Free Complaint - District Court of Delaware - Delaware

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EXHIBIT A

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United States Patent
Baltimore et al.
NUCLEAR FACTORS ASSOCIATED WITH TRANSCRIPTIONAL REGULATION
Inventors: David Baltimore, New York, IVY (US); Ranjan Sen, Cambridge; Phillip A. Sharp, Newton, both of MA (US); Harinder Singh, Chicago, IL (US); Louis Staudt, Silver Springs, MD (US); Jonathan H. Lebowitz, Zionsville, IN (US); Albert S. Baldwin, Jr., Chapel Hill, NC (US); Roger G. Clerc, Binningen (CH); Lynn M. Corcoran, Port Melbourne (AU); Patrick A. Baeuerle, Eichenau (DE); Michael J. Lenardo, Potomac, MD (US); Chen-Ming Fan, San Francisco; Thomas P. Maniatis, Belmont, both of MA (US) Assignees: President & Fellows of Harvard College; Massachusetts Institute of Technology; Whitehead Instittue for Biomedical Research, all of Cambridge, MA (US) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days.

(10) (45)

Patent NO.: US 6,410,516 ~1 Date of Patent: Jun. 25,2002

OTHER PUBLICATIONS Gosh, S. and Baltimore, D., "Activation in vitro of NF-kB by phosphorylation of its inhibitor IkB," Nature, 344(6267): 678-682 (1990). Zabel, U. and Baeurle, P., "Purified Human IkB Can Rapidly Dissociate the Complex of the NF kB Transcription Factor with it Cognate DNA," Cell, 61:255-265 (1990). Haskill, S., et al., "Characterization of an Immediate-Early Gene Induced in Adherent Monocytes That Encodes IkB-like Activity," Cell, 65:1281-1289 (1991). Baldwin, Jr., A.S., and Sharp, P.A., "Two transcription factors, NF-kB and H2TF1, interact with a single regulatory sequence in the class I major histocompatability complex promotor," Proc. Natl. Acad. Sci, USA, 85:723-727 (1988). Bohnlein, E.,, et al., "The Same Inducible Nuclear Proteins Regulates Mitogen Activation of Both the Interleukin-2 ~ e c e ~ t o r - N p h a - ~ e n e Type 1 HIV," Cell, 53327-836 and (1988). Leung, K. and Nable, G.J., "HTLV-1 transactivator induces interleukin-2 receptor expression through an NF-kB-like factor," Nature, 333:776-778 (1988). Ruben, S., et al., "Cellular Transcription Factors and Regulation of IL-2 Receptor Gene Expression by HTLV-1 tax Gene Product," Science, 241:89-92 (1988). Lenardo, J.J., et al., "NF-kB protein purification from bovine spleen: Nucleotide stimulation and binding site specificity," Prod. Natl. Acad. Sci. USA, 85:8825-8829 (1988). Wirth, T. and Baltimore, D., "Nuclear factor NF-kB can interact functionally with its cognate binding site to provide lymphoid-specific promotor function," The EMBO Journal, 7 (10):3109-3113 (1988). Nelsen, B., et al., "The NF-kB-Binding Site Mediates Phorbol Ester-Inducible Transcription in Nonlymphoid Cells," Mol. & Cell Biol., 8:3526-3531 (1988). Ballard, D.W., et al., "HTLV-I Tax Induces Cellular Proteins That Activate the kB Element in the IL-2 Receptor a Gene," Science, 241:1652-1657 (1988). Blanar, M.A., et al., "Nf-kB Binds within a Region Required for B-CellSpecific Expression of the Major Histocompatibility Complex Class I1 Gene Ead," Mol. & Cell. Biol., 9 (2):844-846. Karin, M., et al., "Activation of a Heterologous Promoter in Response to Dexamethasone and Cadmium by Metallothionein Gene 5'Flanking DNA," Cell, 36:371-379 (1984).
\ ,

Appl. No.: 081464,364 Filed:

Jun. 5, 1995 Related U.S. Application Data

Division of application No. 081418,266, filed on Apr. 6, 1995, now Pat. No. 5,804,374, which is a continuation of application No. 071791,898, filed on Nov. 13, 1991, now abandoned, which is a continuation-in-part of application No. 061946,365, filed on Dec. 24, 1986, now abandoned, application No. 081418,266, which is a continuation-in-part of application No. 071341,436, filed on Apr. 21, 1989, now abandoned, and a continuation-in-part of application No. 071318,901, filed on Mar. 3, 1989, now abandoned, and a continuation-in-part of application No. 071280,173, filed on Dec. 5, 1988, now abandoned, and a continuation-in-part of application No. 071162,680, filed on Mar. 1, 1988, now abandoned, and a continuation-in-part of application No. 071155,207, filed on Feb. 12, 1988, now abandoned, and a continuation-in-part of application No. 061817,441, filed on Jan. 9, 1986, now abandoned.

(List continued on next page.) Primary Examiner4avid Guzo (74) Attorney, Agen.nt,or F i r m a a v i d L. Berstein; Matthew P. Vincent; Ropes & Gray (57)

Int. CL7 ........................ A61K 311711; C12N 5110; C12N 15110 U.S. C1. ............................. 514144; 43516; 4351455; 4351325; 4351366; 4351370; 4351372; 4351372.2; 4351372.3 Field of Search ......................... 43516, 172.3, 455, 4351325, 366, 370, 372, 372.2, 372.3; 51412, 44; 935134, 36 References Cited
FOREIGN PATENT DOCUMENTS

ABSTRACT

Constitutive and tissue-specific protein factors which bind to transcriptional regulatory elements of Ig genes (promoter and enhancer) are described. The factors were identified and isolated by an improved assay for protein-DNA binding. Genes encoding factors which positively regulate transcription can be isolated and employed to enhance transription of Ig genes. In particular, NF-kB, the gene encoding NF-kB, IkB and the gene encoding IkB and uses therefor. 203 Claims, 58 Drawing Sheets

