1.  Field of the Invention
The present invention relates generally to molecular biology and medicine and more specifically to Mcl-1 gene regulatory elements, to a variant Mcl-1 polypeptide, the expression of which induces apoptosis of a cell, and to methods of modulating apoptosis of a cell by modulating expression of the variant Mcl-1 polypeptide. 
2.  Background Information
Growth and development of an organism requires that cell proliferation and cell death be tightly regulated such that the organism develops into a normal, healthy individual.  Such regulation ensures that cells are present in the position and at the time they are required, and are removed when their function has been performed or they are no longer necessary or are detrimental to the organism.  A mechanism of programmed cell death, also referred to a apoptosis, has evolved and contributes, in part, to the regulation of development by inducing cells to die at the appropriate time.  Similarly, various self-renewing tissues in an organism, for example, skin and intestinal epithelium in a mammalian organism, are subject to continual replacement.  In order to maintain homeostasis in an organism, programmed cell death acts as a balance to cell proliferation such that the renewing tissue is maintained in a functional form. 
It has become clear that dysregulation of apoptosis is involved in various pathological conditions.  For example, many cancers are characterized by a defect in the apoptotic process, such that the number of dying cells in a tissue is decreased below its normal level, resulting in an imbalance of cell death and cell proliferation and consequent growth of a tumor.  In addition, neurodegenerative diseases are believed to be caused, at least in part, due to the induction of apoptosis, resulting in death of neuronal cells. 
In view of the importance of apoptosis to the health and well being of most organisms, including humans, a great deal of effort has been directed to identifying the cellular molecules and pathways involved in the apoptotic process.  As a result, several gene products that modulate the apoptotic process have been identified, and can be separated generally into two categories, each of which contains members that can function to either inhibit or induce programmed cell death.  One category of gene products that modulate apoptosis includes the members of the Bcl-2 family of proteins.  Bcl-2, is the best characterized member of this family and inhibits apoptosis when overexpressed in cells.  Other members of this gene family include, for example, Bax, Bak, Mcl-1, Bcl-xL, Bcl-xS, and Bad.  Some of these proteins, including Bcl-2, acts to prevent apoptosis, whereas others such as Bax, Bcl-xS and Bak augment apoptosis. 
The second category of gene products that modulate the apoptotic process includes the family of aspartate-specific cysteine proteases (ASCPs; caspases).  These proteases are related genetically to the Ced-3 gene product that was initially shown to be required for programmed cell death in the roundworm, C. elegans.  The ASCPs family of proteases includes, for example, human interleukin-1-xcex2 converting enzyme (ICE), ICH-1L, ICH-1S, CPP32, Mch2, Mch3, ICH-2 and ICErelsup-III.  One common feature of these gene products is that they are cysteine proteases with specificity for substrate cleavage at Asp-X bonds.  Although these proteases generally induce cell death when expressed in cells, several alternative structural forms such as ICExcex4, ICExcex5, ICH-1S and Mch2xcex2 can function to inhibit apoptosis. 
In addition to the Bcl-2 and ASCP gene family members that have been identified, it is likely that other as yet unidentified gene products exist that are important in mammalian cell death.  For example, in addition to Ced-3, another C. elegans gene, Ced-4, exists and also is required for programmed cell death in C. elegans.  However, a mammalian homolog of this protein has not yet been identified.  The physiological control mechanisms that regulate programmed cell death and the mechanisms by which the cell death pathways interact with other physiological processes within the organism also have not yet been fully defined.  The identification of genes and gene products involved in the regulation of apoptosis can provide a means to modulate the apoptotic process in cells, thus allowing the development, for example, of methods for intervening in pathological conditions associated with abnormally increased or decreased levels of apoptosis, including during the growth, development and differentiation of cells in an organism.  Thus, a need exists to identify genes and gene products involved in the apoptotic process in an organism.  The present invention satisfies this need and provides additional advantages. 
