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DEPARTMENT OF HEALTH & H'U51AN SERVICES Public Health Service July 25, 1989 National Institutes of Health Bethesda, Maryland 20892 Building : Room : 1ST (301) 496- 101 5216 FAX: (301) 402-0078 Dr. Harmon McAllister The Council for Tobacco Research-USA, Inc. 900 Third Avenue New York, NY 10022 Dear Dr. McAllister. 2 6 1989, 11] This letter serves to submit a brief outline of the study for which support is sought. I plan to return to Israel in January 1990, after completing a 3 1/2 year training period at the National Institutes of Health. I will head my own laboratory at the Department of Immunology, Lautenberg Center, Jerusalem Medical School. The success of my scientific studies depends much on your financial support and I hope that you will consider my application. If more information is needed or if I can be of assistance in any way, please contact me at (301) 496-5216. Thank you very much for your consideration, I look forward to hearing from you. Sincerely, Miehal Baniyash, Ph.D. Visiting Associate Cell Biology and Metabolism Branch National Institute of Child Health and Human Development IVlB/kmp Enclosure 40000298

REGULATORY MECHANISMS CONTROLLING THE EXPRESSION OF THE T CELL RECEPTOR ZETA CHAIN GENE - SIGNIFICANCE IN T CELL DEVELOP1WENT AND IMMUNODEFICIENCY BACKGROUND AND SIGNIFICANCE T lymphocytes are one of the major cell populations involved in the immune response against infections and malignancies. Impaired T cell function might result in the failure of the immune system to eliminate certain abnormal cell types such as viral infected or tumor cells, thus leading to the development of immunodeficiency, malignancy and other immune disorders. The T cell antigen receptor (TCR) is a multisubunit cell membrane receptor, that serves both antigen recognition and signal transduction functions which are significant in the immune response mediated by T ceils (1-3). Two types of components make up the TCR: 1) elonotypie chains (Tia and # or Ti -t and t) provide the recognition function and antigen/MHC specificity of the receptor; and 2) non-polymorphie chains, "t, 6, ~, collectively termed CD3, f and ,~ chains most likely determine the signaling capacity of the receptor. The functional receptor is defined structurally by non-covalent assembly of a clonotypie heterodimer with a set of CD3 chains, ¢-~ homodimer and/or f--~ heterodimer (4-6). Abnormal expression of TCR chains on the cell surface will lead to an impaired T cell function and immune response abnormalities. The TCR ~ chain is the most recently described subunit and its role in the receptor complex has begun to be illuminated. Structural and functional analyses of mature and variant T cells suggest that the zeta chain plays several unique roles including: 1) in some T cells, the ¢ chain is synthesized in limiting amounts and is required for TCR surface expressions (4-7), 2) the f chain is necessary for normal TCR function, as demonstrated by studies with f deficient T cell hybridomas and with cells from immunodeficiant patients (7,8), 3) f-~" homodimer and heterodlmer function to couple the antigen activated TCR to intracellular signalling pathways (9-12), 4) preliminary results suggest that ¢-¢ and ~-~ of the TCR might be involved in negative selection which is taking place in the thymus during T cell development. Furthermore, when we have recently cloned the f eDNA 03,14) and gene (15) we observed that the ¢ chain has a sequence and predicted structure, different from the CD3 components which are structurally high homologous to each other (3). In addition, we have shown that the f gene is localized to mouse chromosome 1 while the CD3 genes are co-localized to mouse chromosome 9 (3,15). These unique proportions of the TCR zeta chain form the basis for this proposal. The long term goals of the proposal are to eharaeterlze: a) the regulatory mechanisms involved in controlling f gene expression in normal mature T cells and during T cell development;, b) the mechanism which turns zeta expressing T cells into zeta deficient cells; e) possible common regulatory factors that correlate the coordinate expression of the different TCR genes. Studies of regulatory elements and factors involved in the TCR