# Patent

## Title
Promoter sequences for corticotropin releasing-factor receptor CRF2α and method of identifying agents that alter the activity of the promoter sequences

## Abstract

The DNA sequences of human and rat CRF2α receptor promoters are disclosed. Certain functional fragments of the human CRF2α receptor promoter are also disclosed. Further disclosed are a method of identifying functional fragments of human and rat CRF2α receptor promoters and a method of identifying agents that can alter the activity of the human or rat CRF2α receptor promoter.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This invention is a continuation-in-part application of U.S. patent application Ser. No. 09/847,852, filed on Apr. 30, 2001, now abandoned which claims the benefit of provisional patent application Ser. No. 60/201,129, filed on May 2, 2000. This invention also claims the benefit of provisional application Ser. No. 60/338,834, filed on Nov. 12, 2001.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with United States government support awarded by the following agencies: NIH MH40855. The United States has certain rights in this invention.


BACKGROUND OF THE INVENTION
In modern society stress and its consequences are prevalent and result in considerable distress, alterations in physical health and social and occupational functioning. At its extreme, stress can lead to disabling neuropsychiatric problems which include depression, anxiety disorders, post-traumatic stress disorder and other illnesses (Mitchell, 1998; Arborelius et al., 1999). Recent studies demonstrate the potent effects of stress on the body and brain. For example, chronic and intense stress can result in alterations in the region of the brain that plays an important role in memory (McGaugh and Roozendaal, 2002). In addition, stress can negatively impact cardiovascular function, immune function and gastrointestinal physiology (Tache et al., 2001; Beglinger and Degen, 2002; Coste et al., 2002; Vanitallie, 2002).
It is estimated that 10% of the population suffers from depression and another 15% from clinically significant anxiety. This high incidence of stress-related problems is reflected by the fact that approximately 50% of visits to primary care doctors are stress and/or psychologically related.
Current treatments for stress and its disorders are highly sought after and include the traditional anti-anxiety drugs like Valium and Xanax. More recently newer antidepressants like Prozac have been used to treat depression, anxiety and other stress related problems. It is estimated that $6 billion was spent last year in the U.S. on drugs like Prozac. However, these treatments still suffer from lack of efficacy in approximately 30% of individuals and in those that do respond only roughly 50% of them will return to normal function. In addition, these treatments have bothersome side-effects (50% have marked sexual dysfunction) which make treatment with these drugs unacceptable for many individuals. Since depression and anxiety are recurrent and chronic disorders it is important that patients are comfortable taking their medication over a long period of time. Overactivity of the corticotropin-releasing factor CRF system is implicated in depression and anxiety and treatments aimed at this system may be very effective (Reul and Holsboer, 2002). Treatments targeting this system, based on preclinical evidence, offer a completely new and promising approach for treating stress-related illnesses.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention relates to an isolated nucleic acid that contains the full length or a functional fragment of the human or rat CRF2α receptor promoter. By functional fragment, we mean a fragment of the human or rat CRF2α receptor promoter that retains the ability to drive expression of a reporter gene in a host cell to at least twice as that of the house keeping level. The housing keeping level is defined as the expression level of the same reporter gene in the same host cell and under the same conditions without a promoter sequence. Preferably, a functional fragment used in the nucleic acids, vectors, cells and methods of the present invention has the ability to drive expression of a reporter gene to at least three or five times of the house keeping level. The full length human CRF2α receptor promoter is the 3898 bp upstream of the putative transcription start site for the human CRF2α receptor (nucleotides 46 to 3943 of SEQ ID NO:2). The full length rat CRF2α receptor promoter is the 4693 bp upstream of the putative transcription start site for the rat CRF2α receptor (nucleotides 1 to 4693 of SEQ ID NO:1). Examples of functional fragments of the human CRF2α receptor promoter include but are not limited to the 3405 bp (nucleotides 539 to 3943 of SEQ ID NO:2), the 2883 bp (nucleotides 1061 to 3943 of SEQ ID NO:2), the 2346 bp (nucleotides 1598 to 3943 of SEQ ID NO:2), the 1906 bp (nucleotides 2038 to 3943 of SEQ ID NO:2), the 1375 bp (nucleotides 2569 to 3943 of SEQ ID NO:2), the 840 bp (nucleotides 3104 to 3943 of SEQ ID NO:2), the 346 bp (nucleotides 3598 to 3943 of SEQ ID NO:2), the 295 bp (nucleotides 3649 to 3943 of SEQ ID NO:2), the 205 bp (nucleotides 3739 to 3943 of SEQ ID NO:2), and the 104 bp (nucleotides 3840 to 3943 of SEQ ID NO:2) upstream of the putative transcription start site for the human CRF2α receptor.
In another aspect, the present invention relates to a vector that contains a heterologous reporter gene operably linked to the full length or a functional fragment of the human or rat CRF2α receptor promoter. A host cell that contains such a vector is also within the scope of the present invention.
In another aspect, the present invention relates to a method of evaluating the ability of a fragment of the full length human or rat CRF2α receptor to drive transcription. The method involves providing a vector that contains a heterologous reporter gene operably linked to the fragment, introducing the vector into a suitable host cell, and determining the expression level of the reporter gene. The expression level can be determined by measuring the activity of the protein product of the gene. The expression level can also be determined directly by measuring the product of the gene at the mRNA level or the protein level. A negative control should be included for determining the expression level. It is well within the capability of a skilled artisan to set up suitable negative controls for the method of the present invention. For example, a vector that contains the same reporter gene but not operably linked to a promoter can be used as a negative control. Through comparing the expression level of the reporter gene driven by a fragment and that of a negative control, whether the fragment is a functional fragment for purpose of the present invention can be determined.
An isolated nucleic acid that contains a functional fragment of the human or rat CRF2α receptor promoter as determined by the method described above is within the scope of the present invention. Also within the scope of the present invention are a vector that contains a reporter gene operably linked to a functional fragment determined by the method described above and a host cell that contains the vector.
In another aspect, the present invention relates to a method of identifying an agent that can alter the activity of the human or rat CRF2α receptor promoter. The method involves providing a cell that contains a vector in which a reporter gene is operably linked to the full length or a functional fragment of the human or rat CRF2α receptor promoter, exposing the cell to a test agent, and measuring and comparing the reporter gene expression in the cell to that of a control cell that is not exposed to the test agent. A higher or lower expression level in comparison to that of the control cell indicates that the agent can alter the activity of the promoter region of the human or rat CRF2α receptor. Such an agent identified by the method described above is also within the scope of the present invention.
In another aspect, the present invention relates to a method of determining which region of the human or rat CRF2α receptor promoter interacts with a test agent. The method involves providing multiple groups of cells wherein each cell contains a vector in which a reporter gene is operably linked to a fragment of the human or rat CRF2α receptor promoter and wherein the cells of the same group contain the same fragment and the cells in different groups contain different fragments of the human or rat CRF2α receptor promoter, exposing the groups of cells to a test agent, and measuring and comparing the reporter gene expression level of each of the cell groups to that of corresponding control cells that are not exposed to the test agent to determine the effect of the test agent on the promoter activity of different fragments. The effect of the test agent on the promoter activity of different fragments can then be compared.
It is one object of the present invention to identify the promoter region for the human and rat CRF2α receptor.
It is another object of the present invention to provide a method for screening compounds or identifying agents that can alter the activity of the human or rat CRF2α receptor promoter region.
Other objects, advantages and features of the present invention will become apparent after analysis and review of the claims, specification and drawings.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 is the representation of rat and human CRF2 genomic clones.
FIGS. 2A–2H is a comparison of the promoter regions for the rat and human CRF2α receptor gene. In the comparison, the upper sequence is the rat (nucleotides 1506 to 5011 of SEQ ID NO:1) and the lower sequence is the human (SEQ ID NO:2). The arrows denote base +1 (transcription start point) in the rat and human sequences (correspond to nucleotide 4694 of SEQ ID NO:1 and nucleotide 3944 of SEQ ID NO:2, respectively). The promoter fragments are numbered in relation to this. The sequences of the primers used to generate the truncated fragments is denoted in underlined italics, and the identity of the primer is listed below the corresponding sequence.
FIG. 3 shows basal expression from various CRF2α receptor promoter fragments in CHO-K1 cells.
FIG. 4 shows basal expression from various CRF2α receptor promoter fragments in A7R5 cells.
FIG. 5 shows the effects of various treatments on expression from the full-length CRF2α receptor promoter in CHO-K1 cultures.
FIG. 6 shows the effects of various treatments on expression from the full-length CRF2α receptor promoter in A7R5 cultures.
FIG. 7 shows the expression various CRF2α receptor promoter fragments following 10 μM forskolin and 0.25 mM IBMX administration.



