array technology as well as new cloning technology. Vectors with regulated expression of a short hairpin RNA which generates a specific siRNA for silencing a gene will be developed for transgenic analysis of pathways. Additionally, methods will be tested for screening of small libraries of shRNA-lentiviral vectors to identify genes which, when silenced, either inhibit or stimulate tumor development. Furthermore, libraries of retro viral vectors expressing shRNAs will be used in screens to identify (a) genes that modulate the proliferation and/or survival of pRB-deficient cells , (b) genes that modulate the rate of development of a K-ras-driven lung cancer model, and (c) genes important for the differentiation of ES cells. The potential role of short RNAs in transcriptional silencing will be investigated in embryonic stem cells. These processes could be important for epigenetic silencing and genomic stability of cancer cells. ES cells will also be studied for the role of miRNAs in development and proliferation. Changes in the spectrum of microRNAs and siRNAs during T-cell development will be characterized using a cloning technology which requires small amounts of RNAi. Activation of the Arf promoter is an early signal in oncogenic transformation. This promoter is silenced under normal conditions by the E2F3B protein, linking the p19Arf-mdm2-p53 pathway to the p16INK4a-cycD/cdk4-pRB-EdF pathway. The role of E2F3B complexes and other E2F factors in regulation of the Arf promoter will be studied.

Cancer is a major disease burden and one hope of changing this is to develop new treatments through a[unreadable] better understanding of the disease. Recent discoveries concerning the activities of short RNAs in mammals[unreadable] may provide both new insights and new treatments of cancer. Cancer is a disease of gene dysregulation[unreadable] caused by mutations and epigenetic changes. Short RNAs are now known to regulate genes at the levels of[unreadable] mRNA degradation, primarily by siRNAs, mRNA translation, primarily by microRNAs, and mRNA[unreadable] transcription, primarily by repeat-associated short interfering RNAs (rasiRNAs). Recent results from[unreadable] bioinformatic studies indicate that approximately 20% of all mammalian mRNAs are probably regulated by[unreadable] mi RNAs. Furthermore, there is strong and rapidly growing evidence suggesting that changes in miRNA[unreadable] regulation is related to malignant transformation and in fact could be a critical event in oncogenic[unreadable] transformation. Little is known about the potential roles of short RNAs in silencing transcription at the level[unreadable] of chromatin in these cells. It is possible that some of the epigenetic changes and genomic instability[unreadable] common of cancers could be directed by RNAi-related pathways. Part of the revolution of RNA interference[unreadable] is the ability to express short hairpin RNAs from vectors to generate siRNAs which silence a specific gene.[unreadable] First, in collaboration with the Jacks and Lees projects, we will develop lentivirus vectors expressing shRNAs[unreadable] in a regulated fashion using tet-activated Pol II transcription. Second, we will use a very sensitive cloning[unreadable] technology for short RNAs to analyze their expression in T-cell populations as they undergo development.[unreadable] The activities of specific miRNAs will be related to known developmental transitions in this defined pathway.[unreadable] Furthermore, the nature of short RNAs in T-cell lymphomas and other tumor cells will be investigated by[unreadable] collaboration with the other projects. The silencing of repetitive sequences by both chromatin modification[unreadable] and DNA methylation is frequently observed in embryonic stem (ES) cells. We will clone short RNAs from[unreadable] ES cells which have been induced to express high levels of RNA from repetitive sequences to investigate[unreadable] the role of short RNAs in gene silencing at the level of transcription. The relationship between the[unreadable] resilencing of repetitive and unique sequences in these ES cells and the sequences of cloned short RNAs[unreadable] will be investigated. Furthermore, the dependence of these processes on Dicer, Argonaute and other RNAi-related[unreadable] genes will be determined. We will also determine the function of a cluster of miRNAs that is[unreadable] exclusively expressed in ES cells and embryonic tissue. Finally, we have found that both the retinoblastoma[unreadable] (Rb) pathway and the RNAi pathway regulate post-mitotic nuclear division in the intestinal epithelium of C.[unreadable] elegans. We will investigate whether related pathways are important for regulation of cell division in[unreadable] mammalian cells.[unreadable]