gene expression were initiated only recently (3,16,17) as the cloned genes became available. These studies will offer a significant contribution to the understanding of control mechanisms involved in the expression of such a multisubunit complex and particularly to this central receptor of the immune response. Abnormalities in such regulatory mechanisms might play a role in a variety of immune disorders. Performing such studies will be critical for the potential development of future therapeutic tools. STATUS OF PROJECT As I have mentioned above, we have biochemieally and functionally characterized the ¢ chain. In addition, we have cloned the i" eDNA and gene. The ~" gene is characterized and mapped in detail. Recently, we performed some preliminary experiments dealing with the issue of the ¢ gene regulation. These experiments showed the following results: 40000297

A) The f gone contains a strong promoter which does not control the expression of a reporter gone in a tissue specific manner. We do not know yet what is the mechanism and which are the components responsible for the tissue specific expression of the f gone. B) The f gone from mature T cells which express the f chain show a different methylation pattern when compared with the gone isolated from mature T cells which are f deficient. The role of DNA methylation in f gone expression, the exact mapping of the modified sites, and the understanding of how T cells become f deficient, has to be further studied. C) There are indications that the f protein levels vary during thymic development. The regulatory mechanism (transcriptional or post-transcriptional) controlling this expression has to be further analyzed. ~ All the reagents that are required for developing the proposed project are available to us: the ¢ gene and eDNA, eDNAs of the different TCR chains, specific antibodies directed against the different TCR subunits, well characterized T cells such as cells which express ~ chain or are ~" deficient, T cell lines which represent different stages of maturation, different kinds of vectors, etc. This powerful system will enable us to develop and make a progress of the proposed study. SPECIFIC AIMS AND BRIEF METHODOLOGY A. Defining the Type of Mechanism(s) Involved in Regulation of the f Gene Expression Two regulatory mechanisms could control T cell specific ~ gene expression: 1) transcriptional 2) post-transcriptional. The latte.r could operate on three levels:, a)ineorreet RNA splicing in non T cells, b). impaired transport of RNA from the nueleuse into the cytoplasm, e) instability of cytoplasmic RNA in non T cells, which is the most common mechanism responsible for altered levels of RNA. This possibility will be analyzed by performing nuclear run-off experiments using nuclei from T and non-T cells. If a post- transcriptional regulation is involved, detailed studies will be performed to understand this mechanism and to identify the components involved. Preliminary results indicate, that the regulatory mechanism controlling ~ gene expression is most likely to be transcriptional. B. Mapping of Cis-Aeting Elements Regulating the f Gene Expression 1. Candidate sequences will be analy~.ed for their transcriptional function by ligating them to a reporter gene such as: CAT and/or Lueifurase. Such constructs will be transfeeted into mouse mature T and non-T cells and will be compared with control constructs that will contain reporter gene controlled by a known promoter (SV40 or RS¥). By using deletion analysis and ligation of sequences in different orientations, several regions could be identified: promoter, enhancer, silencer, activator, etc. 2. Modification of specific sites in the ¢ gene such as methylation or demethylation will be analyzed by using methylation sensitive and insensitive enzymes to cleave total genomie DNA. If differences are found between T and non T cells,than a careful mapping of the modified sites will be performed. Such DNA modifications might play a role in transcriptional regulation of ¢ gene expression. Assuming eis-aeting elements that regulate £ gene expression will be identified, than the next step will be taken. C. Analysis of St~ecific Trans-Acting Factors..!nteraetlng with f Gene Cis-Aeting Elements Nuclear factors binding to f gene regulatory elements in mature T cells will be analyzed by using 1) foot printing and 2) gel shift assays. Nuclear extracts form non-T cells will be used as control. In conjunction with site directed mutagenesis and competition assays using smaller DNA regions and synthetic oligonueleotides, we will be able to 40000298