DETAILED DESCRIPTION OF THE INVENTION
A. Definition
The term “isolated nucleic acid” used in the specification and claims means a nucleic acid isolated from its natural environment or prepared using synthetic methods such as those known to one of ordinary skill in the art. Complete purification is not required in either case. The nucleic acids of the invention can be isolated and purified from normally associated material in conventional ways such that in the purified preparation the nucleic acid is the predominant species in the preparation. At the very least, the degree of purification is such that the extraneous material in the preparation does not interfere with use of the nucleic acid of the invention in the manner disclosed herein. The nucleic acid is preferably at least about 85% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
Further, an isolated nucleic acid has a structure that is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. An isolated nucleic acid also includes, without limitation, (a) a nucleic acid having a sequence of a naturally occurring genomic or extrachromosomal nucleic acid molecule but which is not flanked by the coding sequences that flank the sequence in its natural position; (b) a nucleic acid incorporated into a vector or into a prokaryote or eukaryote genome such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of clones, e.g., as those occur in a DNA library such as a cDNA or genomic DNA library. An isolated nucleic acid can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. A nucleic acid can be chemically or enzymatically modified and can include so-called non-standard bases such as inosine.
B. In General
The present invention relates to corticotropin-releasing factor (CRF) (Guillemin and Rosenberg, 1955), which is a hormone and neurotransmitter thought to integrate the various electrophysiological, immune, endocrine and behavioral responses to stress (Arborelius et al., 1999; Takahashi, 2001).
Studies in animals demonstrate that antagonism of the CRF system blocks the distress and physical effects related to stress (Takahashi et al., 2001; Bakshi et al., 2002). Studies in humans show that the CRF system in the brain is overactive in patients with depression, anxiety disorders and other neuropsychiatric problems (Nemeroff, 1989; Chappell et al., 1996; Fossey et al., 1996; Bremner et al., 1997; Mitchell, 1998; Baker et al., 1999). In addition, human and animal studies demonstrate that many effective antidepressant treatments decrease brain CRF activity (Veith et al., 1993). Based on these findings the pharmaceutical industry is currently intensively searching for orally administered compounds that will block or reduce the effects of CRF in the brain. Already some compounds have been identified and are in the early stages of human studies (Zobel et al., 2000).
The CRF system is now known to consist of at least seven components. CRF is a neurotransmitter that is released from neurons and has its effects by interacting with CRF receptors located on adjacent brain cells. Urocortin, urocortin II, and urocortin III are other neurotransmitters similar to CRF that also interact with the system (Vaughan et al., 1995; Lewis et al., 2001; Reyes et al., 2001). Once stimulated the receptors activate intracellular processes which mediate the stress effects.
CRF produces its effects by interacting with two different receptors termed CRF1 and CRF2 (Chen et al., 1993; Perrin et al., 1995). There also exists at least three different splice variants of the CRF2 receptor, termed “CRF2α,” “CRF2β” and “CRF2γ” (Lovenber et al., 1995a; Kostich et al., 1998). In addition to CRF1 and CRF2 receptors, there also exists a protein, termed CRF binding protein (CRF-BP), that is found in brain cells and functions to inactivate CRF after it is released (Potter et al., 1991).
While much is known about the biology of CRF, considerably less is understood about CRF1, CRF2 and the binding protein. Most believe that the CRF1 receptor is responsible for mediating the effects of stress and also may be important in depression and anxiety. However, other evidence suggests that CRF2 receptor may also play a critical role in mediating the effects of stress (Bakshi et al., 2002). The pharmaceutical industry has targeted CRF1 for the development of antagonists to block the effects of stress. While interest in CRF2 may exist, small molecule antagonists specific for this receptor remain to be discovered.
The present invention invokes a different therapeutic approach aimed at altering the regulation of the gene encoding the CRF2 receptor and has the potential to be a more effective strategy in the treatment of anxiety, depression and other stress-related problems. This approach is based on the hypothesis that the primary problem in these illnesses is dis-regulation of one or more components of the CRF system. Thus, a treatment aimed at the primary cause of these problems should prove more effective and be without non-specific effects on other systems. For example, drugs that control the regulation of CRF or its receptors would allow greater precision in stress management. Traditional approaches suffer from numerous unwanted effects because receptor antagonists affect all receptors throughout the brain and body and do not selectively interact with those regions that are most important in an illness.
The advantage of understanding and developing drugs to affect regulation of genes that make receptors and other proteins is that they can be directed to alter levels of proteins in specific tissues. For example, the amygdala is located deep in the brain and is thought to be pivotal in mediating the effects of CRF in depression and anxiety. Once the factors that regulate the selective expression of CRF in the amygdala are identified, drugs could be targeted to affect CRF only in this region, leaving other sites (cortex, brain stem, heart, hypothalamus) unaffected.
For the purposes of the present invention we have cloned and identified the promoter region of the rat CRF2 receptor gene. This promoter region of the gene is responsible for determining where in the body and when during development the CRF2 receptor is expressed. This region also controls how much receptor is expressed. Therefore, we envision that the promoter region would be a target for drug development for the treatment of various psychopathologies described above, including depression, generalized anxiety, social anxiety, post traumatic stress and panic disorder. Using the promoter region of the gene in a cell and/or chip based screening assay will allow us to develop methods to identify agents that alter the activity of the promoter region and, thus, affect the expression of the CRF2 receptor. These agents could have significant therapeutic potential in the treatment of various psychopathologies.
C. Human CRF2 Receptor Gene
The clone containing the entire gene for the human CRF2 receptor was obtained from Research Genetics (Huntsville, Ala.). This PAC clone (RP5-1143H19) contained a 127,425 bp insert, which included the first exons for the CRF2α, CRF2β and CRF2γ receptors and remaining 11 exons that are common to all three isoforms (see FIG. 1). The clone contains approximately 42,000 bp upstream of exon 1 of the CRF2α, and approximately 39,000 bp downstream of the final exon.
D. Rat CRF2 Receptor Gene
