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Description  |
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FIELD OF THE INVENTION
This invention relates to oligonucleotide sequences that bind regulatory proteins that affect transcription in response to various molecules, such as cytokines, to DNA constructs comprising the oligonucleotide sequences, cells transfected with
the DNA constructs, and to methods of using the same to provide for the controlled expression of heterologous genes, for the detection and recovery of new regulatory proteins, and for measuring the ability of compounds to act as agonist and antagonists
of gene transcription.
BACKGROUND OF THE INVENTION
In many cellular systems, extracellular signaling molecules, such as polypeptide ligands, bind to receptors on the surface of the cells, thereby triggering an intracellular signaling pathway that ultimately regulates gene transcription within the
cells. For example, cytokines and growth factors, which comprise a large and diverse family of soluble polypeptides that control the growth, differentiation and function of mammalian cells, bind to specific cell surface receptors, that in some way
transduce signals that elicit a specific phenotypic response. A. Miyajama et al., 10 Annu. Rev. Immunol., 295 (1992); M. Aguet et al., 55 Cell, 273 (1988); T. Kishimoto et al., 258 Science, 593 (1992) and A. Ullrich and J. Schlessinger, 61 Cell, 203
(1990). Abundant evidence shows that changes in the transcription rate of specific genes are an important component of this response. This is thought to be a consequence of alterations in the amount or the activity of specific DNA-binding proteins.
In some instances, progress has been made in defining the pathway that leads from a receptor-ligand interaction at the cell surface to changes in the activity of such DNA binding proteins or other nuclear proteins. Ulrich, 61 Cell 203. In this
regard, a common response in surface receptor signaling pathways involves the activation of Ras. L. S. Mulcahy et al., 313 Nature, 241 (1985). Activated Ras then initiates a cascade of serine/threonine phosphorylations through MAP kinases leading to
phosphorylation of DNA binding proteins, thereby changing their transcriptional modulatory activity. S. A. Moodie et al., 260 Science, 1658 (1993); C. A. Lange-Carter et al., 260 Science, 315 (1993); C. S. Hill et al., 73 Cell 395 (1993); H. Gille et
al., 358 Nature, 414 (1992) and R. H. Chen et al., 12 Mol. Cell. Biol., 915 (1992).
Despite these advances, the signal transduction pathways utilized by many growth factors and cytokines to alter gene expression remain unclear. Thus, although known second messengers have been implicated in signal transduction in response to
some of these factors, their role in modulating gene expression remains speculative. Miyajama, 10 Annu. Rev. Immunol., 295 and D. E. Levy and J. E. Darnell, 2 New Biol., 923 (1990). This in turn raises the question of how ligand specific responses
are elicited in such cellular systems. Ullrich, 61 Cell, 203; M. V. Chao, 68 Cell, 995 (1992) and Levy, 2 New Biol., 923.
Progress in resolving these issues has been made recently in the interferon (IFN) system. IFNs .alpha. and .beta. (type I) act as a primary non-specific defense against viral infections. S. Petska and J. A. Langer, 56 Annu. Rev. Biochem.,
727 (1987). IFN.gamma. (type II) has anti-viral properties but also plays a major role in regulation of the immune response. Id. Type I and type II IFNs bind to distinct cell surface receptors and cause rapid alterations in gene expression. Auget,
55 Cell, 273; Uze, 60 Cell, 225; and G. C. Sen and P. Lengyel, 267 J. Biol. Chem., 5017 (1992). Specific sequence elements have been identified in the promoters of genes that respond to IFN.alpha., termed interferon-.alpha. stimulated response
elements (ISREs), that are both necessary and sufficient for regulation by IFN.alpha.. Sen, 267 J. Biol. Chem., 5017. Specifically, activation of the IFN.alpha. receptors stimulates tyrosine phosphorylation of a family of proteins that serve as DNA
binding proteins, and accordingly as transcription regulatory factors via the ISRE. C. Schindler et al., 257 Science, 809 (1992); K. Shuai et al., 258 Science, 1808 (1992) and M. J. Gutch et al., 89 Proc. Natl. Acad. Sci. USA, 11411 (1992). These
DNA binding proteins, generically termed "signal transducers and activators of transcription" (STATs), assemble into a multimeric complex, translocate to the nucleus, and bind cis-acting enhancer elements in the appropriate regulatory regions. D. E.
Levy et al., 3 Genes Dev., 1362 (1989); and D. S. Kessler et al., 4 Genes Dev., 1753 (1990) and Z. Zhong et al., 264 Science, 95 (1994).