determine the specificity of any foot print or gel shift and the exact nueleotides required for binding. Long term goal is to utilize DNA affinity chromatography to isolate and characterize this specific trans-aetive factor(s). This factor(s) will be cloned by screening expression library using the known binding sequences as a probe. High levels of the factor protein will be obtained by using the Baeullo virus system. This will enable us to generate antibodies that will be useful in future studies related to T cell development. D. Studie~of Regulatory Mechanisms Involved in ¢ Deficiency We have analyzed ¢ deficient hybridoma T ceils that lack t mRNA and consequently do not express ¢ chain protein.- Recently, it was published that T cells from some immunodeficient patients lack f chain protein. In both eases, there are low levels of receptors and the T cells are not stimulated by-antigen. Postotranserlptional or transcriptional regulatory mechanisms could be involved is ~ deficiency. Post- transcriptional regulation such as RNA instability, which is a common mechanism for altered levels of RNA, will bo analyzed by using run-off experiments. Rate of RNA synthesis in nuclei from f + T cells will be compared with nuclei from f - T cells, B cells and fibroblasts. If rate and amount of RNA synthesized are the same in f+ and f- cells, than the regulation might be related to RNA instability and this will be further studied. If the rate and amount of RNA synthesis is different in ~+ vs. f- cells, than it is most likely that regulation for f deficiency is transcriptional and the following experiments will be performed: 1. The identified f gone regulatory elements associated with a reporter gone (paragraph B) will be transfeeted into ~- T cells, f+ T cells, B cells and fibroblasts. Together with deletion analysis we could identify the specific elements which are not functional in f- T cells. This might be a result of: a) gone modification; b) appearance of a represser; or e) loss of activator which in all cases, specific gone expression will be abolished. Other regions in the gone might also be involved in this negative regulation. 2. Methylation pattern will be analyzed in DNA from ~+ and ¢- T cells. Preliminary analysis showed that there are differences in the methylation pattern between f gone isolated from f+ T cells vs the gone isolated from f- T cells. The significance of this results in ~" gone transcription is yet unknown. 3. Gel shift analysis will be performed to define the appearance of represser or disappearance of activator in ¢- vs. f+ T cell. In each of these eases different pattern will be observed on the gel shift assay. 4. Long-term experiments will be aimed at isolating and characterizing the specific regulatory factors and to convert f negative cells to f positive cells. If the culprit is a represser DNA binding protein, then the f gone, from which the target sequences have been excised, will be stably transfected into f deficient cells. If the cause is a deficiency of an activator, its gone would be ,cloned and transfeeted into f deficient cells. E. f Gone Expression.During T Cell DeveloPment The coordinate expression of the CD3-~, -t, • genes at the mRNA and protein levels is one of the earliest events in maturation of T cells within the thymus, and proceeds expression of the TCR ~-# or TCI~ "t-t chains (18,19). Thus, immature thymoeytes progress from Ti-CD3+ (intracytoplasmic) to Ti+CD3+ (intracytoplasmie and cell surface) (20). A significant point to be addressed in this study is the characterization of the f chain gone expression during T cell development which has not yet been described. 1. Expression of f protein and f mRNA will be analyzed in fetal thymuses (13-20 days) and will be compared with new born and adult thymuses. Protein will be analyzed by Western blot using anti-zeta antibodies and RNA levels will be analyzed by Northern blot and dot blots, using f-eDNA as a probe. T cell lines which represent different maturation 40000299