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OTHER PUBLICATIONS Davis, N., et al., "Rel-Associated pp40: An Inhibitor of the Re1 Family of Transcription Factors," Science 253: 1268-1271 (1991). Treisman, R., "Transient Accumulation of c-fos RNA Following Serum Stimulation Requires a Conserved 5'Element and c-fos 3' Sequences," Cell, 42:889-902 (1985). Queen, C. and Stafford, J., "Fine Mapping of an Immunoglobulin Gene Activator," Mol. Cel. Biol., 4(6):1042-1049 (1984). Nelson, K. J., et al., "Inducible transcription of the unrearranged K constant region locus is a common feature of pre-B cells and does not require DNA or protein synthesis," Proc. Natl. Acad. Sci. USA, 82:5305-5309 (1985). Foster, J., et al., "An immunoglobulin promoter displays cell-type specificity independently of the enhancer," Nature, 315:423-425 (1985). KO, H.-S., et al., "A Human Protein Specific for the Immunoglobulin Octamer DNA Motif Contains a Functional Homeobox Domain," Cell, 55:135-144 (1988). Sen, R. and Baltimore, D., "Multiple Nuclear Factors Interact with the Immunoglobulin Enhancer Sequences," Cell, 46:705-716 (1986). Nabel, G. and Baltimore, D., An inducible transcription factor activates expression of human immunodeficiency virus in T cells, Nature, 326:711-713 (1987). Baeuerle, P.A and Baltimore, D., "IKB: A Specific Inhibitor of the NF-KB Transcription Factor," Science, 242:54&546 (1988). Baeuerle, P.A. and Baltimore, D., "Activation of DNA-Binding Activity in an Apparently Cytoplasmic Precursor of the NF-KB Transcription Factor," Cell, 53:211-217 (1988). Baeurle, P.A. and Baltimore, D., "Activation of NF-KB: A Transcription Factor Controlling Expression of the Immunoglobuli K Lightshain Gene and of HIV," The Control of Human Retrovirus Gene Expression, Banbury Conference, Cold Spring Harbor, IVY, pp.: 217-226 (1988). Sen, R. and Baltimore, D., Inducibility of K Immunoglobulin Enhancer-Binding Protein NF-KB by a Posttranslational Mechanism, Cell, 47:921-928 (1986). Wall, R., et al., "A labile inhibitor blocks immunoglobulin K-light-chain-gene transcription in a pre-B leukemic cell line," Proc. Natl. Acad. Sci. USA, 83:295-298 (1986). Lenardo, M., et al., "Protein-Binding Sites in Ig Gene Enhancers Determine Transcriptional Activity and Inducibility," Science, 236:1573-1577 (1987). Cross, S. L., et al., "Functionally Distinct NF-KB Binding Sites in the Immunoglobulin K and IL-2 Receptor a Chain Genes," Science, 244:466-469 (1989). Kawakami, K., et al., "Identification and purification of a human immunoglobulin-enhancer-binding protein (NF-KB) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro," Proc. Natl. Acad. Sci. USA, 85:470&4704 (1988). Goodbourn, S., et al., "Human 0-Interferon Gene Expression Is Regulated by an Inducible Enhancer Element," Cell, 41:5O9-52O (1985). Bergman, Y., et al., "Two regulatory elements for immunoglobulin K light chain gene expression," Proc. Natl. Acad. Sci. USA, 81:7041-7045 (1984). Mason, J. O., et al., "Transcription Cell Type Specificity Is Conferred by an Immunoglobulin VH Gene Promoter That Includes a Functional Consensus Sequence," Cell, 41:479-487 (1985).

Fried, M. and Crothers, D. M., "Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis," Nucleic Acids Research, 9(23):6505-6524 (1981). Garner, M. M. and Revzin, A,, "A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system," Nucleic Acids Research, 9(13):3047-3060 (1981). Strauss, F. and Varshavsky, A,, "A Protein Binds to a Satellite DNA Repeat at Three Specific Sites That Would be Brought into Mutualy Proximity by DNA Folding in the Nucleosome," Cell, 37:889-901 (1984). Grosschedi, R. and Baltimore, D., "Cell-Type Specificity of Immunoglobulin Gene Expression is Regulated by at Least Three DNA Sequence Elements," Cell, 41:885-897 (1985). Banerji, J., et al., "A LymphocyteSpecific Cellular Enhancer Is Located Downstream of the Joining Region in Immunoglobulin Heavy Chain Genes," Cell, 33:729-740 (1983). Queen, C. and Baltimore, D., "Immunoglobulin Gene Transcription Is Activated by Downstream Sequence Elements," Cell, 33:741-748 (1983). Church, G. M., et al., "Cell-type-specific contacts to immunoglobulin enhancers in nuclei," Nature, 313:798-801 (1985). Gerster, T., et al., "Cell type-specificity elements of the immunoglobulin heavy chain gene enhancer," EMBO Journal, 6(5):1323-1330 (1987). Landolfi, N. F., et al., "Interaction of cell-type-specific nuclear proteins with immunoglobulin VH promoter region sequences," Nature, 323:548-551 (1986). Staudt, L. M., et al., "A Lymphoid-specific protein binding to the octamer motif of immunoglobulin genes," Nature, 323:640-643 (1986). Fletcher, C., et al., "Purification and Characterizaiton of OTF-1, a Transcription Factor Regulating Cell Cycle Expression of a Human Histone H2b Gene," Cell, 51:773-781 (1987). Scheidereit, C., et al., "Identification and Purification of a Human LymphoidSpecific Octamer-Binding Protein (OTF-2) That Activates Transcription of an Immunoglobulin Promoter In Vitro," Cell, 51:783-793 (1987). Sassone-Corsi, P., et al., "A trans-acting factor is responsible for the simian virus 40 enhancer activity in vitro," Nature, 313:458-463 (1985). Singh, H., et al., "Anuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes," Nature, 319:154-158 (1986). Baldwin, A. and Sharp, P., et al., "Binding of a Nuclear Factor to a Regulatory Sequence in the Promoter of the ~ Mouse H - ~ K Class I Major Histocompatibility Gene," Mol. & Cell. Biol., 7(1):305-313 (1987). Mercola, M., et al., "Transcriptional Enhancer Elements in the Mouse Immunoglobulin Heavy Chain Locus," Science, 221:663-665 (1983). Picard, D. and Schaffner, W., "A lymphocyte-specific enhancer in the mouse immunoglobulin K gene," Nature, 307:80-82 (1984). Mercola, M., et al., "Immunoglobulin Heavy-Chain Enhancer Requires One or More TissueSpecific Factors," Science, 227:266-270 (1985).

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Staudt, L., et al., "Cloning of a Lymphoid-Specific cDNA Encoding a Protein Binding the Regulatory Octamer DNA Motif," Science, 241:577-580 (1988). wu et al, (1988) hrification of the human immunodeficiency virus type 1 enhancer and TAR binding protiens EBP-1 and UBP-1, EMBO J. 7:2117-2129.*

Leonard et al. (1985) Interleukin 2 receptor gene expression in normal human T lymphocytes. Proc. Natl. Acad. Sci. USA 82:6281-6285.* Johnston et al. (1993) Present Status and future prospects for therapies. Science 260:1286-1293.* * cited by examiner

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TTAATGGGTCCACCACAAAACG

0

Fig. 11C

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Fig. 16

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SVEN HIV

Fig. 17

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CTAGCTGGGGACATACAGCAGCTCCTCCAGCTCCAGCAGCTGGTGCTTGTGCCAGGCCAC

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8 4 1 ---------+---------+---------+---------+---------+---------+

CCCCTCCTGGAGAAGTGGCTCAACGATGCAGAGACTATGTCTGTGGACTCAAGCCTGCCC
P P L P L G E E K V W A L Q N R D C A R E D T Y M V S C V G D L S K S P L A P Q

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Fig. 18A
(CONTINU ED)

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GTTACTACCTTATCCTCAGCTGTGGGGACGCTCCACCCCAGCCGGACAGCTGGAGGGGGT
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TCCCTGCCCCGGAATTC

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TACAAGGGACTTTCCGCTGGGGACTTTCCAGGGA ATGTTCCCTGAAAGGCGACCCCTGAAAGGTCCCT