One example of an IFN.alpha.-induced ISRE binding protein complex is ISGF3. T. C. Dale et al., 86 Proc. Natl. Acad. Sci., 1203 (1989) and X-Y. Fu et al., 87 Proc. Natl. Acad. Sci., 8555 (1990). ISGF3 is a complex of 4 binding proteins,
called p48, p84 (STAT1.beta.), p91 (STAT1.alpha.) and p113 (STAT2). Recently, cDNAs encoding the proteins that constitute ISGF3 have been isolated and characterized. X-Y Fu et al., 89 Proc. Natl. Acad. Sci., 7840 (1992); C. Schindler et al., 89
Proc. Natl. Acad. Sci., 7836 (1992) and S. A. Veals et al., 12 Mol. Cell. Biol. 3315 (1992). p48 is the DNA binding component of ISGF3 and has homology to myb. Veals, 12 Mol. Cell. Biol., 3315. p84 and p91 are probably alternatively spliced
products of the same gene and are related to p113. X-Y Fu, 89 Proc. Natl. Acad. Sci., 7840 and Schindler, 89 Proc. Natl. Acad. Sci., 7836. p84, p91 and p113 are novel proteins that contain SH2 and SH3 domains and are found in the cytoplasm of
untreated cells. Schindler, 257 Science, 809 and X. Y. Fu, 70 Cell, 323-335 (1992). Thus, IFN.alpha. treatment of cells results in rapid tyrosine phosphorylation of p84, p91 and p113, causing them to associate and form a heteromeric complex with p48
to form ISGF3, which then translocates to the nucleus and binds to ISREs, stimulating transcription. Id.; Dale, 86 Proc. Natl. Acad. Sci., 1203 and Kessler, 4 Genes Dev., 1753.
Regulation in response to IFN.gamma. is conferred by a distinct sequence from the ISRE, the gamma activated sequence (GAS). T. Decker et al., 10 EMBO J. 927 (1991); K. D. Khan et al., 90 Proc. Natl. Acad. Sci., 6806 (1993) and D. J. Lew et
al., 11 Mol. Cell. Biol., 182 (1991). DNA segments containing just a GAS element can confer an IFN.gamma. response to a heterologous promoter when multimerized Decker et al., 11 Mol. Cell. Biol., 5147-5153 (1991) and Kanno et al., 13 Mol. Cell.
Biol., 3951-3963 (1993). Treatment of cells with IFN.gamma. results in tyrosine phosphorylation of p91 (STAT1.alpha.), which then forms homodimers that translocate to the nucleus and bind to the GAS element. Decker, 10 EMBO J,. 927; K. Shuai et al.,
258 Science, 1808 (1992) and Shuai et al., 76 Cell, 821-828 (1994). Thus the specificity of binding of either IFN.alpha. or IFN.gamma. to their receptors is translated into a specific phosphorylation pattern within a related family of latent
transcription factors (i.e. DNA binding proteins). This pattern of phosphorylation dictates the association state of the proteins, which determines specificity of binding to either an ISRE or a GAS, and the subsequent transcriptional response.
Yet another cytokine, Interleukin-6 (IL-6) plays a major role in the induction of the acute phase response in hepatocytes. The acute phase response is characterized by the dramatic transcriptional upregulation of a distinct set of genes, termed
acute phase response genes. P. C. Heinrich et al, 265 Biochem. J, 621-636 (1990). Studies of the promoter regions of these genes have identified specific DNA sequences that are required for induction of acute phase response genes by IL-6. See D. R.
Kunz et al., 17 Nuc. Acids Res., 1121-1138 (1989); M. Hattori et al., 87 Proc. Natl. Acad. Sci USA, 2364-2368 (1990); K, A. Won and H. Baumann, 10 Mol. Cell. Biol., 3965-3978 (1990) and D. R. Wilson et al., 10 Mol. Cell. Biol., 6181-6191 (1990).
These sequences are termed acute phase response elements (APREs). One type of APRE shows many similarities to the GAS elements that confer induction by IFN.gamma.. Several natural GAS elements mediate transcriptional induction by both IFN.gamma. and
IL-6. Yuan et al., 14 Mol. Cell. Biol., 1657-1668 (1994); Harroch et al., 13 EMBO J., 1942-1949 (1994) and Harroch et al., 269 J. Biol. Chem., 26191-26195 (1994). Proteins that bind to this class of APREs have been characterized and purified. U. M.