stages will be used too. In order to define more efficiently the cellular distribution o£ the f protein and mRNA, in-situ staining with antibodies and in-situ hybridizations with f eDNA will be performed. Antibodies against the different TCR-subunits and eDNA probes will be used for colocalization studies that will follow the expression of the different TCR chains during development. 2. Changes in DNA modification during different stages of T cell development (methylation and demethylation) will be analyzed as described in paragraph B.2. The identification of regulatory elements in the ¢ gene that undergo methylation or demethylation, will be useful for analyzing these regions in T cells isolated from embroyes' thymuses. We will use specific oligonucleotides to amplify a specific modified f gene region by PCR reaction. 3. Changes in specific trans-acting factors will be followed by foot prints and gel shifts. Nuclear extracts isolated from thymocytes of different developmental stages will be analyzed for factors that bind f gene specific regulatory elements. Such studies will define if the appearance or loss of specific trans-acting factor~ during the different developmental stages are the cause for changes in f gene expression. These will advance the understanding of the interplay between different factors and specific elements and their role in T cell development. 4. The ontogenic control mechanism off gene expression (down regulation early in development) will be compared with the mechanism involved in down regulation of f expression in mature ¢ deficient T cells. F. .Comparison Between Regulatory Elements 8nd Factors Involved in the Expression of the CD3 and the ¢ Genes The CD3 chains are expressed early in development on the cell surface. We know from our studies that TCR surface expression requires the ~ chain. Thus, it is most likely that all TCR chains are expressed simultaneously. Our interest is to compare between regulatory components involved in CD3 gene expression with those involved in ¢ gene expression and to understand how different genes are corrdinately expressed to give rise to a functional multisubunit on the T cell surface. Several regulatory elements were already identified as functional in the CD3 gene expression (3). Identification of elements and factors involved in the f gene expression will lead to the following studies: 1) sequence comparison between the different cis-acting elements of the f and CD3 genes; 2) potential CD3 elements will be used as competitors for binding of trans-acting factors to the f specific cis-acting element in gel shift and footprinting assays complete or partial competition might indicate that some regulatory gene regions and trans-acting factors are shared between the CD3 genes and the f gene. Such proteins might be involved in regulating the coordinate temporal expression of the TCR chains; 3) potential candidates for such proteins will be isolated, characterized and cloned. Antibodies will be generated and together with the eDNA probes they will be useful tools for studies analyzing the CD3 and f expression during T cell development. G. Isolation of Rgg¢l~tory Elgm~nts.from the Human ¢ Gene Following the identification of regulatory elements and factors involved in the regulation of mouse f gene expression, a long term goal will be to identify such components in the human f gene. This will be significant in studies analyzing T cells from immunodeficient patients and the= identification of components involved in human f deficiency. The aim of these studies is to revert f- cells to f+ cells and as such to correct the immunodefieiency caused by lacking f expression. This might contribute to the development of therapeutic regimen for ¢ deficiency caused in human. 4 40000300

These investigations will contribute to the understanding of genetic events that commit precursor cells to the T cell lineage and to the specific characterization of ¢ gone regulation and its possible association with immunodeficiency. An in-depth understanding of TCR gone regulation and the definition of factors involved in this process will give insights into important issues including: 1) the relation between immunodeficiency and malignancy; 2) defects in TCR gone regulation and the appearance of T cell malignancies; and 3) potential therapeutic regimens to treat immunodeficiency caused by impaired TCR gone regulation. 40000301