The present invention relates to substantially pure nucleotide sequences that act as regulatory elements for expression of an Mcl-1 gene.  An Mcl-1 gene regulatory element includes at least about twenty contiguous nucleotides of the nucleotide sequence set forth as nucleotides 1495 to 1657 of SEQ ID NO: 1 (positions xe2x88x92162 to +1 of the Mcl-1 gene 5xe2x80x2-flanking sequence as shown in FIG.  3B).  Mcl-1 gene regulatory elements are exemplified herein by a nucleotide sequence that includes nucleotides 1513 to 1564 of SEQ ID NO: 1 (positions xe2x88x92135 to xe2x88x9292 of 3B), as well as by a nucleotide sequence that includes nucleotides 1495 to 1550 of SEQ ID NO: 1 (positions xe2x88x92162 to xe2x88x92107 of FIG.  3B); nucleotides 1495 to 1564 of SEQ ID NO: 1 (positions xe2x88x92162 to xe2x88x9292); nucleotides 1495 to 1606 of SEQ ID NO: 1 (positions xe2x88x9262 to xe2x88x9251); nucleotides 1513 to 1550 of SEQ ID NO: 1 (positions xe2x88x92135 to xe2x88x92107); nucleotides 1513 to 1606 of SEQ ID NO: 1 (positions xe2x88x92135 to xe2x88x9251); nucleotides 1550 to 1657 of SEQ ID NO: 1 (positions xe2x88x92107 to +1); and nucleotides 1606 to 1657 of SEQ ID NO: 1 (positions xe2x88x9251 to +1).  A particularly useful Mcl-1 gene regulatory element is exemplified by the sequence shown as nucleotides 1495 to 1657 in SEQ ID NO: 1, which correspond to position xe2x88x92162 to +1 in FIG.  3B. The present invention also relates to a vector that contains an Mcl-1 gene regulatory element as exemplified above.  The vector can be an expression vector, and can contain a heterologous nucleic acid molecule operatively linked to the Mcl-1 gene regulatory element.  A host cell containing such a vector also is contemplated. 
The present invention also relates to a substantially pure Mcl-1 gene, which is a nucleic acid molecule that encodes an Mcl-1 polypeptide, and includes nucleotides 1727 to 3884 of SEQ ID NO: 1 (see, also, FIG.  1).  Additional nucleic acid molecules of the invention are exemplified by a nucleic acid molecule containing nucleotides 1657 to 3884 of SEQ ID NO: 1; or containing nucleotides 1495 to 3884 of SEQ ID NO: 1; or containing 1 to 8253 of SEQ ID NO: 1 (see, also, FIG.  1).  Nucleotide sequences complementary to the disclosed nucleic acid molecules also are contemplated. 
The present invention further provides a substantially pure polynucleotide encoding the Mcl-1s/xcex94TM amino acid sequence, which is set forth in SEQ ID NO: 3 (see, also, FIG.  4A, lower sequence); as well as a nucleotide sequence complementary to such an encoding polynucleotide.  A polynucleotide of the invention is exemplified by a polynucleotide containing nucleotides 1727 to 2414 of SEQ ID NO: 1 operatively linked to nucleotides 3768 to 3884 of SEQ ID NO: 1, wherein the linked sequence encodes the polypeptide of SEQ ID NO: 3.  A vector, which can be an expression vector, containing such a polynucleotide, which can be a polydeoxyribonucleotide or a polyribonucleotide, also is contemplated, as are host cells that contain such a vector. 
In addition, the present invention relates to oligonucleotides, which contain at least ten contiguous nucleotides of SEQ ID NO: 1 and can hybridize specifically either to a splice junction of the disclosed Mcl-1 gene or to a polynucleotide encoding an Mcl-1 variant polypeptide as disclosed herein.  Nucleotide sequences complementary to such oligonucleotides also are contemplated.  An oligonucleotide that can hybridize specifically to a splice junction of the Mcl-1 gene is exemplified by an oligonucleotide that hybridizes to a portion of SEQ ID NO: 1 that includes nucleotide position 2414, or nucleotide position 2766, or nucleotide position 3013, or nucleotide position 3786 of SEQ ID NO: 1.  In particular, an oligonucleotide of the invention hybridizes specifically to a nucleotide sequence of SEQ ID NO: 1 that includes at least three nucleotides 5xe2x80x2 and contiguous to a specified nucleotide position, and at least three nucleotides 3xe2x80x2 and contiguous to a specified nucleotide position.  As such, the oligonucleotide spans an exon-intron junction of the Mcl-1 gene. 
An oligonucleotide that hybridizes specifically to a polynucleotide encoding an Mcl-1 variant polypeptide as disclosed herein is exemplified by an oligonucleotide that hybridizes specifically to a nucleotide sequence of SEQ ID NO: 1 that includes nucleotides 2412 to 2414 of SEQ ID NO: 1 linked to nucleotides 3768 to 3770 of SEQ ID NO: 1.  Such an oligonucleotide is characterized in that it hybridizes to a nucleic acid molecule containing the splice junction of exon 1 and exon 3 of the Mcl-1 gene.  The oligonucleotides of the invention are useful, for example, as hybridization probes, as primers for a polymerase chain reaction, or as antisense molecules. 