Fig. 25

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GGGGACTTTCC GGGGATTCCCC

Fig. 39

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Figure 43
AACATTGCAACCTTATAAAAAATTAACTATTAACTATTTCGACMTGCCGCAGMGG~TTCTGTGTTTAGGTGCTGGTGGG AAAACACTATCTCCAGCTTGTAGGTTTGAGCATCACCAGAACCACTTGATGMTCACACACAGAACAAGTAGAGG AGGCAACTGTGAATCGTGGGGCTATAAAGCCATCAAGGGATCTGATGMGAACCCGCGAGACGMCCCCCCCACC CCCCACAACAGGATCGGCACCCCAGAGTTCAACAAGTGGCTGACTTTGTTAAAACACTACGTGGGAACCCATAGTC CCGGATCAGTAGTTGCACAGCCCCCTCCCCGACAGACTACACCGCTGTTTGCTGATCCTTGCCCACCCCATGCTCT CCTCCCAGGCCCCCGTTCTGCTCCTCTGTCCTGCGGCGCTGGATTGMCCGCACACWGTCTGCATCTGGCACGM TTCTCATGGGAGCCACGTCATGAGGTACGTGGTTGCACACCTATCACAAGAAGTCTTGCAGTTCTGACTCTCCTGA GCTCGGTGGGAAAGTCTGGATAGTACCTCCCCTCTCCTGCCACWGCAGCCCTCACATTCACMGTTTCCmG CAGGTCTATTGAGTTTCTCTTCAGAGCGAGCCTTTGTCAACACACCTGGAGGGGGGAGTCTCACCTCTCCCCAGC AACTCAGATCAGTGCCTTATTTTTAATGCTCCGGCCCAATCCTGAGGTGCTGCTGGGTTTGTGGGCTGCGTTTTGT TGAACCTCCCCCCTCCCCTCCCAACGCCCTGGcATTTGcAATT~cTGGGATTcmGGGcc~TTcAAGcccA GAGTGAGCAGTAGGATGTGGAGCTCAAAGCAGAGTTGCACCTGCTGACCCCCAGCCTGMTTTGGTTcAcccAGAG ACTACAAGTCAGAAAGGCATGTTTAGAAAGAGGCATGCTAAGGACTGATGGTGGAACGGCCMTTTGTCCCCACCA GCACAGTGGGGAAGGCTGGACAGAGAAGGAAGAGAAGGATCCATAGAGATGTGAACCAGAATCAGTCGTGTTGAGC TCTGGGTATATCACTACATGTTTAACTCTTGCAAGACCGTTTGCCCAGGGCTTTGGTACCACAGGGTTAGAGTTAC ATTAACCACAACCACCAGAGAGGAACTGAGGTTTATGACCCCCCCCCCCCCMGGTTAGATTTCTGCCGAGTATA M T P P P P K V R F L P S I
AAGGGGGGGGAAGGGGGGGGTCCTTGGTTCATTTCCCTTCACTGTGTGACCGAAGTTTTTGCTTTTTCA

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GCCGCCGCCAGCACCAGCAGCCGCGCCGCGCCGCCCCGCCAGCTCCGCCGCCATGCTCAGCGCCCACCGCCCCGCC P P P A P A A A P R R P A S S A A M L S A H R P A

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Figure 43 (continued)
GAGCCGCCCGCCGTGGAGGGCTGCGAGCCGCCGCGCAAGGAACGGAACGGCGGCGGGCTGCTGCCGCCCGACGACCGCC E P P A V E G C E P P R K E R Q G G L L P P D D R H
ACGACAGCGGGCTGGACTCCATGAAGGAGGAGGAGGAGTACAGGCAGCTGGTGCGGGAGCTGGAGGACATCCGCCTGCA D S G L D S M K E E E Y R Q L V R E L E D I R L Q GCCCCGCGAGCCGCCCGCCCGGCCGCACGCCTGGGCCCAGCAGCTCACCGAGGACGGCGACACTTTTCTCCACTTG P R E P P A R P H A W A Q Q L T E D G D T F L H L GCGATCATTCACGAGGAAAAGGCCCTGAGCCTGGAGGTGATCCGGCAGGCCGCTGGGGACGCCGCCTTCCTGMCT A I I H E E K A L S L E V I R Q A A G D A A F L N F Ank. I TCCAGAACAACCTCAGCCAGACTCCGCTCCACCTGGCGGTGATCACGGACCAGGCCGMTCGCCGAGCACCTGCT Q N N L S Q T P L H L A V I T D Q A E I A E H L L Ank. I 1 GAAGGCTGGCTGCGACCTGGATGTCAGGGACTTCCGTGGGMCACCCCGCTCCACATCGCCTGCCAGCAGGGCTCG K A G C D L D V R D F R G N T P L H I A C Q Q G S Ank.
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ACGGCCATACATGTCTCCATTTGGCATCTATTCAAGGATACCTGGCTGTTGTCGMTACCTGCTGTCCTTAGGAGC G H T C L H L A S I Q G Y L A V V E Y L L S L G A Ank. IV AGATGTAAATGCTCAGGAGCCATGCAATGGGGAGAACAGCACTACACTTGGCCGTAGACCTTCAGAAcTcAGAccTG D - V N A Q E P C N G R T A L H L A V D L Q N S D L Ank. V

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Figure 43 (continued)
GTGTCACTTCTGGTGAAACACGGGCCAGATGTGAACAAAGTGACCTACCAGGGCTACTCCCCATACCAGCTTACAT V S L L V K H G P D V N K V T Y Q G Y S P Y Q L T W
GGGCAGAGACAACGCCAGCATACAGGAGCAGCTGAAGCTGCTGACCACAGCTGACCTGCAGATACTGCCCGMGT A E T T P A Y R S S 354
GAGGATGAGGAGAGCAGTGAATCAGAGCCAGAGTTCACAGAGGATGMCTTATGTATGATGACTGCTGTATTGGAG GAAGACAGCTGACATTTTAAAGCAGAGGTTTCTGTGAGAAGTGACTGTGTACATATGTATAGGAAAAAAAGCCTGA
CTTTCTTCATTTAAAAAGAAAGTCTATACTCGAAGGAGWGTACTGAGATACTACACTGCCCAGCCAGGAGC A C A T C A T G C T A A C A G G T T C C A T G C T C T G A C C T G T A C T T A A G A T C

AGTGAACATGCACACCATCTGATAAAGAGCCACGTTATCTMTTTCTCTGCCACATGAGGATMCGGACTGCACGT CCAATGTGCTGTTGTCAGAAATGCGTTTGCGTTTGAGAGCTGCCTTGTGACACTMGTGCTGTGAGGAGTGCTCATCCCCCT CGGTGGCAAGACAGGCTTGCACAAACGTCCCATCTGCTTGmGACTGTGAGGTTGGCATTAGGTTGAGGCACTGCT GTGCCCTGCTCCCTGACCCTGGCTGCTCAGGGTTGAGGAGTCCGACCATGGGAGAGGTGACCTGGCTGCTGGGAGG AAGGTAGCAATGATGTTAACTGTGGGCATTTGGAAACTGTGTGTTTCACACCATGTGTGTCATTTGCTACACTT TTTAGCAACTGTATAGAATGTAAATACTGTACTGTACATCTTTGTTTATMTTATTTTGGTACCTGTGAGATATGTATTTA TTAAAAAAGGCAGATTTCTGTAAAAAA