The rat CRF2 receptor gene was cloned from a Sprague-Dawley rat genomic library constructed in Lambda FIX® II obtained from Stratagene (La Jolla, Calif.). The library was prepared from a partial Sau3A I digest of kidney DNA obtained from male rats (16 months old). The library was probed with a 32P-labelled fragment of the rat CRF2α cDNA (Lovenberg et al., 1995b), which corresponded to bases 1 to 261 of the cDNA (Genbank #U16253). The single positive clone that was obtained was plaque purified, the insert was excised by NotI digestion and subcloned into the pGEM-5Zf(+) vector (Promega, Madison, Wis.). The entire insert was sequenced using the GPS-1 Genome Priming System (New England Biolabs, Beverly, Mass.) which uses randomly interspersed primer binding sites.
The insert was determined to be 14,894 bp long, and the intron/exon junctions were identified by comparison of the insert sequence to that of rat CRF2α (Genbank #U16253), mouse CRF2β (Genbank #U21729) and human CRF2γ (Genbank #AF019381) cDNAs. This revealed that the clone contained the first exons of the CRF2α and second exon (1a) of the CRF2β (FIG. 1). The clone also contained exon 2, which is common to each of the isoforms. In addition, the clone contained a region that corresponds to the first exon of the CRF2γ; however, it lacks the necessary consensus splice site sequences and ATG translation start site to function as an exon.
E. Comparison of Rat and Human CRF2 Gene Sequences
We identified the region of the human CRF2 gene that corresponds to the rat CRF2 genomic clone (see FIG. 1). The promoter region for the CRF2α should be located within the  ̃4000 bp of sequence that lie upstream of the first exon for the CRF2α but downstream of the first CRF2γ exon. We compared the rat and human CRF2 gene sequences in a subregion of this fragment that contains the first 2000 bp immediately upstream of the first CRF2α exon using the BestFit program from the Genetics Computer Group (GCG) Wisconsin Package version 10.0. The gap creation penalty was set at 40 and the gap extension penalty was set at 2. The analysis revealed 70.4% identity between the two sequences (see FIG. 2). It is likely that both mouse and monkey sequences will have greater than 70.4% identity compared to rat and human, respectively.
Transcription factor-binding sites are short sequences of DNA located in promoter regions where transcription factors bind to exert their effect on gene regulation. These sites have been found to confer unique expression properties to genes in other systems and are likely important for the temporal and spatial regulation of the CRF2 receptor gene. They also serve to highlight the basal promoter, which is the region of the CRF2 receptor promoter that is most critical for appropriate developmental and cell-specific expression of the gene.
To identify potential transcription factor binding sites, analysis was performed on 2000 bp of sequence immediately upstream of the first CRF2α exon start site in both the rat and human sequences using MatInspector v2.2 (Quandt et al., 1995), public domain software with the Transfac 4.0 vertebrate matrices (Heinemeyer et al., 1999). The threshold levels were set at 1.0 for core similarity and 0.9 for matrix similarity. This identified 152 and 146 potential transcription factor binding sites in the human and rat CRF2α promoter regions, respectively.
Numerous potential transcription factor binding sites are present within any given promoter sequence. Very few of these are ultimately functionally relevant. A comparison between the same promoter from two different species allows one to identify those elements that are conserved and therefore likely to be critical for the appropriate functioning of the gene. Comparison of the human and rat results revealed 51 putative binding sites that were conserved in terms of location and orientation within the two sequences. These transcription factor-binding sites are listed in Table 1. The location in the table refers to the position of the sequence upstream of the putative transcription start site (+1 in FIG. 2). By convention, positions upstream of the transcription start site are preceded by a minus symbol. The plus and minus symbols in parentheses following the location refer to the sense and antisense strands, respectively. Because these sites are conserved between rat and human we feel they may constitute important regulatory elements.
F. Preparation of CRF2α Receptor Promoter Constructs
The minimal promoter fragment within the human and rat CRF2α receptor genes that confers the correct temporal and spatial expression of the CRF2α receptor will be subcloned into an expression vector that contains either the firefly luciferase (pGL3-basic Promega, Madison, Wis.) or enhanced green fluorescent protein as a reporter (Clontech, Palo Alto, Calif.).
i. Human CRF2α Receptor Promoter
To obtain the fragment corresponding to the promoter region of the CRF2α gene, it was necessary to first subclone into an intermediate vector, pRL-null (Promega, Madison, Wis.) prior to subcloning into the reporter construct that will be used to transfect cells. A 4040 bp fragment of the human CRF2 gene corresponding to the promoter region of the CRF2α receptor (see FIG. 1) was excised with the restriction enzymes NarI and NdeI. The fragment was subcloned into the vector pRL-null that had been digested with the same two enzymes. This insert was then removed from the pRL-null construct with XhoI and EcoRi and subcloned into the pEGFP-1 vector that had been digested with the same two enzymes. We also subcloned this fragment into a luciferase reporter, pGL-3 basic (Promega). The insert was removed from pRL-null with EcoIcRI and SalI and inserted into pGL3-basic that had been digested with SmaI and XhoI.
We focused on the first 2000 bp in our sequence comparison and found a 70.4% identity between the rat and human sequence. Although we will initially examine a fragment containing 3898 bp of sequence, we know that a smaller fragment that has been deleted from the 5′ end will function as the basal promoter. Using a common reverse (3′) primer that ended 36 bp downstream of the putative transcription start point (TSP), we generated sequentially smaller fragments of the CRF2α promoter region through PCR with several forward (5′) primers. The putative TSP has been clearly identified in FIG. 2 and it's relative location is +1. Please note that in this standard nomenclature system there is no zero position. The constructs generated were from −3898, −3405, −2883, −2346, −1906, −1375, −840, −346, −295, −205, and −104 bp relative to the putative TSP through +36 bp (referred to as the −3898, −3405, −2883, −2346, −1906, −1375, −840, −346, −295, −205, and −104 constructs respectively). Our goal is to define the basal promoter, which in some instances has been found to be shorter than 500 bp.
ii. Rat CRF2α Receptor Promoter
A 4693 bp fragment corresponding to the promoter region of the rat CRF2α receptor (see FIG. 1) can be obtained by digestion with HindIII and BsrBI. This can be subcloned into the HindIII and SmaI sites of the pEGFP-1 vector. This fragment can also be subcloned into a luciferase reporter, pGL-3 basic (Promega). To generate smaller fragments of the rat CRF2α promoter, a strategy identical to that described for the human CRF2α promoter can be used. The ability of each fragment to drive transcription can be determined as described below.
Table 1. Location of conserved putative transcription factor binding sites. Numbering is in relation to the putative transcription start sites noted as +1 in FIG. 2. The (+) and (−) indicate that the sequence is present in the sense or antisense strand, respectively.