The present invention also relates to a substantially pure Mcl-1s/xcex94TM polypeptide, which has an amino acid sequence as set forth in SEQ ID NO: 3, as well as to peptide portions of Mcl-1s/xcex94TM that contain at least three contiguous amino acids of the sequence set forth as amino acids 228 to 271 of SEQ ID NO: 3.  As disclosed herein, the sequence set forth as amino acids 228 to 271 of SEQ ID NO: 3 are unique to Mcl-1/xcex94TM, and are not found in the full length Mcl-1 polypeptide, or in other proteins as determined by a database search.  As such, the invention also relates to antibodies that can interact specifically with an epitope of Mcl-1s/xcex94TM (SEQ ID NO: 3), but not with an epitope of the full length Mcl-1 polypeptide (SEQ ID NO: 2). 
The present invention further relates to a method of expressing a nucleic acid molecule in a cell by introducing into the cell the Mcl-1 gene regulatory element as disclosed herein, such that a nucleic acid molecule that is operatively linked to the Mcl-1 gene regulatory element is expressed in the cell.  In one embodiment, the Mcl-1 gene regulatory element integrates into a region of genomic DNA in the cell, such that an endogenous nucleic acid sequence to which the Mcl-1 gene regulatory element is operatively linked is expressed.  Such a method allows the identification of nucleic acid molecules that can be expressed in a cell from the Mcl-1 gene regulatory element, and further allows the identification of a function of the expressed nucleic acid molecules in a cell in which they otherwise might not be expressed. 
In another embodiment, the Mcl-1 gene regulatory element is operatively linked to a heterologous nucleic acid molecule prior to its introduction into the cell, then, following introduction into the cell, the heterologous nucleic acid molecule is expressed from the Mcl-1 gene regulatory in the cell.  Such a method provides a means for the selective expression of the heterologous nucleic acid molecule in a cell, which can be, for example, a hematopoeitic cell or a cell is involved in a pathologic condition.  In particular, the heterologous nucleic acid molecule can be expressed in a cell that also expresses an endogenous Mcl-1 gene product. 
Additionally, the present invention relates to a method of identifying an agent that can modulate expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element.  Such a method can be performed, for example, by contacting under suitable conditions an Mcl-1 gene regulatory element, at least a first protein that can interact specifically with the regulatory element, and an agent to be tested; and detecting a change in the ability to form a complex between the Mcl-1 gene regulatory element and the first protein.  The agent can be, for example, a nucleotide sequence, a peptide, a peptidomimetic, or a small organic molecule such as a pharmaceutical agent.  Suitable conditions for performing such a method can be provided by performing the method in a reaction mixture in vitro or in a cell.  A change in complex formation, which can be manifest, for example, by the formation of a complex, by the disruption of a complex, or by altered stability of a complex, can be detected directly by detecting, for example, a change in the electrophoretic mobility of the complex (or of components of the complex), or indirectly by detecting, for example, a change in the expression of a reporter nucleotide sequence expressed from the regulatory element. 
In one embodiment, at least the first protein and the regulatory element interact specifically to form a complex in the absence of the agent.  The agent can alter a specific interaction of the protein with the regulatory element, for example, by disrupting a complex of the first protein and the regulatory element, or by stabilizing such a complex.  Where the agent disrupts the complex, it can be useful for decreasing the expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element.  Where the agent stabilizes the complex, for example, by altering or inducing an alteration of a component of the complex such as by effecting phosphorylation of a component of the complex, the agent can be useful for increasing the expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element. 
In another embodiment, at least a second protein interacts specifically with a complex formed between the first protein and the regulatory element.  Such a method allows the identification of an agent that alters a specific interaction of the second protein with the complex, thereby identifying an agent that can modulate expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element.  The second protein can be, for example, a kinase that can phosphorylate the first protein or another component of a complex comprising the first protein and the regulatory element, and the agent can inhibit a specific interaction of the kinase with complex comprising the first protein, Where phosphorylation of the first protein is involved in expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element, the agent can be useful for decreasing such expression. 
In yet another embodiment, the first protein does not interact specifically with the regulatory element in the absence of the agent.  Such a method can be useful to identify an agent that induces a specific interaction of the first protein and the regulatory element to form a complex, thereby identifying an agent that can increase expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element. 
In another embodiment, the first protein and the regulatory element are contacted with a compound that is known to affect expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element.  The compound can be a compound that inhibits expression of the nucleic acid molecule from the regulatory element, for example, an ERK inhibitor.  Such a method can be used to identify an agent that alleviates the inhibition of expression due to the compound, thereby identifying an agent that, for example, can increase expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element in the presence of the inhibitor. 