REFERENCES I. Marrack P & Kappler J (1986) Adv. Immunol. 38, 1-24. 2. Weiss A, Imboden J, Hardy K, Manger B, Terhorst C & Stobo J (1986) Ann. Rev. Immunol. 4, 593. 3. Clevers, Hans, Alarcron B, Wileman T & Terhorst C (1988) Ann. Rev. Immunol. 6, 629. 4. Minami Y, Weissman AM, Samelson LE & Klausner RD (1987) Prec. NatL Acad. Sci. USA 84, 2688-2692. 5. Baniyash M, Garcia-Morales P, Bonifacino JS, Samelson LE & Klausner RD (1988) J. Biol. Chem. 263, 9874-9878. 6. Bonifacino JS, Chen C, Lippincott-Schwartz J, Ashweil JD & Klausner RD (1988) Proc. Natl. Acad. Sci. USA 85, 6929-6933. 7. Sussman J J, Bonifacino JS, Lippincott-Schwartz J, Weissman AM, Salto T, Klausner RD & Ashwell JD (1988) Cell 52, 85-95. 8. Alacron B, Regueiro JR, Arnaiz-Villena A & Terhorst C (1988) The New Engl. J. Med. 319, 1203-1208. 9. Samelson LE, Patei MD, Weissman AM, Harford JB & Klausner RD (1986) Cell 46, 1083- 1090. 10. Patel MD, Samelson LE & Klausner RD (1987) J. Biol. Chem. 262, 5831-5837. 11. Baniyash M, Garcia-Morales P, Luong E, Samelson LE & Klausner RD (1988) L Biol. Chem. 263, 18225-18230. 12. Mercep M, Bonifacino JS, Garcia-Morales P, Samelson LE, Klausner RD & Ashwell JD (1988) Science 242, 571-569. 13. Weissman AM, Baniyash M, Hou D, Samelson LE, Burgess WH & Klausner RD 0988) Science 239, 1018-1021. 14. Weissman AM, Hou D, Orloff DG, Modi WS, Senanez H, O'Brien SY & Klausner RD (1988) Proc. Natl. Acad. Sci. USA, in press. 15. Baniyash M, Hsu VW, Seldin MF & Klausner RD (1989) J. Biol. Chem., in press. 16. Luria S, Gross G, Horowitz H & Givol D (1987) EMBO J. 6, 3307-3312. 17. McDougal S, Peterson CL, Calame K (1988) Science 241,205-208. 18. Van Dongen JJM, Quertermous T, Bartram CR, Gold DP, Wolvers-Tetters ILM, Marieke Comans-Bitter W, Hooijkaas H, Adriaansen HJ, Deklin A, Raghavachar A, Ganser A, Duby AD, Seidman JG, Van den Elsen P & Terhorst C (1987) J. Immunoi. 138, 1260- 1269. 19. Davis M & Bjorkman P (1988) Natur~ 334, 395-400. 20. Alacron B, Berkhout B, Breitmeyer J & Terhorst C (1988) J. Biol. Chem. 263, 2953-2961. 6 40000302

ANNEAL DIRECT COSTS OF THE STUDY AS PROPOSED AND ANTICIPIATED DURATION I am starting to conduct my own laboratory on January 1, 1990 at the Lawtenberg Center, Jerusalem University Medical School, Israel. The space which will be available for me to carry out the proposed project is a laboratory of about 60 meters square. It has standard equipment for work with biological materials as laminar flow hood, refrigerator,- 20°C freezer, -70°C freezer, liquid nitrogen, table top centrifuge, etc. In addition, there are institutional and departmental facilities which are available for this project; tissue culture incubators, cold rooms, ultracentrifuges, radioactive counters, PCR-machine, fluorocytometer, computer service, etc. Office with typing facilities is available. Permanent equipment which will be needed includes: UV table and lamp, speed vacuum centrifuge, camera, horizontal and vertical electrophoresis apparatus for DNA and protein electrophoresis and gel dryer. Annual Direct Costs of the Study: A. Principal Investigator. "/0% Technician: 100% $0 per year $20,000 per year Be Consumables; disposable tissue culture flasks, test tubes, filters, plates. For bacterial work; petri dishes, centrifuge tubes, flasks. Nitrocellulose and nytran filters, DEAE paper, elutips. Kits such as: mutagenesis kit, sequencing kit. Radioisotopes Films Oligonucleotides and Peptides Restriction enzymes and other enzymes Commercial Antibodies Various chemicals such as: CsCI, GITC, Agarose, Phenol, Chloroform, Dextran-Sulfate, etc. Media (fetal calf serum + antibiotics) for tissue culture work, and media for growing bacteria TOTAL = $25,000 per year C. Mice and Rabbits: $2,500 per year Permanent equipment; camera, UV table, horizontal and vertical gel electrophoresis apparatus, sequencing gel apparatus, ultracentrifuge rotor (Beckman) TOTAL = $I0,000 per year E. Travel $2,500 per year TOTAL AMOUNT REQUESTED: $60,000 per year The anticipated duration of this study is three years. 40000303