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IgNF-A (NF-A1) NUCLEAR FACTORS ASSOCIATED WITH IgNF-A binds to DNA sequences in the upstream regions TRANSCRIPTIONAL REGULATION of both the murine heavy and kappa light chain gene Related Applications promoters and also to the murine heavy chain gene enhancer. , m i s application is a division of application ser,N ~ s The binding is sequence specific and is probably mediated a sequence A7TTGCAT, present 081418,266 filed Apr. 6, 1995, U.S. Pat. No. 5,804,374 in all three transcriptional elements. A factor with binding N , which is a continuation of U,S, ser, ~ 071791,898, filed Nov. 13,1991, abandoned which is a continuation-in-part of specificity similar to IgNF-A is also present in human ~ e ~ indicating that lgNF-A may be tissue U.S. Ser. No. 061946,365, filed Dee. 24, 1986, abandoned and of U.S. Ser. No. 071318,901, filed Mar. 3, 1989, abanThe E factors are expressed in all cell types and bind to doned and of U.S. Ser. No. 071162,680, filed Mar. 1, 1988, the light and heavy chain enhancers. abandoned and of U.S. Ser. No. 071341,436, filed Apr. 21, lgNF-B (NF-A2) 1989, abandoned and of U.S. Ser. No. 061817,441, filed Jan. lgNF-B exhibits the same sequence-s~ecificit~ k N F as 9, 1986, abandoned and of U.S. Ser. No. 071155,207, filed Feb. 12, 1988, abandoned and of U.S. Ser. No. 071280,173, A; it binds upstream regi0ns murine and light chain gene promoters and murine chain gene filed Dee. 5, 1988, abandoned. All of the above applications enhancer. This factor, however, is lymphoid specific; it is are incorporated herein by reference in their entirety. restricted to B and T cells. NF-KB (Previously Kappa-3) GOVERNMENT SUPPORT NF-KB binds e&s%ely 'to the kappa light chain gene The work leading to this invention was supported in part 20 enhancer (the sequence TGGGGATTCCCA). Initial work by a grant from the National Cancer Institute. The Governprovided evidence that NF-kB is specific to B-lymphocytes ment has certain rights in this invention. (B-cells) and also to be B-cell stage specific. NF-kB was originally defected because it stimulates transcription of BACKGROUND OF THE INVENTION 25 genes encoding kappa immunoglobulins in B lymphocytes. Trans-acting factors that mediate B cell specific transcripA, described herein, it has subsequently been shown that of immunoglobulin genes have been postulated transcription factor NF-kB, previously thought to be limited based On an of the expression of in its cellular distribution, is, in fact, present and inducible Ig gene in lymphoid and Ilonin many, if not all, cell types and that it acts as an intracTwo cell-s~ecific, transcri~- 30 ellular messenger capable of playing a broad role in gene lymphoid tional regulatory elements have been identified. One element regulation as a mediator of inducible signal transduction, It is located in the intron between the variable and constant has now been demonstrated that NF-kB has a central role in regions of both and light chain genes and acts regulation of intercellular signals in many cell types, For as a transcriptional enhancer. The second element is found example, NF-kB has not been shown to positively regulate upstream of both chain and light chain gene 35 the human 0-interferon (0-IFN) gene in many, if not all, cell promoters. This element directs lymphoid-specific transcriptypes, A, described below, it is now clear not only that tion even in the presence of viral enhancers. NF-kB is not tissue specific in nature, but also that in the and human light chain Promoters the wide number of types of cells in which it is present, it serves octamer sequence P d T T G C m approximately 70 base pairs the important function of acting as an intracellular transhas upstream from the site of initiation. Heavy chain gene 40 ducer of external influences, N F - ~ been shown to Promoters contain the identical sequence in inverted interact with a virus inducible element, called PRDII, in the orientation, ATGCAAAT, at the same position. This element 0-IFN gene and to be highly induced by virus infection or appears to be required for the efficient utilization of Ig treatment of cells with double-stranded RNA. In addition, Promoters in B cells. The high degree of sequence and NF-kB controls expression of the human immunodeficiency positional conservation of this element as well as its appar- 4s virus (HIV), ent functional requirement suggests its interaction with a A, further described, it has been shown that a precursor sequence-specific transcription factor but no such factor has of N F - is present in a variety of cells, that the N F - ~ ~ been identified. precursor in cytosolic fractions is inhibited in its DNA binding activity and that inhibition can be removed by DISCLOSURE OF THE INVENTION so appropriate stimulation, which also results in translocation This invention pertains to human lymphoid-cell nuclear of NF-KB to the nucleus. A protein inhibitor of NF-KB, factors which bind to gene elements associated with regudesignated IkB, has been shown to be present in the cytosol lation of the transcription of Ig genes and to methods for and to convert NF-KB into an inactive form in a reversible, identification and for isolation of such factors. The factors saturable and specific reaction. Release of active NF-kB are involved in the regulation of transcription of Ig genes. ss from the IkB-NF-kB complex has been shown to result from The invention also pertains to the nucleic acid encoding the stimulation of cells by a variety of agents, such as bacterial regulatory factors, to methods of cloning factor-encoding lipopolysaccharide, extracellular polypeptides and chemical genes and to methods of altering transcription of Ig genes in agents, such as phorbel esters, which stimulate intracellular lymphoid cells or lymphoid derived cells, such as hybriphosphokinases. IkB and NF-KB appear to be present in a doma cells, by transfecting or infecting cells with nucleic 60 stoichiometric complex and dissociation of the two complex acid encoding the factors. components results in two events: activation (appearance of Four different factors which bind to transciptional reguNF-KB binding activity) and translocation of NF-KB to the latory DNA elements of Ig genes were identified and isonucleus. lated in nuclear extracts of lymphoid cells. Two of the Identification and Isolation of the Transcriptional Regulafactors, IgNF-Aand E, are constitutive; two IgNF-B and K-3 65 tory Factors (hereinafter NF-KB) are lymphoid cell specific. Each factor The transcription regulatory factors of the present invenis described below. tion were identified and isolated by means of a modified