The present invention also relates to a method of identifying an agent that can modulate expression of a nucleic acid molecule operatively linked to an Mcl-1 gene regulatory element.  Such a method can be performed, for example, by contacting under suitable conditions an agent and an Mcl-1 gene regulatory element, which is operatively linked to a reporter nucleotide sequence, and detecting an effect on expression of the reporter nucleotide sequence due to the agent.  Expression of the reporter nucleotide sequence can be detected, for example, by detecting an RNA transcript of the reporter nucleotide sequence, or by detecting a polypeptide encoded by the reporter nucleotide sequence.  A polypeptide reporter, for example, a xcex2-lactamase, chloramphenicol acetyltransferase, adenosine deaminase, aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycin-B phosphotransferase, thymidine kinase, xcex2-galactosidase, luciferase or xanthine guanine phosphoribosyltransferase polypeptide or the like, and can be detected, for example, by detecting radioactivity, luminescence, chemiluminescence, fluorescence, enzymatic activity, or specific binding due to the reporter polypeptide. 
The present invention also relates to a method of inhibiting Mcl-1 gene expression in a cell by introducing an Mcl-1 gene regulatory element as disclosed herein into the cell.  The introduced regulatory element can, for example, compete with an endogenous Mcl-1 gene regulatory element for one or more cellular proteins that bind specifically to the regulatory element, thereby modulating transcription of the endogenous Mcl-1 gene.  Since, as disclosed herein, an Mcl-1 gene alternatively can encode a full length anti-apoptotic Mcl-1 polypeptide and a truncated pro-apoptotic Mcl-1s/xcex94TM polypeptide, such a method can be useful for inducing apoptosis of the cell or for increasing viability of the cell, depending upon which Mcl-1 isoform was being expressed in the cell. 
The present invention further relates to a method of modulating apoptosis in a cell by introducing into the cell an Mcl-1 gene.  In one embodiment, the method is directed to inhibiting apoptosis of the cell by providing for the expression of an Mcl-1 polypeptide encoded by exons 1, 2 and 3 of the Mcl-1 gene in the cell, for example, a neuronal cell.  In a second embodiment, the method is directed to inducing apoptosis by providing for expression of an Mcl-1s/xcex94TM variant polypeptide encoded by exons 1 and 3 of the Mcl-1 gene in the cell, thereby inducing apoptosis of the cell, for example, a tumor cell.  As such, the invention also provides a method of treating a pathologic condition by inhibiting or inducing apoptosis in cells involved in the pathologic condition. 
The present invention also relates to a method of inducing apoptosis of a cell by expressing the Mcl-1s/xcex94TM polypeptide in the cell.  Such a method can be performed, for example, by introducing a polynucleotide encoding the Mcl-1s/xcex94TM polypeptide into the cell, and expressing the pro-apoptotic polypeptide.  Such a method also can be performed an oligonucleotide that spans a portion of an intron and a portion of exon 2 into the cell.  Such an oligonucleotide can hybridize specifically, for example, to an endogenous Mcl-1 gene transcript in the cell, particularly to a splice junction involved in splicing of exon 2 into the mature mRNA, such splicing of exon 2 is inhibited, and the Mcl-1s/xcex94TM polypeptide is expressed in the cell.  As such, the invention also provides a method of treating a pathologic condition by inducing apoptosis in cells involved in the pathologic condition. 
The present invention further relates to a method of identifying a cellular factor that can be involved in splicing of an Mcl-1 gene transcript.  Such a method can be performed, for example, by contacting a cellular extract with an oligonucleotide that spans an Mcl-1 gene intron-exon splice junction, and detecting a cellular factor that binds specifically to the oligonucleotide, thereby identifying a cellular factor that can be involved in splicing of the Mcl-1 gene transcript.  Such a method can be useful, for example, to identify a cellular factor involved in splicing exon 1 of the Mcl-1 gene transcript to exon 3 of the Mcl-1 gene transcript. 
In addition, the present invention relates to a method of identifying an agent that induces expression of the Mcl-1s/xcex94TM polypeptide in a cell.  Such a method can be performed, for example, by contacting a cell with the agent, and identifying the expression of the Mcl-1s/xcex94TM polypeptide or a ribonucleic acid molecule encoding the polypeptide in the cell.  An agent identified using such a method can be useful for inducing apoptosis of a cell, and can be useful for treating a pathologic condition such as cancer by inducing apoptosis in cells involved in the condition. 
The present invention also relates to a method of identifying a cell that expresses the Mcl-1s/xcex94TM polypeptide by contacting the cell with a reagent that interacts specifically with the Mcl-1s/xcex94TM polypeptide or with a ribonucleic acid molecule encoding the polypeptide.  Such a reagent can be, for example, an antibody that binds specifically to Mcl-1s/xcex94TM, or an oligonucleotide binds specifically to a nucleic acid molecule encoding Mcl-1s/xcex94TM.