Telomerase Promoter Sequences for Screening Telomerase Modulators

Abstract

Telomerase reverse transcriptase is part of the telomerase complex responsible for maintaining telomere length and increasing the replicative capacity of progenitor cells. Telomerase activity is turned off in mature differentiated cells, but is turned back on again in hyperplastic diseases, including many cancers. This disclosure provides regulatory elements that promote transcription in cells that express telomerase reverse transcriptase (TERT). The disclosure also provides systems using TERT promoter sequences for identifying compounds that can be used to modulate telomerase expression

1 . A method for identifying a compound that binds to human genomic DNA upstream of the translation initiation site for telomerase reverse transcriptase, comprising:
 a) combining a compound with a polynucleotide comprising 15 or more consecutive nucleotides of SEQ. ID NO:1 between residue 44 and residue 15375; and   b) determining if the compound binds said polynucleotide.   
     
     
         2 . The method of  claim 1 , wherein said polynucleotide comprises 50 or more consecutive nucleotides of SEQ. ID NO:1 between the Alu sequences and the translation initiation site (position 13545). 
     
     
         3 . The method of  claim 1 , wherein said polynucleotide:
 a) comprises the sequence from position −117 to position −36 relative to the translation initiation site of SEQ. ID NO:1;   b) comprises the sequence from position −239 to position −36 relative to the translation initiation site of SEQ. ID NO:1;   c) comprises the sequence from position −117 to position +1 relative to the translation initiation site of SEQ. ID NO:1;   d) comprises the sequence from position −239 to position +1 relative to the translation initiation site of SEQ. ID NO:1; or   e) hybridizes with a polynucleotide complementary to a sequence having feature a), b), c), or d) under stringent conditions, and has the characteristic of binding a compound that can modulate telomerase expression.   
     
     
         4 . The method of  claim 1 , wherein said polynucleotide comprises a binding site for c-Myc, c-Myb, SRY, HNF-3β, HNF-5, TFIID-MBP, NFκB, or E2F. 
     
     
         5 . The method of  claim 1 , wherein said polynucleotide comprises a TATA box, a CAAT box, or an E box. 
     
     
         6 . The method of  claim 1 , which is a method for screening compounds that may modulate telomerase expression, wherein a compound that binds to human genomic DNA upstream of the translation start codon for telomerase reverse transcriptase is identified as capable of modulating telomerase expression. 
     
     
         7 . The method of  claim 6 , whereby a compound is identified that enhances expression of telomerase reverse transcriptase. 
     
     
         8 . The method of  claim 6  whereby a compound is identified that inhibits expression of telomerase reverse transcriptase. 
     
     
         9 . The method of  claim 1 , which is a method for screening a protein that binds a transcription recognition sequence within SEQ. ID NO:1. 
     
     
         10 . The method of  claim 1 , which is a method for screening on oligonucleotide that hybridizes to a transcription recognition sequence within SEQ. ID NO:1. 
     
     
         11 . The method of  claim 1 , which is a high throughput method for screening small molecule drugs for their ability to modulate telomerase expression. 
     
     
         12 . The method of  claim 2 , wherein said nucleotides of SEQ. ID NO:1 are operably linked to a heterologous reporter sequence such as luciferase, β-glucuronidase, β-galactosidase, chloramphenicol acetyl transferase, or green fluorescent protein. 
     
     
         13 . The method of  claim 12 , comprising measuring binding of the compound to the polynucleotide by determining expression of said reporter sequence in the presence and absence of the compound. 
     
     
         14 . The method of  claim 1 , comprising measuring binding of the compound using a promoter affinity column or by immunoprecipitation. 
     
     
         15 . The method of  claim 1 , comprising measuring binding of the compound to the polynucleotide by UV or chemical crosslinking. 
     
     
         16 . The method of  claim 1 , comprising measuring binding of the compound to the polynucleotide by a mobility shift DNA binding assay, a methylation or uracil interference assay, DNAse or hydroxyl radical footprint analysis, or fluorescence polarization. 
     
     
         17 . A method of obtaining a compound for regulating transcription of telomerase reverse transcriptase in a cell, comprising producing a compound identified as being able to modulate telomerase expression by way of the method of  claim 6 . 
     
     
         18 . A polynucleotide for use in a screening method according to  claim 1 , comprising 50 or more consecutive nucleotides of SEQ. ID NO:1 between residue 44 and residue 15375, which can bind an activator or inhibitor of the expression of human telomerase reverse transcriptase. 
     
     
         19 . The polynucleotide of  claim 18 , which comprises a recognition element listed in Table 1. 
     
     
         20 . The polynucleotide of  claim 18 , bound to an activator or inhibitor of the expression of human telomerase reverse transcriptase.