Position (strand) of Binding Site











Binding Site Name
Rat
Human







AP1FJ_Q2
−1870 (+)
−1771 (+)



AP1FJ_Q2
−1574 (+)
−1468 (+)



AP1FJ_Q2
−1542 (−)
−1544 (−)



AP1FJ_Q2
−1299 (−)
−1093 (−)



AP1FJ_Q2
 −434 (−)
 −654 (−)



AP1FJ_Q2
 −109 (−)
 −189 (−)



AP1_Q2
−1564 (−)
−1544 (−)



AP1_Q2
 −434 (−)
 −654 (−)



AP1_Q2
 −109 (−)
 −189 (−)



AP4_Q5
−1679 (+)
−1631 (+)



AP4_Q5
−1679 (−)
−1631 (−)



AP4_Q5
 −269 (−)
 −280 (−)



CREB_02
 −108 (−)
 −188 (−)



DELTAEF1_01
−1986 (+)
−1956 (+)



DELTAEF1_01
−1812 (+)
−1916 (+)



DELTAEF1_01
 −899 (+)
 −877 (+)



DELTAEF1_01
 −189 (−)
 −250 (−)



E47_02
 −901 (−)
 −879 (−)



GATA1_02
−1663 (+)
−1667 (+)



GATA1_02
 −601 (+)
 −711 (+)



GATA1_03
 −512 (+)
 −711 (+)



GATA1_03
 −273 (−)
 −629 (−)



GATA1_04
 −600 (+)
 −710 (+)



GATA1_04
 −551 (−)
 −629 (−)



GATA1_05
 −510 (+)
 −709 (+)



GATA_C
 −508 (+)
 −707 (+)



GC_01
 −41 (−)
 −42 (−)



GKLF_01
−1836 (−)
−1851 (−)



IK2_01
−1974 (−)
−1986 (−)



IK2_01
−1857 (+)
−1967 (+)



IK2_01
−1709 (−)
−1817 (−)



IK2_01
−1210 (−)
−1232 (−)



IK2_01
−1004 (+)
−1103 (+)



IK2_01
 −314 (−)
 −296 (−)



LMO2COM_01
 −899 (−)
 −877 (−)



LMO2COM_02
 −549 (−)
 −627 (−)



MYOD_01
 −899 (−)
 −877 (−)



MYOD_Q6
 −898 (+)
 −876 (+)



MZF1_01
−1400 (+)
−1321 (+)



MZF1_01
−1345 (−)
−1228 (−)



MZF1_01
 −889 (−)
 −852 (−)



MZF1_01
 −310 (−)
 −203 (−)



NF1_06
 −210 (+)
 −20 (+)



NFAT_Q6
−1274 (−)
−1356 (−)



NFAT_Q6
 −719 (+)
 −829 (+)



NFAT_Q6
 −177 (−)
 −432 (−)



NFY_01
 −25 (−)
 −25 (−)



NFY_Q6
 −22 (−)
 −22 (−)



NKX25_01
−1283 (−)
 −489 (−)



S8_01
 −509 (−)
 −708 (−)



SP1_06
 −40 (−)
 −41 (−)