a

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DNA binding assay. The assay has general applicability for regulating or influencing transduction, by NF-KB, of extracellular signals into specific patterns of gene expression and, analysis of protein DNA interactions in eukaryotic cells. In thus, of regulating NF-KB-mediated gene expression in the performing the assay, DNA probes embodying the relevant cells and systems in which it occurs. DNA elements or segments thereof are incubated with In particular, the present invention relates to a method of cellular nuclear extracts. The incubation is performed under s regulating (enhancing or diminishing) the activity of NF-KB conditions which allows the formation of protein-DNA in cells in which it is present and capable of acting as an complexes. Protein-DNA complexes are resolved from intracellular messenger, as well as to substances or compouncomplexed DNA by electrophoresis through polyacrylasition useful in such a method. Such methods and compomide gels in low ionic strength buffers. In order to minimize binding of protein in a sequence nonspecific fashion, a 10 sitions are designed to make use of the role of NF-KB as a mediator in the expression of genes in a variety of cell types. competitor DNA species can be added to the incubation The expression of a gene having a NF-KB binding recognimixture of the extract and DNA probe. In the present work tion sequence can be regulated, either positively or with eukaryotic cells the addition of alternating copolymer negatively, to provide for increased or decreased production duplex poly(d1-dC)-poly(d1-dC) as a competitor DNA species provided for an enhancement of sensitivity in the IS of the protein whose expression is mediated by NF-KB. NF-KB-mediated gene expression can also be selectively detection of specific protein-DNA complexes and facilitated regulated by altering the binding domain of NF-KB in such detection of the regulatory factors described herein. a manner that binding specificity andlor affinity are modiThis invention pertains to the transcriptional regulatory fied. In addition, genes which do not normally possess factors, the genes encoding the four factors associated with transcriptional regulation, reagents (e.g., oligonucleotide 20 NF-KB binding recognition sequences can be placed under the control of NF-KB by inserting an NF-KB binding site in probes, antibodies) which include or are reactive with the an appropriate position, to produce a construct which is then genes or the encoded factors and uses for the genes, factors regulated by NF-KB. As a result of the present invention, and reagents. It further relates to NF-KB inhibitors, includcellular interactions between NF-KB and a gene or genes ing isolated IkB, the gene encoding IkB and agents or drugs which enhance or block the activity of NF-KB or of the 2s whose expression is mediated by NF-KB activity and which have, for example, medical implications (e.g., NF-KBI NF-KB inhibitor (e.g., IkB). cytokine interactions; NF-KBIHTLV-I tax gene product The invention also pertains to a method of cloning DNA interactions) can be altered or modified. encoding the transcriptional regulatory factors or other Genes encoding the regulatory factors can be used to alter related transcriptional regulatory factors. The method involves screening for expression of the part of the binding 30 cellular transcription. For example, positive acting lymphoid specific factors involved in Ig gene transcription can be protein with binding-site DNA probes. Identification and inserted into Ig-producing cells in multiple copies to cloning of the genes can also be accomplished by convenenhance Ig production. Genes encoding tissue specific factional techniques. For example, the desired factor can be tors can be used in conjunction with genes encoding conpurified from crude cellular nuclear extracts. Aportion of the protein can then be sequenced and with the sequence 3 s stitutive factors, where such combinations are determined necessary or desirable. Modified genes, created by, for information, oligonucleotide probes can be constructed and example, mutagenesis techniques, may also be used. used to identify the gene coding the factor in a cDNAlibrary. Further, the sequence-specific DNA binding domain of the Alternatively, the polymerase chain reaction (PCR) can be factors can be used to direct a hybrid or altered protein to the used to identify genes encoding transcriptional regulatory 40 specific binding site. factors. DNA sequences complementary to regions of the factorThe present invention further relates to a method of encoding genes can be used as DNAprobes to determine the inducing expression of a gene in a cell. In the method, a gene presence of DNA encoding the factors for diagnostic purof interest (i.e., one to be expressed) is linked to the enhancer poses and to help identify other genes encoding transcripsequence containing the NF-KB binding site in such a manner that expression of the gene of interest is under the 4s tional regulatory factors. Antibodies can be raised against the factors and used as probes for factor expression. In influence of the enhancer sequence. The resulting construct addition, the cloned genes permit development of assays to includes the kappa enhancer or a kappa enhancer portion screen for agonists or antagonists of gene expression andlor containing at least the NF-KB binding site, the gene of of the factors themselves. Further, because the binding site interest, and a promoter appropriate for the gene of interest. Cells are transfected with the construct and, at an appropri- so for NF-kB in the kappa gene is clearly defined, an assay for blockers or inhibitors of binding is available, as is an assay ate time, exposed to an appropriate inducer of NF-KB, to determinte whether active NF-kB is present. resulting in induction of NF-KB and expression of the gene of interest. BRIEF DESCRIPTION OF THE DRAWINGS The subject invention further relates to methods of reguFIG. 1 A is a schematic depiction of the 5' region of the lating (inducing or preventing) activation of NF-KB, con- ss MOPC 41 V, gene segment; FIG. 1B is an autoradiograph trolling expression of the immunoglobulin kappa light chain of gel electrophoresis DNA binding assays with the SfaNIgene and of other genes whose expression is controlled by SfaNI K promoter fragment of the MOPC 41 V, gene; and NF-KB (e.g., HIV). FIG. 1 C is an autoradiograph of gel electrophoresis DNA As a result of this finding, it is now possible to alter or modify the activity of NF-KB as an intracellular messenger 60 binding assay with overlapping K promoter fragments. FIGS. 2A-2B show autoradiographs of binding compeand, as a result, to alter or modify the effect of a variety of tition analysis in nuclear extracts of human (a) EW and (b) external influences, referred to as inducing substances, HeLa nuclear extracts. whose messages are transduced within cells through NF-KB FIG. 3 shows the results of DNase I foot printing analysis activity. Alteration or modification, whether to enhance or reduce NF-KB activity or to change its binding activity (e.g., 65 of factor-DNA complexes. FIG. 4A shows the nucleotide sequences of actual and affinity, specificity), is referred to herein as regulation of putative binding sites of IgNF-A, FIG. 4B is an autoradioNF-KB activity. The present invention relates to a method of

