Promoter sequences for urocortin II and the use thereof

A method for screening for an agent that may alter the activity of human urocortin II promoter, the method comprising the steps of:

• (a) providing a nucleic acid that comprises a human urocortin II promoter sequence operably linked to a reporter gene, wherein the promoter sequence is 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);
• and
• (b) subjecting the nucleic acid to conditions suitable for the promoter sequence to drive the expression of the reporter gene in the presence of the test agent;
• (c) evaluating the expression of the reporter gene compared to a control nucleic acid that is exposed to the same conditions but without the test agent wherein a higher or lower expression than that of the control nucleic acid indicates that the agent may alter human urocortin II promoter activity.

A method for determining whether a fragment of the −3527 bp upstream of the transcription start site of human urocortin II gene is functional under a set of conditions of interest, the method comprising the steps of:

• (a) providing a nucleic acid that comprises the fragment and a heterologous reporter gene operably linked to the fragment;
• (b) subjecting the nucleic acid to the set of conditions of interest;
• (c) measuring the expression level of the reporter gene;
• and
• (d) comparing the expression level to a suitable negative control wherein a higher than negative control expression level indicates that the fragment is functional.

A method of determining which region of the human urocortin II promoter interacts with an agent that is known to alter the activity of the promoter, the method comprising the steps of:

• (a) providing multiple groups of nucleic acids in which a reporter gene is operably linked to a fragment of the 3527 bp upstream of the transcription start site of the human urocortin II gene promoter and wherein the nucleic acids of the same group contain the same fragment and the nucleic acids in different groups contain different fragments;
• (b) subjecting the nucleic acids to conditions suitable for the fragments to drive the expression of the reporter gene in the presence of the agent;
• (c) measuring and comparing the reporter gene expression level of each of the nucleic groups to that of corresponding control cells that are not exposed to the agent to determine the effect of the agent on the promoter activity of different fragments;
• and
• (d) comparing the effect of the agent on the promoter activity of different fragments.

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.

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.