G. Production of Transfected Cell Lines

In one embodiment, the present invention is a transfected cell line. One preferred method of creating such a cell line is described as follows: The constructs described above containing the human or rat promoter fragments placed upstream of the firefly luciferase gene are used to transfect immortalized cell lines. The constructs are transfected into CHO-K1 and A7R5 cell lines using lipofectamine 2000 (Life Technologies, Rockville, Md.). The CHO-K1 cells are not known to express CRF receptors whereas A7R5 cells, derived from aortic cells, have been demonstrated to express CRF receptors. Both cell lines can be maintained at 37° C. with 5% CO2 in DMEM supplemented with 10% fetal bovine serum. Primary cultures of the central nervous system, as well as additional immortalized cell lines, are also appropriate for these transfections. To control for transfection efficiency, the cells are also co-transfected with the pRL-TK vector (Promega, Madison, Wis.). The pRL-TK vector contains the Renilla luciferase gene downstream of the herpes simplex virus thymidine kinase promoter, a promoter which provides low to moderate levels of expression. Cell lysates are assayed for total protein using the BCA assay (Pierce, Rockford, Ill.) to standardize for the protein extraction. The level of reporter gene expression from a standardized amount of cell extract is quantified by measuring luciferase activity using a luminometer (EG&G Wallac, Gaithersburg, Md.) and the dual-luciferase reporter assay system (Promega, Madison, Wis.). Firefly luciferase activity reflects CRF2α receptor promoter activity and Renilla luciferase activity is used to normalize data between experiments.
H. Characterization of Basal Expression from CRF2α Receptor Promoter Fragments
Using the methods described above, transient transfections of CHO-K1 and A7R5 cultures were assayed for reporter gene expression (See FIG. 3 and FIG. 4). In these experiments, three basic controls were utilized. The cultures referred to as pGL-3 basic were transfected with a pGL-3 firefly luciferase reporter construct that did not contain an experimental promoter, and with the pRL-TK vector. These cultures should demonstrate a very low level of expression and may be considered a negative control. The cultures referred to as pGL-3 control were transfected with a construct containing the firefly luciferase reporter downstream of the SV40 viral promoter as well as the pRL-TK vector. These cultures should demonstrate a very high level of expression and may be considered a positive control. Finally, the cultures referred to as unrelated DNA were transfected with a construct containing 1916 bp of DNA sequence upstream of the firefly reporter gene and with the pRL-TK vector. The 1916 bp of this construct were a random DNA sequence with the final 21 bp most 3′ being identical to our putative promoter constructs. These cultures were intended to demonstrate the specificity of our promoter constructs.
Our results in the CHO-K1 cultures indicate that the −840, −346, −295, −205, −104 constructs have the highest levels of expression of the CRF2α promoter constructs (See FIG. 3). Distal regions of the CRF2α promoter appear to exert an inhibitory influence that is gradually unmasked as the length of the promoter is shortened, reaching a plateau beginning with the −840 construct that appears to last through the shortest promoter construct. 1-way ANOVA revealed a significant difference amongst constructs (F=124, P<0.0001). Planned pairwise comparisons using Student's t-tests indicated that all the constructs were significantly higher than the pGL3-basic control construct (*, p<0.0001). Examination of the mean indicates that our lowest level of expression (−2883 construct) is 378% greater than housekeeping levels of expression (pGL-3 basic) (mean −2883=0.330±0.009, mean pGL3 basic=0.069±0.004), and is 4% of the expression elicited by the viral SV40 promoter (pGL-3 control) (mean −2883=0.330±0.009, mean pGL3 control=7.433±0.401). Our highest level of expression (−840 construct) is 7657% greater than expression from the promoterless control vector (pGL-3 basic) (mean −840=5.353±0.596, mean pGL3 basic=0.069±0.004), and is 72% of the strong expression from the SV40 promoter (pGL-3 control) (mean −840=5.353±0.596, mean pGL3 control=7.433±0.401). Furthermore, an unrelated human chromosomal DNA sequence (unrelated) was not able to drive expression above background (FIG. 3). Thus, the CRF2α promoter constructs function and are appropriate tools to monitor CRF2α specific transcription.
Our results in the A7R5 cultures indicate that the pattern of expression for the various constructs is very similar to that seen with the CHO-K1 cells (See FIG. 4). Distal regions of the CRF2α promoter appear to exert an inhibitory influence that is gradually unmasked as the length of the promoter is shortened. Expression reaches a plateau that begins with the −840 construct and appears to last through the smallest promoter construct. 1-way ANOVA revealed a significant difference amongst constructs (F=221.9, P<0.0001). Planned pairwise comparisons using Student's t-tests indicated that all the constructs were significantly higher than the pGL3-basic control construct (*, p<0.0001 for all cases). Examination of the means indicate that our lowest level of expression (−2883 construct) is 354% greater than expression from the promoterless control vector (pGL3-basic) (mean −2883=0.473±0.011, mean pGL3-basic=0.104±0.016), and is 5% of the strong expression elicited by the SV40 promoter (pGL3-control) (mean −2883=0.473±0.011, mean pGL3-control=9.038±0.610). Our highest level of expression (−840 construct) is 9412% greater than expression from the promoterless control vector (pGL3-basic) (mean −840=9.903±0.532, mean pGL3-basic=0.104±0.016), and is 9.6% greater than the strong expression from the SV40 promoter (pGL3-control) (mean −840=9.903±0.532, mean pGL3-control=9.038±0.610). Furthermore, the unrelated human chromosomal DNA sequence (unrelated) was unable to drive expression above background. This data in the A7R5 cultures provides further evidence that our CRF2α promoter constructs function and are appropriate tools to monitor CRF2α specific transcription.
