Promoter sequences for urocortin II and the use thereof

Abstract

Various human urocortin II promoter sequences are disclosed. Nucleic acids and host cells that contain the promoter sequences are also disclosed. Further disclosed are various methods involving the use of these sequences. Also disclosed are methods of modulating the activity of a human urocortin II promoter sequence and methods of modulating urocortin II expression in a cell.

1 . An isolated nucleic acid comprising a human urocortin II gene promoter sequence selected from the 3527 bp upstream of the transcription start site (nucleotides 1 to 3527 of SEQ ID NO:1), the 3030 bp upstream of the transcription start site (nucleotides 498 to 3527 of SEQ ID NO:1), the 2451 bp upstream of the transcription start site (nucleotides 1077 to 3527 of SEQ ID NO:1), the 1987 bp upstream of the transcription start site (nucleotides 1541 to 3527 of SEQ ID NO:1), the 1426 bp upstream of the transcription start site (nucleotides 2102 to 3527 of SEQ ID NO:1), the 931 bp upstream of the transcription start site (nucleotides 2597 to 3527 of SEQ ID NO:1), the 533 bp upstream of the transcription start site (nucleotides 2995 to 3527 of SEQ ID NO:1), the 380 bp upstream of the transcription start site (nucleotides 3148 to 3527 of SEQ ID NO:1), or the 195 bp upstream of the transcription start site (nucleotides 3333 to 3527 of SEQ ID NO:1).  
     
     
         2 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 3527 bp upstream of the transcription start site (nucleotides 1 to 3527 of SEQ ID NO:1).  
     
     
         3 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 3030 bp upstream of the transcription start site (nucleotides 498 to 3527 of SEQ ID NO:1).  
     
     
         4 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 2451 bp upstream of the transcription start site (nucleotides 1077 to 3527 of SEQ ID NO:1).  
     
     
         5 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 1987 bp upstream of the transcription start site (nucleotides 1541 to 3527 of SEQ ID NO:1).  
     
     
         6 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 1426 bp upstream of the transcription start site (nucleotides 2102 to 3527 of SEQ ID NO:1).  
     
     
         7 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 931 bp upstream of the transcription start site (nucleotides 2597 to 3527 of SEQ ID NO:1).  
     
     
         8 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 533 bp upstream of the transcription start site (nucleotides 2995 to 3527 of SEQ ID NO:1).  
     
     
         9 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 380 bp upstream of the transcription start site (nucleotides 3148 to 3527 of SEQ ID NO:1).  
     
     
         10 . The isolated nucleic acid of  claim 1  wherein the nucleic acid comprises the 195 bp upstream of the transcription start site (nucleotides 3333 to 3527 of SEQ ID NO:1).  
     
     
         11 . A DNA construct comprising the nucleic acid of  claim 1  operably linked to a heterologous reporter gene.  
     
     
         12 . The DNA construct of  claim 11 , wherein the construct is an expression vector.  
     
     
         13 . A host cell comprising the vector of  claim 12 .  
     
     
         14 . The host cell of  claim 13 , wherein the host cell is a primarily cultured central nervous system cell.  
     
     
         15 . The host cell of  claim 13 , wherein the host cell is a cell of an immortalized cell line.  
     
     
         16 . A method for screening for an agent that can alter the activity of human urocortin II promoter through a CRE or GRE site, or determining whether an agent that is known to alter the activity of human urocortin II promoter alters the activity through a CRE or GRE site, the method comprising the steps of: 
 (a) providing a first human urocortin II promoter sequence that contains at least one CRE or GRE site;    (b) providing a second human urocortin II promoter sequence that is the same as the first sequence except that the CRE or GRE site is mutated; and    (c) determining and comparing the effect of the agent on the promoter activity of the first and second sequence wherein a difference in effect indicates that the agent can alter the human urocortin II gene promoter activity through a CRE or GRE site.    
     
     
         17 . A method for screening for an agent that can affect the modulation of the activity of human urocortin II promoter by cAMP level, glucocorticoid receptor activity or corticotropin-releasing factor (CRF) receptor activity, the method comprising the steps of: 
 (a) providing a host cell that comprises a human urocortin II promoter sequence and a reporter gene operably linked to the promoter sequence wherein the expression of the reporter gene controlled by the promoter sequence can be modulated by cellular cAMP level, cellular glucocorticoid receptor activity or cellular CRF receptor activity;    (b) changing the cellular cAMP level, the cellular glucocorticoid receptor activity or the cellular CRF receptor activity;    (c) exposing the cell to a test agent; and    (d) determining the expression level of the reporter gene and comparing the expression level to that of a control cell that is not exposed to the test agent wherein a higher or lower than control expression indicates that the test agent can affect the modulation of the human urocortin II gene promoter activity by cAMP level, glucocorticoid receptor activity or CRF receptor activity.    
     
     
         18 . A method for modulating the promoter activity of a human urocortin II promoter sequence that contains at least one cAMP response element (CRE) or glucocorticoid response element (GRE), the method comprising the step of: 
 modulating the CRE or GRE activity sufficiently to modulate the promoter activity.    
     
     
         19 . The method of  claim 18  wherein the promoter activity is cellular promoter activity and regulating the CRE or GRE activity is accomplished through modulating cellular cAMP level or cellular glutocorticoid receptor activity.