DESCRIPTION (provided by applicant): Changes in the levels of microRNAs and effects of over-expression of microRNAs have already been related to human malignancy. However, the mechanisms by which microRNAs control cellular states and how this control is altered under stress conditions and in stationary verses proliferating cells have not been investigated. We present preliminary data indicating that microRNA-mediated regulation is important during the cell's response to stress and propose to study this relationship in normal and malignant cells. Preliminary bioinformatic analysis suggests that many mRNAs that are targets of microRNA regulation also contain conserved sites for RNA binding proteins that are known to control translation and mRNA stability during stress. mRNAs targeted by both microRNAs and stress-related RNA binding proteins can be preferentially expressed during stress. We propose to characterize the differential binding of proteins to Argonaute/microRNA complexes under stress and non-stress conditions using Stable Isotope Labeling with Amino acids in Cell culture (SILAC). We present preliminary evidence detecting differences in bound proteins under these two conditions. These proteins will be analyzed for their roles in cellular processes such as translational regulation, subcellular targeting, and mRNA stability. We further propose to identify the total set of mRNAs targeted by microRNAs by selective immunoprecipitation of Argonaute bound mRNAs from Dicer- negative embryonic stem cells, thus deficient in endogenous microRNAs that have been transfected with a single microRNA. In total, these experiments should reveal the importance of microRNA- regulation during stress conditions and could identify drug targets that could be used to preferentially inhibit/kill tumor cells undergoing stress-associated tumorigenesis. Gene regulation by microRNAs could also change during tumorigenesis if the target sites in 3'UTRs disappear. We have discovered that a large number of genes is expressed with short 3'UTRs during proliferation and longer 3'UTRs in quiescent cells. This shift was observed when arrays were used to compare mRNA expression in resting CD4-T cells and receptor stimulated CD4 cells. Further bioinformatic analysis shows this shift occurs in most resting verses proliferating tissues and in tumor verses normal tissue. The long 3'UTRs in resting cells almost certainly mediate enhanced microRNA regulation because they contain conserved seed target sites. We proposed to continue this analysis by investigating the nature of the factors controlling the proliferation-dependent shift, the importance of this change in microRNA control of the malignant phenotype, and how this shift can be modulated to induce more microRNA regulation in cancer cells. PUBLIC HEALTH RELEVANCE: The burden of cancer in public health is apparent in both human suffering and the cost of healthcare. The proposed research will provide the basis for new therapeutics to better treat cancer and thus improve public health.

siRNAs silence or suppress specific genes utilizing an endogenous pathway common to all cells. Since particular tumor cells are exquisitely sensitive to the silencing of certain genes, siRNAs offer the prospective of a general gene-specific approach to the treatment of cancer. The major barrier to the use of siRNAs therapeutically is delivery of these agents to the cytoplasm of tumor cells. This program will explore the design of multifunctional nanoparticles which will enhance the pharmacokinetics of siRNAs, target them to tumor cells, promote their uptake by cells, and their release into the cytoplasm. Two complementary approaches will be explored to enhance the therapeutic potential of siRNA. In one project, siRNAs will be attached to the surface of nanoparticles (semiconductor quantum dots) together with: peptides targeted to specific tumor cells, polyethylene glycol to limit uptake by the reticuloendothelial system, and agents to promote endosomal escape. Identification of novel targeting peptides to glioblastoma, lung adenocarcinoma and their stem cells, and peptides that facilitate transport across the blood-brain barrier will be done in collaboration with the Ruoslahti lab. siRNA may require protection from serum and tissue nucleases. Therefore, in a second project in collaboration with the Langer lab, siRNAs will be incorporated into polymeric nanoparticles that enhance their uptake and release into the cytoplasm. Specifically, a combinatorial polymer library will be screened in a high-throughput format to identify materials for efficient, non-toxic delivery to tumors. Because this is a new therapeutic approach where several basic technologies must be explored, we will concentrate in this program on testing the effectiveness of siRNAs in two murine tumor models: a glioblastoma generated by activation of a ROS tyrosine kinase receptor oncogene, and a murine lung adenocarcinoma generated by activation of the K-ras oncogene. There are several advantages in using these models: the tumors develop in the context of their normal organ tissue, the tumors are more homogenous than human cancers, and the biology of the tumors'response to treatment, such as the role of stem cells, can be explored. The project therefore combines sophisticated tumor models with two complementary nanoparticle-based approaches to cancer therapy. If successful, this research could be direcly translated to human disease and could revolutionize cancer treatment.