Wegenka, et al., 14 Mol. Cell. Biol., 3186-3196 (1994); U. M. Wegenka et al., 13 Mol. Cell. Biol., 276-288 (1993); T. Ito et al., 17 Nuc. Acids Res., 9425-9435 (1989) and Hattori, 87 Proc. Natl. Acad. Sci. USA, 2364-2368. A cDNA clone encoding
the IL-6-induced APRE-binding protein has been isolated (Zhong, 264 Science, 95; Akira et al., 77 Cell 63-71 (1994); Zhong, et al., 91 Proc. Natl. Acad. Sci., 4806-4810 (1994); Raz et al., 269 J. Biol. Chem., 24391-24395 (1994)), and was found to
encode a protein that shows considerable homology to p91 (STAT1.alpha.. For this reason the protein is termed STAT3. Like STAT1.alpha., STAT3 is a latent transcription factor that is activated to bind DNA by rapid tyrosine phosphorylation. In some
cells, IL-6 also activates STAT1.alpha.. In these cells, heterodimers of STAT1.alpha. and STAT3 form in addition to homodimers of STAT1.alpha. and homodimers or STAT3. Sadowski et al., 261 Science, 1739-1744 (1993); Raz et al., 269 J. Biol. Chem.,
24391-24395 (1994) and Zhong et al., 264 Science, 95-98 (1994).
Although IFN.gamma. and IL-6 activate STAT proteins that can bind to similar sequences (GAS/APREs), they regulate distinct sets of genes. This suggests that there is specificity with respect to the response elements in some of these genes, such
that they respond only to one of these cytokines. In addition, many cytokines other than IFN.gamma. and IL-6 cause the rapid activation of GAS-binding protein complexes. See O. Silvennionen et al., 261 Science, 1736 (1993); H. B. Sadowski et al., 261
Science, 1739 (1993); A. C. Larner et al., 261 Science, 1730 (1993); D. Finbloom et al., 14 Mol. Cell. Biol., 2113-2118 (1994); D. Meyer et al., 269 J. Biol. Chem., 4701-4704 (1994); Lamb, et al., 83 Blood 2063-2071 (1994); and Tian, et al., 84 Blood
1760-1764 (1994). Some of these complexes contain STAT1.alpha. and/or STAT3, and some contain uncharacterized proteins. These cytokines also regulate sets of genes distinct from each other and from those regulated by IFN.gamma. and IL-6. It is
therefore possible that distinct classes of GAS-like sequences exist that are selective in their ability to respond to various cytokines. Accordingly, specific DNA sequences that show selectivity with respect to their ability to bind cytokine-activated
proteins, including STAT proteins, would be useful tools allowing the responses mediated by different cytokine-activated DNA-binding proteins to be assayed selectively.
The disclosures of the above-cited references are hereby incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
The present invention is directed to oligonucleotide sequences comprising DNA regulatory elements that bind, either directly or indirectly, to activated transcriptional regulatory proteins, preferably STAT proteins, in response to signaling
molecules, including cytokines such as interferon gamma (IFN.gamma.), interleukin 4 (IL-4), interleukin 6 (IL-6), and granulocyte-macrophage colony stimulating factor (GM-CSF). Surprisingly, individual point mutations in the Ly6E GAS regulatory element
result in discrete regulatory elements that bind a variety of activated transcriptional regulatory proteins and/or specifically bind a single type or class of activated transcriptional regulatory protein. Accordingly, the regulatory elements of the
present invention can be used in transcriptional assays to discover agonists or antagonists of a signaling molecule, such as IFN.gamma., and its cognate transcriptional regulatory protein, STAT1.alpha..
In particular, the present invention provides oligonucleotide sequences comprising regulatory elements of the nucleotide sequence TATTCCTGGAAGT (SEQ ID NO. 1), TATTCCGGTAAGT (SEQ ID NO. 2), TCTTCCTGTAAGT (SEQ ID NO. 3), TATTCTTGTAAGT (SEQ ID NO.
4), TATTCCTGTTAGT (SEQ ID NO. 5), TATTCCCGTAAGT (SEQ ID NO. 6), TATTCCTATAAGT (SEQ ID NO. 7), TATTCCTGTCAGT (SEQ ID NO. 8), TATACCTGTAAGT (SEQ ID NO. 9), TATGCCTGTAAGT (SEQ ID NO. 10), TATTCCTTTAAGT (SEQ ID NO. 11), TATTCCTCTAAGT (SEQ ID NO. 12),
TATTCCTGCAAGT (SEQ ID NO. 13), or TATTCCTGTACGT (SEQ ID NO. 14). These oligonucleotide sequences can be double stranded, including their complement.
The present invention also provides a DNA construct comprising the regulatory elements of the oligonucleotide sequences described above operably linked to a promoter, which promoter is operably linked to a heterologous gene, wherein the DNA
construct is linked in such a manner that the heterologous gene is under the transcriptional control of the oligonucleotide sequence and promoter. Also provided is a host cell transfected with this DNA construct.
The present invention also provides a method for the controlled expression of a heterologous gene of interest comprising culturing the transfected host cells containing an appropriate transcriptional regulatory protein(s) in the presence of a
signaling molecule. Preferably, the signaling molecule in this method comprises a cytokine and the transcriptional regulatory protein comprises a STAT protein.