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graph of binding assays with various DNA probes of three Ig transcriptional control elements. FIG. 5A shows the DNA sequence of the promoter region of MODC41; FIG. 5B shows an autoradiograph of RNA transcript generalized in whole cell extracts made from human B lymphoma cell lines RAMOS and EW and from HeLa cells from the indicated templates. FIG. 6 shows an autoradiograph of RNA transcripts from templates containing an upstream deletion. FIG. 7 is a radioautograph of the binding of B cell nuclear extract to the MOPC-41 K promoter region showing the IgNF-A and IgNF-B complexes. FIG. 8 shows the binding of T cell and nonlymphoid cell nuclear extracts to the MOPC-41 K promoter region. FIG. 9A shows a restriction map of the p-enhancer; FIG. 9B shows an autoradiograph binding assay carried out with p-enhancer fragments. FIG. 10A shows a restriction map of the p300 fragment; FIG. 10B shows complexes formed by various subfragments ofp300; FIG. 10C is a restriction map of the relevant region; FIG. 10E and 10D show competition binding assays with the subfragment p70. FIG. 11A and 11B show location of binding sites in p50 and p70 by the methylation interference technique; FIG. 11C provides a summary of these results. FIG. 12A and 12B show an autoradiograph of binding complexes formed withp50 andp70 in B-cell and non B-cell extracts. FIG. 13A is a restriction map of K enhancer; FIG. 13B shows an autoradiograph of binding assays with K-enhancer fragments; FIGS. 13C and 13D show an autoradiograph of competition assays with K-enhancer fragments. FIG. 14 shows location of NK-KBbinding by methylation interference experiments. FIG. 15A shows binding analysis of NK-KB in various lymphoid and non-lymphoid cells; FIG. 15B shows the binding analysis of NK-KB in cells at various stages of B-cell differentiation. FIG. 16 shows the hgtll-EBNA-1 (hEB) recombinant and the oriP probe. FIG. 1 7 shows the sequence of the DNA probe used to screen for an H2TF1 and NF-KB expression. FIG. 18Ashows the nucleotide sequence of the oct-2 gene derived from cDNA and the predicted amino acid sequence of encoded proteins. FIG. 18B shows the nucleotide sequence of the 3' terminus and predicted the amino acid sequence of the C-terminus derived from clone pass-3. FIG. 18C is a schematic representation of the amino acid sequence deduced from oct-2 gene derived cDNA. FIG. 19 is a schematic representation of expression plasmid pBS-ATG-oct-2. FIG. 20 shows amino acid sequence alignment of the DNA binding domain of oct-2 factor with homeo-boxes of several other genes. FIGS. 21A-21B show the electrophoretic mobility shift analysis of (A) extracts derived from 10213 cells before and after simulation with bacterial lipopolysaccharide (LPS) and (B) extracts derived from PD, an Abelson murine leukemia virus transformed pre-B cell line before and after stimulation with LPS. FIGS. 22A-22B show the effect (A) of cycloheximide on LPS stimulation of 70213 cells and (B) of anisomycin on LPS stimulation of 70213 cells. FIGS. 23A-23B show the effect of phorbol 12-myristate13-acetate (PMA) on NF-B in 70213 cells. FIGS. 24A-24C shows the induction of NFKB in a human lymphoma and in HeLa cells. FIG. 25 is a representation of binding sites for the NF-kB transcription factor in the immunoglobulin kappa light chain enhancer and the HIV enhancer. Boxes indicate the binding sites for NF-kB (B); other regulatory sites are referred to as E l , E2 and E3 and Spl. Dots indicate guanosine residues in the kappa enhancer whose methylation interfered with binding of NF-kB. FIGS. 26A-26B are a characterization of the NF-kB protein. FIG. 26Arepresents determination of the molecular weight of NF-kB. Nuclear extract (300 ug of protein) from TPA-stimulated 70213 pre-B cells was denatured and sub. jected to reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein in the molecular weight fractions indicated by dashed lines was eluted and renatured prior to mobility shift assays as described. A fluorogram of a native gel is shown. The filled arrowhead indicates the position of a specific protein DNA-complex only detected in the 62-55 kDa fraction with a wild type (wt) but not with a mutant (mu) kappa enhancer fragment. The open arrowhead indicates the position of unbound DNA-fragments. FIG. 26B is a representation of glycerol gradient centrifugation of NF-kB. Nuclear extract (400 ug of protein) from TPAstimulated 70213 cells was subjected to ultracentrifugation on a continuous 10-30% glycerol gradient for 20 hours at 150,000xg in buffer D(+). Co-sedimented molecular weight standards (ovalbumin, 45 kDa; bovine serum albumin, 67 kDa; immunoglobulin G, 158 kDa; thyroglobulin monomer, 330 kDa and dimer 660 kDa) were detected in the fractions by SDS-PAGE, followed by Coomassie Blue staining. The distribution of NF-kB activity was determined by electrophoretic mobility shift assays using an end-labelled kappa enhancer fragment. Fluorograms of native gels are shown. The specificity of binding was tested using a kappa enhancer fragment with a mutation in the NF-kB binding site. FIGS 27A-27C represent detection of a cytosolic precursor of NF-kB. FIG. 27A represents analysis of subcellular fractions for NF-kB DNA-binding activity. Nuclear extracts (N), cytosolic (C) and postnuclear membrane fractions (P) from control and TPA-stimulated 70213 pre-B cells were analyzed by gel-shift assays. The filled arrowhead indicates the position of the specific protein-DNA complex seen only with a wild type but not with a mutant kappa enhancer fragment. FIG. 27B represents activation of a cytosolic NF-kB precursor after treatment with dissociating agents. Subcellular fractions were treated with 25% formamide followed by dilution and addition of 0.2% sodium desoxycholate as described. FIG. 27C represents detection of a cytosolic NF-kB precursor after denaturation, SDS-PAGE and renaturation of protein. Nuclear extract (N) and cytosolic fraction (C) from unstimulated (control) 70213 cells was subjected to the treatment outlined in FIG. 26A. For details of illustration, see FIG. 27A. FIG. 28 represents analysis of subcellular fractions for DNA-binding activity of the TPA-inducible transcription factor AP-1. Equal cell-equivalents of nuclear extracts (N) and cytosolic fractions (C) from 70213 and HeLa cells were used in mobility shift assays. AP-1 specific DNA-binding activity was detected using an end-labeled EcoRI-Hind111 fragment from the yeast HIS 4 promoter containing three binding sites for GCN4 recognized by mammalian AP-1. The three protein-DNA complexes seen on shorter exposures of the fluorogram are indicated by filled arrowheads and the position of unbound DNA-fragment by an open arrowhead.