Nearly all cancer therapies are limited by their ability to precisely deliver a lethal dose of a therapeutic agent to tumor cells while sparing normal tissue. Recent progress in nanotechnology has resulted in nanomaterials with remarkable biological and material properties that can be leveraged for enhanced cancer diagnosis and therapy. These include targeting of tumors via ligands that direct nanoparticles to receptors in the tumor microenvironment, providing electromagnetic properties for imaging and sensing tumors, and delivery chemotherapeutic agents to tumor cells in vivo. RNA interference offers an attractive means to silence expression of genes with extraordinary specificity, particularly for the subset of genes considered undruggable. However, systemic delivery of siRNAs has been challenging due to pharmacokinetic properties resulting from their small size, the requirement for delivery of these agents into the cytosol of target cells, and their susceptibility to serum nucleases. To date, approaches for delivery of siRNAs have primarily focused on chemical modifications, carrier development using polymers, antibodies, aptamers, and peptides with limited success. The goal of this project is to assemble a multidisciplinary team to improve the management of ovarian cancer by delivering therapeutic siRNAs. As the most lethal gynecologic malignancy with 31% five-year survival rates, new therapies are desperately needed. This malignancy disseminates in the peritoneal cavity and delivery of therapeutic agents to this compartment has shown to prolong survival. siRNAs can be designed to silence most, if not all genes and thus could be used to block pathways that induce cell death in cancer cells as compared to normal cells. We propose to continue the development and testing of nanoparticles for the delivery of siRNAs to treat ovarian cancer. The specific aims are: 1 Identification and testing by nanoparticle delivery of siRNAs to specific genes that when silenced induce cell death of subsets of ovarian cancer cells in the peritoneal cavity, 2 Development of modular nanomaterials that will target the delivery of siRNAs to ovarian tumors and 3 Development of safe and effective nanoparticles composed of novel biomaterials to deliver siRNA to ovarian cancer cells in the peritoneal cavity.

CORE SUMMARY (See instnjctions); The Core supports two activities that are important to all of the projects. The first of these is the engagement of Dr. Arjun Bhutkar as a half-time Bioinformatics Specialist (Bio-sp) for analysis of results from microarrays and deep sequencing of RNA preparation. Dr. Bhutkar has significant expertise and experience in the analysis of large databases from microarray formats and from deep RNA sequencing formats (see CV). He has collaborated with students and fellows in all three projects. As a member of this Program, Dr. Bhutkar will also help design and analyze experiments where crosslinking is used to characterize protein-DNA complexes, Protein-RNA complexes, and RNA-RNA complexes. He will also help adapt deep sequencing to define the transcriptome of cells. Over time this will displace the use of microarray in characterization of cancer cells. Because computation needs to be designed into the experimental protocol at the onset, it is important to have a committed and knowledgeable Bio-sp available to the three projects. Core Specific Aim 1. Addition of Dr. Bhutkar as a Bioinformatics Specialist will facilitate for all three projects (a) the analysis of microarray results in comparison of tumor cells, (b) processing of large data sets from high-throughput sequencing of DNA sequences bound to proteins, and (c) complexes of coding and non-coding RNAs bound to proteins and RNAs. Inclusion of Dr. Bhutkar's expertise in the design of experiments as well as analysis will reduce the cost of the use of expensive and limited sequencing devices such as illumina Genome Analyzer and produce more valid results. The second activity of the Core that brings three projects together and is cost-effective is the sharing of common equipment, services, and materials. The use of this pool of equipment requires the interactions of investigators on a daily basis and is effective because all three labs occupy adjacent space on the same floor. All of the equipment supported by the Core is in shared space, 3,200 sq. ft. These Core funds are supervised by an assistant in the group and their purchase and repair are responsive to the needs of the three projects. Core Specific Aim 2. To cover the expenses of some shared equipment, services and materials that are necessary for the three projects and facilitate the interactions of investigators in the group in a cost-effective manner.