The present invention further provides a method for detecting the presence of an activated transcriptional regulatory protein, such as a novel STAT protein, in a sample comprising contacting the sample with an oligonucleotide sequence as
described above under conditions where the transcriptional regulatory protein is activated and binds with the oligonucleotide sequence to form a complex, and detecting the presence of the complex in the sample. Thereafter, the complex can be separated
from the sample, and the transcriptional regulatory protein isolated from the regulatory element.
Further, the present invention provides a method for measuring the ability of a compound to act as an agonist of gene transcription comprising (a) contacting the compound with a transfected host cell as described above under conditions in which
the heterologous gene is capable of being expressed in response to the compound, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound. Alternatively, the present
invention also provides a method for measuring the ability of a compound to act as an antagonist of gene transcription comprising (a) contacting the compound with a transfected host cell as described above in the presence of a predetermined amount of a
signaling molecule under conditions in which the heterologous gene is expressed in response to the signaling molecule, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the presence of the
signaling molecule, but the absence of the compound. In both these methods, the heterologous gene may be any appropriate reporter gene such as the gene for luciferase, chloramphenicol acetyl transferase, green fluorescent protein or
.beta.-galactosidase.
Further yet, the present invention provides a method for selectively measuring the ability of a compound to agonize or antagonize the induction of STAT heterodimers comprising (a) contacting the compound with a host cell according to claim 19
under conditions in which the heterologous gene is capable of being expressed in response to the compound, wherein the host cell is transfected with a DNA construct comprising a single copy of an oligonucleotide sequence comprising a regulatory element
that that is capable of selectively binding to an activated STAT heterodimer, and (b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound.
These and various other advantages and features of novelty which characterize the invention are pointed out with particularity in the claims annexed hereto and forming a part hereof. However, for a better understanding of the invention, its
advantages, and objects obtained by its use, reference should be had to the accompanying drawings and descriptive matter, in which there is illustrated and described preferred embodiments of the invention.
DEFINITIONS
For the purposes of this invention:
"Oligonucleotide" or "DNA" molecule or sequence refers to a molecule comprised of the deoxyribonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in either single-stranded form or a double-stranded helix, and comprises or
includes a "regulatory element" according to the present invention, as that term is defined herein. The exact size, strandedness and orientation (i.e. 3' to 5', or 5' to 3') will depend upon many factors, which, in turn, depend upon the ultimate
function and use of the oligonucleotides of the present invention. Thus, the term "oligonucleotide" or "DNA" includes double-stranded DNA found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA.
As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction.
"Regulatory element" refers to a deoxyribonucleotide sequence comprising the whole, or a portion of, an oligonucleotide sequence to which an activated transcriptional regulatory protein, or a complex comprising one or more activated
transcriptional regulatory proteins, binds so as to transcriptionally modulate the expression of an associated gene or genes, including heterologous genes.
"Signaling molecule" refers to an extracellular polypeptide, oligosaccaride or other non-peptidyl molecule, in either a free or bound form, that interacts with a receptor at or near the surface of a cell. This interaction in turn triggers an
intracellular pathway which includes the activation of one or more transcriptional regulatory proteins that bind to a regulatory element, thereby transcriptionally modulating the expression of an associated gene or genes. As used herein, "signaling
molecule" includes naturally occurring molecules, such as cytokines, peptidyl and non-peptidyl hormones, antibodies, cell-surface antigens, or synthetic mimics of any of these signaling molecules, or compounds that mimic the action of any of these
signaling molecules.
"Cytokines" refer to a diverse grouping of soluble polypeptides, including growth factors and hormones, that control the growth, differentiation and function of cells in such a manner as to ultimately elicit a phenotypic response in an organism.
Preferred cytokines useful with the regulatory elements and associated methods of the present invention include IFN.gamma., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, Epo, Tpo, GM-CSF, Oncostatin M, growth
hormone, G-CSF, LIF, EGF, CNTF and PDGF.
"Transcriptional regulatory protein" refers to cytoplasmic or nuclear proteins that, when activated, bind the regulatory elements/oligonucleotide sequences of the present invention either directly, or indirectly through a complex of
transcriptional regulatory proteins or other adapter proteins, to transcriptionally modulate the activity of an associated gene or genes. Thus, transcriptional regulatory proteins can bind directly to the DNA regulatory elements of the present
invention, or can bind indirectly to the regulatory elements by binding to another protein, which in turn binds to or is bound to a DNA regulatory element of the present invention. See e.g., S. A. Veals et al., 13 Molec. Cell. Biol., 196-206 (1993).