I. Characterization of Inducible Expression from Full-Length CRF2α Promoter
A stated goal for the constructs is the ability to identify agents that can alter expression of the CRF2α gene. Therefore, we designed experiments to demonstrate this ability. Using methods previously described in this application, CHO-K1 cultures were transfected with the −3898 construct and the pRL-TK internal control construct. These cultures were then treated with either CRF (1 μM), urocortin (1 μM), dexamethasone (1 μM), forskolin (10 μM), or the appropriate control at the time of transfection. The control for CRF and urocortin was culture media whereas the control for dexamethasone and forskolin was the culture media with the amount of DMSO required to solubilize these compounds. CRF and urocortin are ligands for the CRF receptors, dexamethasone stimulates the glucocorticoid pathway and forskolin increases intracellular cAMP levels. Twenty-four hours following transfection and treatment, the cultures were harvested a processed for luciferase assay as described previously in this application.
Statistical analysis was done on results from CHO-K1 cultures stimulated with the various compounds (See FIG. 5). Following demonstration of a main effect of treatment in a 1-factor ANOVA (F=668.1, *, p<0.0001), post-hoc analysis with Newman-Kuels multiple comparison test indicated that treatment of CHO-K1 cultures with CRF or urocortin did not significantly change expression compared with the untreated control cultures. This was expected because CHO-K1 cells do not express CRF receptors. Dexamethasone also does not appear to alter expression in the full-length CRF2α promoter compared with the DMSO control cultures. However, forskolin treatment significantly lowers expression (*, p<0.001) compared with the DMSO control cultures (forskolin mean=0.302±0.012, DMSO control mean=0.411±0.004). This finding suggests that altering intracellular cAMP levels affects expression from the CRF2α promoter. It should be noted that IBMX, an antagonist of phosphodiesterase activity, was not given to cultures receiving forskolin. The prolonged exposure to forskolin (24 hours) without IBMX may have lead to increased phosphodiesterase activity within the A7R5 and CHO-K1 cultures resulting in below normal levels of cAMP. Nonetheless, the results demonstrate the constructs ability to monitor agent induced changes in expression from the CRF2α receptor promoter.
Using a similar experimental paradigm, we treated A7R5 cultures to determine what agents may alter expression from the CRF2α promoter. In addition to treating the cultures with CRF and urocortin (1 μM each), A7R5 cultures also were treated with either of the antagonists alone D-Phe or DMP696 (1 μM each), CRF plus D-Phe (1 μM each), CRF plus DMP696 (1 μM each), urocortin plus D-Phe (1 μM each), and with urocortin plus DMP696 (1 μM each). D-Phe is a non-selective CRF receptor antagonist, blocking both CRF1 and CRF2 receptors, whereas DMP696 is specific to CRF1 receptors. A7R5 cultures are known to express CRF2 receptors and should be a highly appropriate cell type to monitor CRF2α receptor expression.
Analysis of the CRF and urocortin experiments with separate 2-factor ANOVAs revealed significant main effects of agonist and antagonist treatment and there was a significant agonist by antagonist interaction (See FIG. 6). Post-hoc analysis with a Bonferroni posttest revealed that CRF and urocortin significantly decrease expression from the −3898 construct as compared to the untreated control cultures in the A7R5 cultures (*, p<0.001 for both cases). The addition of the non-selective CRF receptor antagonist, D-Phe, with either CRF or urocortin brought expression from the full length promoter back to the levels seen in the untreated controls. However, the CRF1 receptor antagonist, DMP696, did not affect either the CRF or urocortin induced reduction in expression from the full length promoter. These results demonstrate that either CRF or urocortin can reduce expression from the −3898 CRF2α receptor promoter construct within A7R5 cultures in a CRF2 receptor dependent manner.
In a separate experiment the effects of forskolin and dexamethasone were compared to DMSO control A7R5 cultures. Analysis with a 1-factor ANOVA revealed a significant main effect of treatment, and post-hoc analysis indicated that both forskolin and dexamethasone significantly reduced expression (FIG. 6, +, p<0.001) compared with the DMSO control cultures. These findings are consistent with those seen in the CHO-K1 cultures.
J. Characterization of Forskolin Induced Expression from CRF2α Receptor Promoter Fragments
In the present assay system each active test agent may produce its effect by interacting with one or more regulatory elements, or corresponding transcription factors, that are present in the promoter region. One of the advantages of having a series of constructs that contain sequentially smaller fragments of the promoter is that these constructs can be used to identify the region of the promoter where a test compound may be exerting its effect. This is achieved by identifying which of the promoter constructs respond to the test compound and which do not.