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FIG. 29 represents results of electrophoretic mobility shift ficity was tested with a mutant fragment (kB mu, right panels). The inactive cytosolic NF-KB precursor (lower analysis of subcellular fraction of 70213 cells. panel) was activated formamide treatment (Fa; FIG. 30 shows the effect of denaturation and renaturation pane'). of kB-specific DNA-binding activity in nuclear extracts and FIGS. 36A-36B show the reversibility and kinetics of the 5 cytosolic fractions of 70213 cells. inactivation of NF-kB. FIG. 36A: The effect of DOC treatFIGS. 31A-31B show the effects of dissociating agents on ment on in vitro inactivated NF-kB, NF-kB contained in the activity of NF-kB in subcellular fractions of 70213 cells. nuclear extracts from TPA-stimulated 70213 cells (N TPA; FIG. 31A: cell-free activation of a NF-kB precursor in the 1.1 pg of protein) was inactivated by addition of a gel cytosolic fraction by desoxycholate. FIG. 31B: cell-free lo filtration fraction containing I ~ B (2.5 pg protein), A activation of a NF-kB precursor in the cytosolic fraction by duplicate sample was treated after the inhibition reaction formamide and by a combined treatment with formamide with 0.8% DOC followed by addition of DNA binding and desoxycholate. reaction mixture containing 0.7% NP-40. Samples were analyzed by EMSA. In the fluorograms shown, the filled FIG. 32 shows the effect of TPA stimulation on the arrowhead indicates the position of the NF-kB-DNA comsubcellular distribution of NF-kB in 70213 cells. on the l5 plex and the open arrowhead the position of unbound DNA FIG, 33 shows the effect of TPA probe. FIG. 36B: A titration and kinetic analysis of the in subcellular distribution of NF-kB in HeLa cells. vitro inactivation of NF-kB. NF-kB contained in nuclear 34A-34B show of DNA-ce11u1ose chromaextracts from TPA-treated 70213 cells (2.2 pg of protein) tOgra~h~ c~tOsO1. C~tOsO1 was prepared was incubated with increasing amounts (0.25 to 2.25 pg of from unstimulated 70213 pre-B cells and protein concentra- 20 protein) of a gel filtration fraction containing IkB. After the tions determined. In the fluorograms of native gels shown, DNA binding reaction, samples were analyzed by EMSA, the filled arrowheads indicate the position of the NF-kB-k The 32~-radioactivity the NF-kB-DNA complexes visuin enhancer fragment complex and the open arrowheads the alized by fluorography was determined by liquid scintillaposition of unbound DNA probe. FIG. 34A: Release of tion counting. All reactions were performed in triplicates. DOC-independent NF-kB activity. Equal proportions of 25 The bars represent standard deviations. load, flow-through (FT), washings, and eluates were anaFIGS. 37A-37B show the specificity of IkB. Nuclear lyzed by EMSA, with (+) or without (-) excess DOC. The extracts from unstimulated (Co) or TPA-treated cells were 32P-radioactivity in the NF-kB-DNA complexes was incubated with 5 pl of buffer G (-) or with 5 pl of a gel counted by liquid scintillation and the percentage of NF-kB filtration fraction containing IkB (+) (A, in the presence of activity recovered in the various fractions was calculated. 30 150 mM NaCl). After DNAbinding reactions, samples were FIG. 34B: Release of an inhibitory activity. NF-kB conanalyzed by EMSA. FIG. 37A: Influence of IkB on the DNA tained in the 0.2M NaCl fraction (31 ng of protein) or NF-kB binding activity of various nuclear factors. The probes were: in a nuclear extract from TPA-treated 70213 cells (1.1 pg of NF-kB; H2TF1, an oligonucleotide subcloned into pUC protein) was incubated under non dissociating conditions containing the H2TF1 binding site from the H-2 promoter; with the indicated amounts (in microliters) of either cytosol 35 OCTA, an oligonucleotide subcloned into pUC containing which was DOC-treated but not passed over DNA-cellulose the common binding site for the ubiquitous (upper filled (lanes 4 to 6 and 13 to 15) or the flow-through fraction arrowhead) and lymphoid-specific (lower filled arrowhead) (referred to as NF-kB-depleted cytosol; lanes 7 to 9 and 16 octamer-binding proteins; NF-pE1; NF-kE2; and AP-1, to 18). EcoRI-Hind111 fragment of the yeast HIS4 promoter conFIGS. 35A-35C shows-characterization of IkB and its 40 taining three binding sites recognized by mammalian AP-11 complex with NF-kB. In the fluorograms shown, the filled jun. In the fluorograms shown, filled arrowheads indicate the positions of specific protein-DNA complexes. Open arrowarrowheads indicate the position of the NF-kB-DNA cornplex and the open arrowheads the position of free DNA heads indicate the positions of uncomplexed DNA fragprobe. FIG. 35A: For size determination of IkB, the flowments. FIG. 37B: Interaction of IkB with NF-kB from through from the DNA-cellulose column was passed over a 45 different cell lines. The filled arrowheads indicate the posi(3-200 Sephadex column. Portions of fractions were incutions of the NF-kB-DNA complexes from the various cell bated with NF-kB contained in nuclear extracts from P A lines and the open arrowhead indicates the position of stimulated 70213 cells (N TPA), and analyzed by EMSA. v, uncomplexed DNA probe. void volume; P, fraction where remaining NF-kB precursor FIGS. 38A-38B show the presence of NF-kB in enucle(FIG. 34A, lane 4) peaked after gel filtration as assayed with so ated cells. FIG. 38A: Phase contrast and fluoresence microsexcess DOC in the absence of added NF-kB; I, fraction copy of enucleated HeLa cells. From 612 cells counted on where the inhibiting activity peaked. FIG. 35B: The effect of photographic prints, 63 showed nuclear staining. A repretrypsin treatment on the inhibiting activity of IkB. NF-kB in sentative micrograph is shown. The arrow indicates a cell a nuclear extract (lane 1) was incubated with a fraction that retained its nucleus. FIG. 38B: Analysis of complete and containing inhibitor (lane 2) without any addition (-; lane 3) ss enucleated cells for NF-kB activity. Total cell extracts (1.2 or with bovine pancreas trypsin inhibitor (TI; lane 4), trypsin pg of protein) from control (Co) and TPA-treated complete that had been incubated with BPTI (T+TI; lane 5), or with and enucleated cells were analyzed by EMSAwith a labeled trypsin alone (T; lane 6). Samples were then used in the k enhancer fragment (kB) or HIS4 promoter fragment (APinhibitor assay. FIG. 35C: Glycerol gradient sedimentation I), 3 pg of poly(d1-dC), 1pg of BSA, 1.2% NP-40 and the of NF-kB and its complex with IkB. Nuclear extract from 60 binding buffer in a final volume of 20 pl. In lanes 5 to 8, TPA-stimulated 70213 cells (N TPA) and cytosol from extracts were treated with DOC followed by the addition of unstimulated cells (C Co) were subjected to sedimentation the DNA binding mixture to give final concentrations of through a glycerol gradient. Cosedimented size markers 0.8% DOC and 1.2% NP-40. Samples were analyzed by were ovalbumin (45 kD), BSA (67 kD), immunoglobulin G EMSA. In the fluorograms shown, the filled arrowheads (158 kD) and thyroglobulin (330 and 660 kD). NF-kB 65 indicate the positions of specific protein-DNA complexes activity was detected in the fractions by EMSA with a wild and the open arrowheads the positions of uncomplexed DNA type k enhancer fragment (kB wt, left panels). The speciprobe.