As used herein, transcriptional regulatory proteins, include, but are not limited to, those proteins referred to in the art as STAT proteins (Z. Zhong et al., 264 Science, 95) STF proteins (C. Schindler et al., 13 EMBO J., 1350 (1994)), Mammary
Gland-Specific Nuclear Factor (M. Schmidt-Ney et al., 6 Mol. Endochronol., 1988 (1992); Wakao, et al., 13 EMBO J. 2182-2191 (1994); and H. Wakao et al., 267 J. Biol. Chem., 16365 (1992)), APRF (Wegenka, 13 Mol. Cell Bio., 276), GHIF (Mayer, 269 J. Biol. Chem., 4701); IL-4 STAT (Hou, et al., 265 Science 1701-1706 (1994)), GHSF and EPOSF (Finbloom, 14 Mol. Cell Bio., 2113), as well as to all substantially homologous analogs and allelic variations thereof.
"Transcriptionally modulate the expression of an associated gene or genes" means to change the rate of transcription of such gene or genes.
"STAT protein" refers to those transcriptional regulatory proteins designated as "Signal Transducers and Activators of Transcription" (STAT) by Dr. J. E. Darnell of Rockefeller University. See Zhong, 264 Science 95. As used herein, STAT
proteins include the p91 (STAT1.alpha.), p84 (STAT1.beta.), p113 (STAT2) proteins and the STAT-associated p48 family of proteins. S. A. Veals et al., 12 Mol. Cell. Biol., 3315 (1992). Further, STAT proteins also include a binding protein designated as
STAT3 (Zhong, et al., 91 Proc. Natl. Acad. Sci., 4806-4810 (1994); Zhong, 264 Science 95), and a binding protein designated as STAT4 (Id.). In addition, MGF is now renamed STAT 5 (Gouilleux et al., 13 EMBO J. 4361-4369 (1994)), and IL-4-STAT is
referred to as STAT6 by some investigators (Ihle et al., 11 Trends Genet., 69 (1995)). Also included are substantially homologous analogs and allelic variations of all of the above STAT proteins.
"Activate", "activated", "activation" or derivatives thereof, means that one or more transcriptional regulatory proteins within a cell are modified post-translationally, or are constituitively active, such that they can bind directly or
indirectly to DNA regulatory elements/oligonucleotide sequences of the present invention in a sequence-specific manner. This modification will typically comprises phosphorylation of the transcriptional regulatory proteins via a variety of mechanisms,
including, but not limited to activation by various protein kinases. See, e.g. (Shuai, 258 Science 1808 and P. Cohen, 17 TIBS, 408 (1992)).
"DNA construct" refers to any genetic element, including, but not limited to, plasmids, vectors, chromosomes and viruses, that incorporate the oligonucleotide sequences of the present invention. For example, the DNA construct can be a vector
comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.
"Promoter" refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of the present invention, the promoter
is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will
be found a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain
"TATA" boxes and "CCAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
"Gene" refers to a nucleic acid molecule, the sequence of which includes all the information required for the normal regulated production of a particular protein. A "heterologous" region of a DNA construct (i.e. a heterologous gene) is an
identifiable segment of DNA within a larger DNA construct that is not found in association with the other genetic components of the construct in nature. Thus, when the heterologous gene encodes a mammalian gene, the gene will usually be flanked by a
promoter that does not flank the structural genomic DNA in the genome of the source organism.
A promoter of a DNA construct, including an oligonucleotide sequence according to the present invention, is "operably linked" to a heterologous gene when the presence of the promoter influences transcription from the heterologous gene, including
genes for reporter sequences such as luciferase, chloramphenicol acetyl transferase, .beta.-galactosidase and secreted placental alkaline phosphatase. Operably linked sequences may also include two segments that are transcribed onto the same RNA
transcript. Thus, two sequences, such as a promoter and a "reporter sequence" are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence. In order to be "operably linked" it is not necessary
that two sequences be immediately adjacent to one another.
A host cell has been "transfected" by exogenous or heterologous DNA (e.g. a DNA construct) when such DNA has been introduced inside the cell. The transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up
the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transfecting DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stablely transfected cell is one in which the
transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a
population of daughter cells containing the transfecting DNA.
"Host cell" refers to a cell line that expresses, either normally or after transfection of the requisite cDNAs, the relevant receptor components for a given signaling molecule, signaling (e.g., kinase) proteins, transcriptional regulatory
proteins, and accessory factors such that, upon binding of the signaling molecule to the cell surface, transcriptional regulatory protein-mediated gene transcription is affected. Preferably, the host cell line is responsive to cytokines, such that the
host cell line expresses, either normally or after transfection of the requisite cDNAs, the relevant cytokine receptor components, JAK proteins, STAT proteins, and accessory factors such that, upon cytokine binding to the cell surface, STAT-mediated gene
transcription is affected.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention may be further illustrated by reference to the accompanying Drawings wherein:
FIG. 1 is a summary of the oligonucleotide competition data for complexes containing transcriptional regulatory proteins activated by IFN.gamma., IL-6, GM-CSF and IL-4. The wildtype sequence is recited at the top of the figure (SEQ ID No. 77).