MicroRNAs are predicted to regulate a majority of genes in human cells and both overexpression and loss of expression of some miRNAs are correlated with malignant phenotypes in different cancer cells. A decrease in miRNA activity is most commonly observed and may be important for the plasticity of tumor cells to undergo transitions between differentiation states and to grow in different niches. In Aim 1 of this Project we plan to investigate the consequences of either partial or total loss of microRNAs in control of the stability of differentiation and quiescent states of tumor cells. Tumor cells from a mouse sarcoma cell line and from transformed mesenchymal stem cells have been isolated that are null for Dicer activity and missing miRNAs. Preliminary results show that Rb-type proteins regulate expression of miRNAs (see Project 3). The ability of these cells to undergo differentiation, survive stress and generate tumors in xenograft challenges will be explored. The plasticity of cell state due to the loss of miRNAs in the presence or absence of Rb-type proteins is being investigated in collaboration with Lees lab as outlined in Project 3. The expression of non-coding small RNAs in these Dicer null cells will also be investigated using deep sequencing technology. Preliminary results indicate that all significant miRNAs decrease by 100 fold in Dicer null cells. Aim 2 of this Project is designed to investigate the potential roles of RNAi related processes in control of transcriptional and RNA processing in the nucleus of normal and malignant cells. The activities of the RNAi-related small non-coding RNAs are probably mediated by Argonaute proteins. An objective is to define the roles of Argonaute-type proteins in the nucleus and their association with genomic sequences. Transcriptional silencing by RNAi-related small RNAs in non-vertebrate organisms is mediated by recognition of nascent RNA. In Aim 3 of this Project we will investigate the roles of long non-coding RNAs in regulation of transcription and RNA-related processes in normal and tumor cells. A majority of the promoters for genes expressed in embryonic stem cells have a second divergent polymerase paused in the antisense direction about 250 base pairs upstream of the sense polymerase. Elongation by this anti-sense polymerase is controlled by P-TEFb (Positive Transcription Elongation Factor b) and can produce RNAs thousands of nucleotides long. Establishing the roles of these and other types of long non-coding RNAs in cancer is also an objective. In particular, we will collaborate with the Jacks lab in investigating long noncoding RNAs and cancer.

Project Summary ? Project 2 This project has three aims addressing new aspects of transcription regulation. The first aim investigates the processes responsible for variation among cell states, such as those found in tumors, with a focus on the role of microRNAs (miRNAs). Data from single cell RNA sequencing provides new windows into these processes. In a cell population some genes vary in mRNA expression while others are uniform. Networks of co-varying genes define a range of possible transitional cell states. Elucidation of the structure of these networks will provide insights into the heterogeneity of cancers. The relationship between miRNA activity in embryonic cells and networks of pluripotent genes will be investigated in Aim 1, Definition of biological variation in gene networks due to microRNAs, with future focus on collaborations with the Jacks and Lees labs. The second aim investigates mechanisms underlying transcriptional variation. A liquid-liquid phase or, more specifically, gel-sol phase transition model is proposed as central to interactions between enhancers and promoters. This quantitative model predicts highly cooperative transitions for changes in the valences of interactions between macromolecules. The model is motivated by the high sensitivity of super-enhancers to inhibitors of general transcription factors. These inhibitors are now being tested in treatment of cancer. The relationship between super-enhancers and miRNA expression provides an experimental context to investigate the model, particularly the activity of super-enhancers in the efficient processing of primary miRNA and how human cancers with poor survival gain super-enhancers at genes for oncogenic miRNAs. Investigation in Aim 2, Regulation of transcription and RNA processing by super-enhancers and gel-sol phase transition biology, may enable identification of new cancer therapy targets. The third aim is a collaboration with Jackie Lees' lab analyzing the role of inhibition of the arginine dimethylase PRMT5. Lees' lab has found that glioblastoma cells vary dramatically in sensitivity to a small-molecule inhibitor of PRMT5. Establishing the bases of this differential sensitivity could allow identification of patients who would therapeutically benefit from PRMT5 inhibition. PRMT5 dimethylates residues in Sm proteins that comprise the snRNPs critical for spliceosome function. Analysis of RNA-seq data following inhibition of PRMT5 revealed a dramatic increase in ?detained? introns for a subset of genes and a decrease in the corresponding mRNAs. This will be investigated in Aim 3, Tumor sensitivity to PRMT5 inhibition through splicing regulation and snRNP.

The main goal of this RAISE Award project is to discover fundamental aspects of gene regulation in mammals. Many diseased cellular states (including cancer and autoimmunity) are associated with aberrant regulation of transcription of genes regulated by super-enhancers (SEs), large clusters of enhancers that regulate the transcription of genes important for cell type specific processes in both healthy and diseased (e.g., cancer) states.. Therefore, the proposed fundamental studies are of significance to the design of therapies for diseases that have a large toll on human health. The immediate potential impact of the studies concerns inhibitors of SEs that are currently being tested in clinical trials to treat cancer and other diseases. This effort also has an important training component. Undergraduate research is an integral part of labs at MIT. Formal mechanisms such as the MIT Undergraduate Opportunities Program (UROP), the MIT Summer Research Program and Amgen Scholars Program for underrepresented minorities, will be used to recruit undergraduates to the PI's laboratories. One PI will also participate in ACCESS, a weekend at MIT for underrepresented minority students designed to make them aware of opportunities for graduate study. The impact of this research on science and technology will be disseminated to the broader scientific community through the production of a video learning module targeted at both the broad community of citizens and specifically the K-12 educational audience. MIT has been at the epicenter of research at the convergence of the physical, life, and engineering sciences. The PIs will teach courses wherein this interdisciplinary work will be highlighted.