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9
FIG. 39 is a diagram showing the location of positive regulatory domain I1 (PRDII) within the interferon gene regulatory element (IRE) and a comparison of the nucleotide sequences of the PRDII site, KB site, and the H2TF1 site. FIGS. 40A-40B show the results of assays demonstrating that NF-KB binds to PRDII in vitro. FIG. 40Ashows the results of mobility shift electrophoresis assays using as radiolabelled probe an IRE DNA fragment (IRE, lanes 1-3, 10-14, 20-24, 30 and 31), an oligonucleotide containing two copies of the PRDII sequence (PRDII,, lanes 4-6) or a KB site oligonucleotide (KB, lanes 7-9, 15-19,25-29,32 and 33). Assays contained either no protein (4,lanes 1, 4 and 7); 5 pg of unstimulated Jurkat nuclear extract (-, lanes 2 , 5 and 8) or 5 pg of nuclear extract from Jurkat cells stimulated with PHA and PMA(+, lanes 3, 6, 9 and 10-29). Competitions used either the KB oligonucleotide (KB; lanes 10-19) or the PRDII oligonucleotide (PRD II,, lanes 2&29) in the ng amounts shown above each lane. Cytosol (8 pg) from unstimulated Jurkat cells was tested either before (-, lanes 30 and 32) or following treatment with 0.8% deoxycholate (DC, lanes 31 and 33). FIG. 40B shows that mutations within PRDII that reduce 0-IFN induction in vivo decrease the affinity of NF-KB for PRDII in vitro. IRE sequences bearing the mutations indicated (for positions refer to FIG. 39) were tested. The mutations were previously shown to have either high (+) or low (-) inducibility. Goodbourn and Maniatis, Pvoc. Natl. Acad. Sci. USA, 85:1447-1451 (1988). Binding and competition were carried out, as described for FIG. 2A, using 5 pg of nuclear extract from virus infected 70213 cells. Competitor was 10 ng of either the wild-type (WT) or mutant (MUT) KB oligonucleotide (lanes 7,8). FIGS. 41A-41B demonstrate the functional interchangeability of PRDII and NF-KB in vivo. FIG. 41Ais an autoradiogram showing the results of CAT assays of extracts prepared from L929 and S194 myeloma cells transfected with the reporter genes illustrated in FIG. 41B. FIG. 41B is a diagram of the reporter genes containing multiple copies of PRDII or KB. TWOor four PRDII sites [(P), and (P),, respectively] were inserted upstream of the truncated -41 human 0-globin promoter1CAT fusion gene (-410) using an oligonucleotide containing two copies of PRDII (PRDIIx2, as described in the Exemplification). Two copies of a synthetic wild-type KB site, or mutant KBsite (B and B-, respectively) were inserted upstream of the mouse c-fos promoter1CAT fusion gene in which the promoter was truncated to nucleotide -56 (A56). FIGS. 42A-42C demonstrate that virus infection activates binding of NF-KB and gene expression in B lymphocytes and fibroblasts. FIG. 42A represents the results of a binding assay which shows complexes formed with the Ig KB site using 5 pg of nuclear extract from unstimulated cells (lanes 1, 9 and 17) and 5 pg (lanes 2-6, 10-14 and 18-22) or 1pg (lanes 7 , 8 , 15, 16, 23 and 24) of nuclear extract from cells after virus infection. Extracts were prepared from Namalwa cells (lanes 1-8), 70213 cells (lanes 7-16), and L929 cells (lanes 17-28). Competitions used either 5 or 20 ng of the wild-type (WT) or mutant (MUT)KB oligonucleotide. GTP stimulation was tested by addition directly to the binding assay to a final concentration of 3 mM. Cytosol was obtained from L929 cells either prior to (lanes 25,26) or after (lanes 27,28) viral treatment and tested before (-) and after (DC) treatment with deoxycholate. FIG. 42B presents a comparison of methylation interference footprints of virus-induced complexes from Namalwa and L929 cell extracts with NF-KB complexes derived from PHAPMA-stimulated Jurkat cells. The cleavage pattern resulting from methylation of guanine or adenine residues is shown for DNA extracted from the free probe (F) or the DNA-protein complex (B) observed following mobility shift electrophoresis. The sequence of the KBsite presented at the sides and methylated residues that interfere with binding are indicated by a solid circle. FIG. 42C presents results of Northern blot analyses of 70213 cells treated with various inducers. The upper panel shows the induction (from 0 to 20 hr as indicated) of K (K) mRNAby treatment with 50 nglml PMA(PMA, lanes 1-4), 15 pglml LPS (LPS, lanes 5-8) or Sendai virus (V, lanes 9-12). The lower panel shows the same blot hybridized to a 0-IFN DNA fragment. FIG. 43 is the nucleotide sequence and the amino acid sequence of IkB-a. CLONE DEPOSITS Clones hh3 and h3-1 were deposited (Feb. 12, 1988) at the AMerican Type Culture Collection (12301 Parklawn Drive; Rockville, Md. 20852), under the terms of the Budapest Treaty. They were assigned ATCC Designationals 67629 and 67630, respectively. Upon issue of a U.S. patent from the subject application, all restrictions upon the availability of these clones will be irrevocably removed. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the identification, isolation and characterization of human transcriptional regulatory factors, to genes encoding the factors, methods of isolating DNA encoding transcriptional regulatory factors and the encoded factors, uses of the DNA, encoded factors, and antibodies against the encoded factors, inhibitors of the transcriptional regulatory factors. In particular, it relates to the transcriptional regulatory factor NF-kB (previously designated Kappa-3); its inhibitor; IkB, DNA encoding each, methods of altering interactions of NF-kB and IkB and methods of regulating the activity of NF-kB. As described herein, NF-kB, was initially thought to be a B-cell specific factor involved in immunoglobulin gene regulation and has since been shown to be inducible in many, if not all, cell types and to act as an intracellular transducer or mediator of a variety of external influences. The following is a description of the discovery and characterization of four transcriptional regulatory factors, assessment of the function of NF-kB and its role, in many cell types, as an intracellular mediator or transducer of a variety of external influences, discovery of the NF-kB inhibitor IkB and demonstration that NF-kB and IkB exist in the cytoplasm as a NF-kB-IkB complex whose dissociation results in activation of NF-kB and its translocation into the nucleus. The followinn is also a a description of the uses of the genes, regulatory factors and related products and reagents. The transcriptional regulatory factors described herein can be broadly classified as constitutive (non-lymphoid) or tissue (lymphoid) specific. All factors are believed to play a role in transcription of immunoglobulin (Ig) genes. Constitutive factors, which are present in non-lymphoid cells, may have a role in regulating transcription of genes other than Ig genes; lymphoid-specific factors might also play a role in regulating transcription of genes in addition to Ig genes. Four transcriptional regulatory factors were identified, as described below and in the Examples. The presence of constitutive factors rendered the detection of tissue specific

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factors more dificult. A sensitive DNA binding assay, described below, was employed in all studies to facilitate detection of tissue specific factors. The characteristics of the transcriptional regulatory factors IgNF-A, E, IgNFB and Kappa-3 (or NF-KB) are summarized in Table 1 below. TABLE 1

Ig Regulatory Sequence Factor Designation IgNF-A (NF-A1) E factors IgNF-B (NF-A2) Kappa-3 (NF-KB) Promoter
V, V,

Enhancer
UE

KE
-

Lymphoid

Nonlymphoid

+
-

+ +
-

+ + +
-

+ + + +

+ +
-

+
-

+
-

+

+

Factor Ig NF-A As indicated in Table 1, IgNF-A binds to Ig regulatory DNAelements in the region of mouse heavy and kappa light chain gene promoters and also to mouse heavy chain gene enhancer. DNAase I footprint analysis indicates that the binding is mediated by the octamer sequence (ATTTGCAT) which occurs in mouse and human light chain gene promoters approximately 70 base pairs upstream from the site of initation and in heavy chain gene promoters at about the same position (in inverted sequence). Deletion or disruption of the IgNF-A binding site in Ig promoters significantly reduces the level of accurately initiated transcripts in vivo. See, e.g., Bergman, Y. et al. PNAS USA 8 1 7041-7045 (1984); Mason, J. 0. et al. Cell 41 479-487 (1985). As demonstrated in Example 2, this also occurs in an in vitro transcription system. IgNF-A appears to be a positive transacting factor. The IgNF-A binding site appears to be a functional component of the B-cell-specific Ig promoter. For example, sequences from this promoter containing the IgNF-A binding site specify accurate transcription in B-cells but not in Hela cells. IgNF-Ahowever, may not be restricted to B-cells because a factor was detected in Hela cell extracts which generated complexes with similar mobilities and sequence specificity (as tested by competition analysis). Interestingly, the Ig octamer motif in the IgNF-Abinding site has recently been shown to be present in the upstream region (about 225 bp) of vertebrate U1 and U2 snRNA genes. More importantly, this element dramatically stimulates (20 to 50 fold) transcription of U2 snRNA genes in Xenopus oocytes. Therefore, IgNF-A may be a constitutive activator protein that also functions in the high level expression of U1 and U2 snRNA genes in vertebrate cells. The presence of an IgNF-A binding site in the mouse heavy chain enhancer suggests the additional involvement of IgNF-Ain enhancer function. It is known that deletion of an 80 bp region of the enhancer containing the putative binding site reduces activity approximately tenfold. The occupation of the binding site, in vivo, has been inferred from the fact that the G residue in the enhancer octamer is protected from dimethyl sulfate modification only in cell of the B lineage. Furthermore, IgNF-A also binds in a sequence-specific manner to the SV40 enhancer (J. Weinberger, personal communication), which contains the Ig octamer motif,

thereby strengthening the notion that the factor participates in enhancer function. E Factors The E factors are constitutive factors which bind to the Ig light and heavy chain enhancer. Factor Ig NF-B Factor IgNF-B binds to the same regulatory elements as IgNF-A. Indeed, the binding site for IgNF-B appears to be the octamer motif. In contrast to IgNF-A, IgNF-B is lymphoid cell specific. It was. found in nuclear extracts from pre-B, mature B and myeloma cell lines and in nuclear extracts from some T cell lymphomas. IgNF-B was undetectable in nuclear extracts of several n