Each subsequent oligonucleotide is given a designation in the left column consisting of the only nucleotide position which differs from the WT sequence, followed by the nucleotide occurring at the designated position. For example, the oligonucleotide
designated 2G differs from the WT sequence by having a G at position 2. The ability of each sequence to compete with the Ly6E GAS element (termed the "wild type, WT" sequence) for binding to transcriptional regulatory proteins activated by either
IFN-.gamma., IL-6, GM-CSF or IL-4 is shown by the number in the appropriate column;
FIG. 2 is an example of an oligonucleotide competition experiment. Nuclear extracts from cells treated with either IFN-.gamma., IL-6, IL-4 or GM-CSF were incubated with the indicated amounts of oligonucleotide 8T (SEQ ID NO. 11) prior to the
addition of radiolabeled Ly6E GAS element probe. Complexes were then separated on a non-denaturing polyacrylamide gel, which was then exposed to X-ray film. A reproduction of the resulting autoradiogram is shown. A and B indicate the positions of the
complexes of transcriptional regulatory proteins that correspond to STAT1.alpha. and STAT3 respectively;
FIG. 3 is a graph showing transcriptional induction mediated by a single Ly6E GAS element and selected regulatory element-containing oligonucleotide sequences of the present invention in response to cytokines. Cells were transfected with
reporter plasmids containing a single copy of the inventive regulatory elements or the wild type Ly6E GAS element driving expression of luciferase. Cells were treated with either IFN-.gamma., IL-6, LIF or OSM as indicated, and induction of transcription
over untreated cells determined as described in the Examples. The TK luc control reporter lacks a regulatory element;
FIG. 4 is a graph showing transcriptional induction mediated by multiple regulatory elements in response to cytokines. Cells were transfected with reporter plasmids containing four copies of either the Ly6E GAS element or the 7C or 8A
oligonucleotides containing regulatory elements of the present invention, treated with the indicated cytokines, and fold inductions calculated as described in the Examples;
FIG. 5 is a comparison of the in vitro binding affinities of the Ly6E GAS element and the 7C, 8A, 4G, and 8T oligonucleotides containing regulatory elements of the present invention for STAT1.alpha., with the transcriptional induction in response
to IFN-.gamma. from constructs containing 4 copies of the same elements. The first column lists the regulatory elements studied. The next column indicates the ability of each element to bind to STAT1.alpha. in vitro (data taken from FIG. 1). The
last column shows the fold induction of luciferase activity in response to IFN-.gamma. in cells transfected with reporter plasmids containing four copies of the listed regulatory elements; and
FIG. 6 is a series of gel panels showing tyrosine phosphorylation of STAT1.alpha. and STAT3 in response to cytokines. Cells were treated with either IFN-.gamma., LIF, IL-6 or OSM for the indicated time, and lysates prepared. Lysates were
immunoprecipitated with either a STAT1 antisera (top panels) or a STAT3 antisera (bottom panels). After resolution on SDS-polyacrylamide gels and blotting, proteins were detected with antisera directed against either phosphotyrosine, STAT1 or STAT3 as
indicated at the left of FIG. 6.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
The present inventors have discovered a series of DNA regulatory elements (i.e. response elements) that in response to various signaling molecules, bind, either directly or indirectly, to activated transcriptional regulatory proteins, and
accordingly, transcriptionally modulate the expression of one or more genes operably linked with such regulatory elements. In this regard, the inventors have surprisingly discovered that selected point mutations in the Ly6E GAS response element can
transform an element from one that binds with a variety of cytokine-induced, activated transcriptional regulatory proteins to a regulatory element that selectively binds just one type or class of cytokine-induced, activated transcriptional regulatory
proteins. For example, the sequence TATACCTGTAAGT (SEQ ID NO. 9) yields a regulatory element that is selective for a STAT1.alpha. transcriptional regulatory protein, while the sequence TATTCCCGTAAGT (SEQ ID NO. 6) yields a regulatory element that is
selective for the STAT1.alpha. and STAT3 transcriptional regulatory proteins. On the other hand, nucleotide sequences such as TATTCCTGGAAGT (SEQ ID NO. 1) yield a regulatory element that can mediate transcriptional induction in response to a variety of
different cytokine-induced transcriptional regulatory proteins, including the STAT1.alpha. and STAT3 proteins, as well as those transcriptional regulatory protein(s) that are activated by the IL-4, IL-13 and GM-CSF cytokines.