Super-enhancers are occupied by an unusually high density of interacting molecules, and are able to drive higher levels of transcription than typical enhancers. Several lines of evidence suggest that they form via cooperative processes, and SEs are far more vulnerable than typical enhancers to perturbation of components that are commonly associated with most enhancers. Recently, the PIs proposed that a phase separated multi-molecular assembly regulates the formation and function of SEs (Cell, 2017). They suggested that some puzzles associated with SE function are consistent with such a model. These results provide just a starting point to explore the role of phase separation in gene control in mammals. By bringing together sophisticated theoretical studies (rooted in statistical physics) and biological experiments, the PIs now aim to study their novel proposal with the goal of developing a conceptual framework for understanding gene regulation in mammals, and why SEs evolved to regulate key genes. The mechanistic insights thus gleaned will also apply to diverse processes in eukaryotic cells that are mediated by phase separated membraneless organelles. By bringing together approaches rooted in physics and biology, the PIs aim to address the following significant questions: 1] What are the fundamental physical and biological principles that determine how SEs form and function to regulate gene transcription in mammals? 2] Why have genes with the most prominent roles in cell identity evolved to be regulated by SEs? 3] Why do cancer cells have SE-regulated oncogenes, and why are these so vulnerable to drugs that inhibit certain transcriptional regulators? In order to take steps toward answering these questions, the PIs propose to study two major topics: 1] Understanding the nature of the phase transition and its implications for gene regulation. 2] Understanding how super-enhancers nucleate and form.

This RAISE project is being jointly funded by the Physics of Living Systems program in the Division of Physics in the Mathematical and Physical Sciences Directorate, by the Cellular Cluster in the Division of Molecular and Cellular Biosciences in the Biosciences Directorate, and by the Office of Integrative Activities.

Project Summary Ovarian carcinoma is the fifth deadliest cancer among women in the United States. In spite of advances in surgical resection and platinum-taxane combination therapy over the past several decades, cure rates remain relatively low (~30%) and a majority of women diagnosed with advanced ovarian cancer will die with drug-resistant disease within 5 years. The long-term goal of this project, ?RNA-Binding Proteins as Molecular Integrators that Control the Response of HGSOC to Anti-Cancer Therapies?, is to identify specific RNA-binding proteins that, together with their upstream protein kinase regulators, control the resistance and sensitivity of high-grade serous ovarian cancers to these clinically used first line anti-cancer therapies. The project involves: (1) a detailed computational analysis that queries pre-existing publically available RNA expression data using RNA-BP recognition motifs to identify specific RNA binding proteins whose mRNA targets are up- or down- regulated in ovarian cancer patients with chemo-sensitive versus chemo-resistant tumors; and (2) an independent CRISPR-interference and CRISPR-activation genome-wide screen for RNA-BPs whose manipulation alters the resistance and sensitivity of ovarian cancer cells to platinum and taxane agents in vitro, and in vivo using cell line xenografts and human PDX ovarian cancer mouse models. The RNA-BPs identify by these two complimentary approaches, together with a collection of RNA-BPs that we have already identified in previous experiments and preliminary data, are then directly validated for their effects on drug resistance in these model systems, and the identity of their bound RNAs and their effects on gene expression determined using CLIP technologies and RNA-Seq. In selected cases the importance of specific phosphorylation sites on RNA-BP function is examined to further elucidate the molecular basis for anti-cancer drug resistance through pathway-specific regulation of RNA-BP action. The project builds on a broad foundation of expertise and related work from all of the co-Investigators laboratories. Expected outcomes from the studies include the identification of specific RNA-BPs and upstream regulatory kinase pathways whose targeting can prevent or reverse the resistance of ovarian cancers to current clinically used front-lime therapeutics; the elucidation of new molecular circuits that control gene expression in cancers after chemotherapy treatment; and the creation of a suite of web-based tools available to the entire scientific community that can be used to query any set of differentially expressed genes for RNA-BP-based regulation, particularly in a form that is optimized for analysis of new and existing cancer patient RNA expression datasets.