As a proof of this idea, we have examined the ability of forskolin to affect expression from all of the truncated fragments of the CRF2α promoter. In these experiments, CHO-K1 cultures were transiently transfected with the CRF2α promoter constructs. At the time of transfection, the cultures were either given vehicle or forskolin (10 μM) with IBMX (0.25 mM). Following twenty-four hours, the cultures were harvested for the luciferase assay as described above. 2-way ANOVA indicates there is a significant main effect of treatment and construct (See FIG. 7), and there is also a significant treatment by construct interaction (*, p<0.0001 for all three). Post-hoc analysis with a Bonferroni posttest revealed a significant forskolin-induced increase in expression when compared to the respective vehicle control cultures for three of the constructs (−840, −346 and −295; *, p<0.001). All other constructs did not show a significant forskolin-induced change in expression compared with the respective vehicle controls. These results suggest that a forskolin-induced increase in expression from the CRF2α promoter is mediated through regulatory element(s) located somewhere between −840 and −205 bp relative to the putative TSP. These results demonstrate the ability of these promoter constructs to identify agents that alter expression driven by the promoter, and the ability of the same constructs to facilitate identification of the region of the promoter where these agents exert an effect.
K. Production of Transgenic Mice
In another embodiment, the present invention is a transgenic mouse comprising a heterologous promotor sequence for corticotropin releasing hormone receptors CRF2α. In one preferred embodiment, the transgenic mouse would be created as follows: Once potential therapeutic agents are identified in our cell culture model we will test their ability to alter CRF2 receptor promoter activity in transgenic animals. Reporter constructs that consist of the basal CRF2α receptor promoter placed upstream of the enhanced green fluorescent protein or β-galactosidase will be used to generate transgenic mice. The procedure for generating the enhanced green flourescent construct has already been described, and the procedure for generating the β-galactosidase construct was identical to that used to make the firefly luciferase construct. These animals will allow us to confirm the appropriate spatial and temporal expression of the CRF2α receptor promoter.
The reporter constructs will be identical to those described above and will preferably consist of 3898 bp of human CRF2α receptor promoter or 4693 bp of rat CRF2α receptor promoter fused to the coding region of EGFP or β-galactosidase. Transgenic animals will be generated using standard techniques. The preferred technique would involve the microinjection of 100 copies of the promoter-reporter construct into the male pronucleus of a fertilized egg. Injected eggs are then transplanted into pseudo-pregnant females and the progeny from these transplantations examined for the presence of the CRF2α receptor promoter-reporter construct (called “the transgene”). Animals containing the transgene will be identified by extracting DNA from a small amount of tail tissue and probing this DNA with a segment of the EGFP or β-galactosidase gene, which is not normally found in the mammalian genome. Animals that contain the CRF2α receptor promoter-reporter transgene will be mated to normal animals so that transgenic lines are established. Preferably, we will generate three transgenic lines that contain the transgene in three separate sites within the genome. In this way we will verify that the expression patterns we observe are a result of EGPF or β-galactosidase expression from our promoter segment and are not due to site insertion effects.
To confirm the appropriate function and expression of the CRF2α receptor promoter-reporter transgene, the following will preferably be performed: Brain tissue sections will be taken from transgenic animals beginning in late embryonic development and extending at five-day intervals into adulthood (postnatal day 60). Sections will then be observed under 488 nm light or 420 nm light to identify those brain cells that express EGFP or β-galactosidase, respectively. The pattern of reporter expression will be compared with the normal pattern of CRF2α receptor expression. The expression of the CRF2α receptor promoter transgene should overlap with expression of the endogenous CRF2α receptor gene both temporally (i.e., it should begin to expressed when CRF2α receptor is first expressed) and spatially (i.e., expression of the transgene should be confined to those cells within septum and ventromedial hypothalamus that normally express CRF2α receptor).
L. Use of Transformed Cell Lines and Transgenic Animals
Cells transfected with CRF2α receptor promoter regions fused to a reporter construct will allow the testing of potential therapeutics. Pharmacologically relevant amounts of candidate small molecules will be applied to the transfected cells in the media and the influence of these molecules on reporter gene expression levels will be assessed by the methods discussed above. These experiments will be replicated at least 10 times and any small molecule that yields a statistically significant difference in expression will be considered a positive find. The level of reporter expression after treatment with a specific candidate drug will enable the determination of the degree to which the drug is influencing CRF2α receptor activity.
Candidates that increase the expression of CRF2 promoter-reporter activity can then be further tested in vivo. Transgenic animals will be treated with the candidate drug to determine whether CRF2α promoter-reporter transgene levels are elevated in the same way and to the same degree as that found in the cells lines. Adverse drug effects can also be determined with these animals.
If the drug behaves similarly in vivo and there are no signs of significant toxicity, then the drug could be tested in a variety of animal models that are predictive of antidepressant or anti-anxiety activity. If the candidates are active in these tests they could serve as therapeutic agents in psychiatric disorders, such as depression.
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## Claims