The regulatory elements according to the present invention are selected from the nucleotide sequences TATTCCTGGAAGT (SEQ ID NO. 1), TATTCCGGTAAGT (SEQ ID NO. 2), TCTTCCTGTAAGT (SEQ ID NO. 3), TATTCTTGTAAGT (SEQ ID NO. 4), TATTCCTGTTAGT (SEQ ID
NO. 5), TATTCCCGTAAGT (SEQ ID NO. 6), TATTCCTATAAGT (SEQ ID NO. 7), TATTCCTGTCAGT (SEQ ID NO. 8), TATACCTGTAAGT (SEQ ID NO. 9), TATGCCTGTAAGT (SEQ ID NO. 10), TATTCCTTTAAGT (SEQ ID NO. 11), TATTCCTCTAAGT (SEQ ID NO. 12), TATTCCTGCAAGT (SEQ ID NO. 13), or
TATTCCTGTACGT (SEQ ID NO. 14). In this regard, SEQ ID NOs 9-14 comprise STAT1.alpha. protein selective regulatory elements, SEQ ID NOs 1-5 comprise regulatory elements that bind a variety of transcriptional regulatory proteins, and SEQ ID NOs 6-8
comprise regulatory elements that selectively bind the STAT1.alpha. and STAT3 transcriptional regulatory proteins. Furthermore, these regulatory elements alone, or with additional flanking nucleotide sequences, can form various oligonucleotide
sequences according to the present invention. In this regard, it is preferable that such nucleotide sequences comprise between 13 and 200 nucleotides, including the regulatory elements of the present invention. However, sequences in excess of 200
nucleotides that contain the regulatory elements of the present invention, that are capable of binding activated transcriptional regulatory proteins, and of transcriptionally modulating the expression of one or more genes thereby, are considered to be
within the scope of the present invention.
The oligonucleotide sequences of the present invention can also comprise multimers of two or more "units" of the basic regulatory elements. In this regard, such multimer oligonucleotide sequences can, as a practical matter, contain from about 2
to 15 units of the same or different regulatory elements according to the present invention. However, theoretically, there is no limit to the number of regulatory elements within such a multimer oligonucleotide sequence. Such multimeric oligonucleotide
sequences are useful as probes for detecting, isolating and/or purifying transcriptional regulatory proteins. Further, when used in a DNA construct, including a promoter and heterologous gene, according to the present invention, a multimer of the
regulatory elements can enhance the expression of the gene from the DNA construct in response to various cytokines or other signaling molecules.
A variety of signaling molecules activate transcriptional regulatory proteins that bind directly or indirectly to the regulatory elements/oligonucleotide sequences of the present invention. Nonlimiting examples of such signaling molecules
include polypeptides such as cytokines and antibodies, and cell-surface antigens, oligosaccarides typically found at or near the surface of cell, non-peptidyl molecules such as TUBag4 (P. Constant et al., 264 Science, 267 (1994)) and synthetic mimics any
of these molecules, in both their free and bound forms. Thus, the present invention includes cell to cell or cell to substrate transcriptional regulatory protein activation via signaling molecules bound to or near the surface of a cell or other
substrate.
Preferably, the signaling molecules according to the present invention comprise cytokines that activate transcriptional regulatory proteins that bind to the regulatory elements/oligonucleotide sequences of the present invention. Examples of such
cytokines include, but are not limited to, Interleukins 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13 and 15 (IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13 and IL-15), granulocyte-macrophage colony stimulating factor (GM-CSF), granuloctyte
colony stimulating factor (G-CSF), colony stimulating factor 1 (CSF-1), interferons alpha, beta, and gamma (IFN.alpha., IFN.beta., IFN.gamma.), epidermal growth factor (EGF), platelet derived growth factor (PDGF), leukemia inhibitory factor (LIF),
Oncostatin-M, nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), erythropoietin (Epo), thrombopoietin (Tpo), growth hormone and prolactin. Particularly preferred cytokines according to the present
invention include, but are not limited to, IFN.gamma., IL-4, IL-6, GM-CSF, Oncostatin-M, G-CSF, LIF, EGF, PDGF, Epo, Tpo and CNTF.
The regulatory elements and/or oligonucleotide sequences of the present invention will also prove useful in detecting, isolating and purifying new transcriptional regulatory proteins that display binding specificity to the regulatory
elements/oligonucleotide sequences of the present invention. Further, it is contemplated that these regulatory elements/oligonucleotide sequences will prove particularly useful in the discovery of novel STAT proteins or STAT-related transcriptional
regulatory proteins. In this regard, detection of such novel transcriptional regulatory proteins can be accomplished with the following technique. Cells, such as HepG2 cells, are treated with an appropriate cytokine, for example, with IFN.gamma. for
15 minutes to induce the activation of one or more transcriptional regulatory proteins. Extracts of the nucleus and cytoplasm of these cells are then prepared using conventional methods and tested for binding to the regulatory elements/oligonucleotide
sequences by an electrophoretic mobility shift assay, in comparison with untreated cells that will show little or no specific binding as described in Levy, 3 Genes Dev., 1362 and Kessler, 4 Genes Dev., 1753, the disclosures of which are herein
incorporated by reference. Furthermore, DNA regulatory element binding activity may also be stimulated in vitro by treating a cytoplasmic extract, supplemented with cell membranes, with a signaling molecule, such as cytokine.