1. An isolated nucleic acid consisting of a polynucleotide selected from the group consisting of nucleotides 46 to 3943 of SEQ ID NO:2 and a fragment of nucleotides 46 to 3943 of SEQ ID NO:2 that comprises nucleotides 3840 to 3943 of SEQ ID NO:2.

2. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 46 to 3943 of SEQ ID NO:2.

3. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 539 to 3943 of SEQ ID NO:2.

4. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 1061 to 3943 of SEQ ID NO:2.

5. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 1598 to 3943 of SEQ ID NO:2.

6. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 1588 to 3943 of SEQ ID NO:2.

7. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 2569 to 3943 of SEQ ID NO:2.

8. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 3104 to 3943 of SEQ ID NO:2.

9. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 3598 to 3943 of SEQ ID NO:2.

10. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 3469 to 3943 of SEQ ID NO:2.

11. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 3739 to 3943 of SEQ ID NO:2.

12. The isolated nucleic acid of claim 1 wherein the nucleic acid consists of nucleotides 3839 to 3943 of SEQ ID NO:2.

13. A vector comprising:
a nucleic acid that comprises nucleotides 3840 to 3943 of SEQ ID NO:2; and
a heterologous reporter gene operably linked to the nucleic acid.

14. A host cell comprising the vector of claim 13.

15. An isolated nucleic acid consisting of a functional fragment of nucleotides 46–3943 of SEQ ID NO:2 identified by a method comprising the steps of:
(a) providing a vector that comprises a fragment of nucleotides 46–3943 of SEQ ID NO:2 and a heterologous reporter gene wherein the reporter gene is operably linked to the fragment;
(b) introducing the vector into a suitable host cell;
(c) measuring the reporter gene expression level;
(d) comparing the gene expression level to a suitable negative control; and
(e) identifying the fragment as a functional fragment if the gene expression level is at least twice as that of the negative control.

16. A vector comprising:
a nucleic acid that comprises a functional fragment of nucleotides 46 to 3943 of SEQ ID NO:2 identified according to claim 15; and
a heterologous reporter gene operably linked to the nucleic acid.

17. A host cell comprising the vector of claim 16.