If an antibody specific for a transcriptional regulatory protein is available, it can be used to specifically interfere with the binding of the regulatory element of the present invention to the activated transcriptional regulatory protein,
thereby assisting in the identification of the transcriptional regulatory protein. Furthermore, an unknown transcriptional regulatory protein identified or purified using the regulatory elements/oligonucleotide sequences of the present invention can be
used to immunize animals to prepare an antibody specific for the transcriptional regulatory protein using methods well known in the art. See, e.g., E. Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1988), the disclosure of which is herein incorporated by reference.
Thus, the regulatory elements/nucleotide sequences of the present invention thus can serve as a "probe", similar to those used in a variety of nucleic acid detection systems well known in the art, except that the probes of the present invention
are used to detect proteins, rather than a nucleic acid sequences, which specifically bind to the regulatory elements/oligonucleotide sequences of the present invention.
The sensitivity of such a nucleic acid detection assay can be increased by altering the manner in which a signal is detected by an observer. For example, assay sensitivity can be increased through the use of labeled oligonucleotide sequences
using a wide variety of detectable labels, including, without limitation, enzyme labels, radioisotopic labels, fluorescent labels, and modified bases. See, e.g., U.S. Pat. Nos. 4,581,333, 4,358,535, 4,446,237, 4,582,789, and 4,563,417, as well as
European Patent Application Nos. EP 144914 and EP 119448, the disclosures of which are herein incorporated by reference. Thus, DNA probes according to the present invention preferably include the regulatory elements alone, or as part of a longer
oligonucleotide sequence of the present invention, labeled with a detectable label, such as a radioisotope, an enzyme, a fluorescent label, a chemical label, or a modified base.
Thus, the present invention provides a method for detecting the presence of novel transcriptional regulatory proteins in a sample. Such samples are preferably biological samples, including, but not limited to, cells, cell culture supernatant,
cell or tissue extracts, or particular fractions thereof, and other biological fluids such as blood, sera, urine, saliva, etc. Binding of the probe containing the regulatory elements/oligonucleotide sequences of the present invention to a transcriptional
regulatory protein in the sample may be detected by any appropriate means known in the art. For example, direct or indirect, or competitive binding assays may be used. In such assays, association of the labeled probe with the proteinaceous material of
the sample is then detected. In a preferred embodiment, the oligonucleotide sequence is modified by the incorporation of a radioactivity labeled nucleotide.
Once detected, the novel transcriptional regulatory protein can be separated and purified from the probe-protein complex by any of a variety of techniques well known to those of skill in the art. For example, such isolation and purification can
be based on affinity chromatography, which relies on the interaction of the protein to be purified with an immobilized ligand. In the present invention, a regulatory element and/or oligonucleotide sequence of the present invention immobilized on a
support would serve as the immobilized ligand, which in turn would be used to isolate and purify a novel transcriptional regulatory protein from a sample.
In a preferred embodiment, the regulatory element/oligonucleotide sequence of the present invention is immobilized on a solid support or carrier. As used herein "solid phase carrier or support" refers to any support capable of binding the
oligonucleotide sequences/DNA regulatory elements of the present invention. Well known supports, or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses,
and magnetite. Methods for coupling nucleic acids to the solid phase, the solid phase substances useful in these methods, and the means for elution of the proteins from the bound ligand, are well known to those of skill in the art.
In addition to the specific methods described above, purification steps prior to affinity separation may also include one or more additional methods, such as ammonium sulfate precipitation, size exclusion chromatography (gel filtration), ion
exchange chromatography, differential precipitation and the like, all well known in the art. Also useful is the method known as hydrophobic interaction chromatography (HIC) which is based on the interaction between the solute and the gel that is
hydrophobic. Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures. Elution from HIC supports can be effected by alterations
in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene glycol. General principles of HIC are described in U.S. Pat. Nos. 3,917,527 and 4,000,098. Purification of specific proteins using HIC is
described in the U.S. Pat. Nos.: 4,332,717; 4,771,128; 4,743,680; 4,894,439; 4,908,434; and 4,920,196, the disclosures of which are herein incorporated by reference.
The regulatory elements/oligonucleotide sequences of the present invention may be included in a recombinant DNA construct which contains a regulatory element/oligonucleotide sequence operably linked to a promoter and a heterologous gene.
Typically the heterologous gene comprises a reporter sequence, such as the gene for luciferase. In this regard, a recombinant DNA construct, such as a reporter plasmid according to the present invention, can be constructed using conventional molecular
biology, microbiology, and recombinant DNA techniques well known to those skill in the art. Such techniques are explained fully in the literature, including Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A
Practical Approach," Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nuclei | | |
