Part:BBa_K404106

phTERT promoter

phTERT promoter
BioBrick Nr.	K404106
RFC standard	RFC 10
Requirement	pSB1C3
Source	synthetic
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

(telos, end; mere, part)
Providing the tumorspecific promoter phTERT with the “Virus Vonstruction Kit”, the iGEM Freiburg_Bioware team 2010 ensures a layer of specificity and safety to the recombinant viral vector system.
At the 3´end of the human chromosomes the DNA sequence” TTAGGG” can be found in a repetitively manner. The DNA polymerase cannot elongate at this overhanging regions because the overhangs do not provide any template strand for DNA polymerases. In most somatic cells, except for germ cells, the telomerase activity is shut off (Cech 2000). Human telomerase is a reverse transcriptase which synthesizes telomeric DNA sequences. The ribonucleoprotein consists of two subunits – the catalytic protein subunit human telomerase reverse transcriptase which catalyzes the polymerization of the nucleotides onto the chromosomes and the RNA component which serve as the template for the synthesis of the telomeric repeat sequences (Wick et al. 1999).
Telomerase activation is a critical step in human tumorigenesis. About 85 ± 90% of several human tumors show telomerase activity, while healthy tissue is telomerase negative which means that the protein subunit hTERT is absent in these cells (Cech, 2000), but can be reactivated in cancer cells.
Human telomerase reverse transcriptase is driven by the phTERT promoter. Several factors were identified to regulate the hTERT promoter positively and negatively (Kyo et al. 2008). The TATA-less promoter is characterized by enriched GC regions at the putative transcription start region. These are binding sites for the zinc finger transcription factor Sp1, which are clustered upstream of the startcodon (Wick et al. 1999). Since Sp1 is ubiquitously expressed in a wide range of somatic cells, it is not a strong candidate for cancer-induced gene expression. Within the core promoter (approximately 260 bases upstream of the startcodon) further transcription factor binding sites can be found: The so-called E-boxes (CACGTG) bind the basic helix-loop-helix zipper (bHLHZ) encoded by the Myc familiy (Kyo et al. 2008). Thy c-myc oncogenes are activated by the MAP-kinase pathway induced by EGF-R binding ligands (Maida et al. 2002). A critical factor for regulating hTERT expression is the Activating Enhancer-binding Protein 2 (AP-2) which was found to be an activator of cancer-specific gene expression. Activation of the hTERT promoter was increased by hypoxia described in Nishi et al., 2004 by binding of the HIF-1 alpha transcription factor to the HRE binding sites, still activation cannot be verified in every cancer type. AP-1 acts as a repressor on transcriptional gene expression in humans demonstrated in Takakura, Kyo, Inoue, Wright, & Shay, 2005. Since the palindromic consensus sequence cannot be found in the phTERT sequence submitted, no repression on gene transcription should be observed.

For analysis of the submitted BioBrick part, the phTERT promoter was tested for functional biological activity in different cell lines driving gene expression of several genes of interest. The idea of the iGEM team Freiburg_Bioware 2010 was to produce viral particles encapsidating the transgene expression cassette flanked by the inverted terminal repeats (ITRs). The left ITR is followed by the tumor-specific promoter phTERT, which regulates the gene expression of mVenus, mCherry and the prodrug convertase thymidine kinase fused to the guanylatekinase. Three different human cell lines, AAV-293, HT1080 and A431, were transduced with the viral vectors containing the genes coding for different proteins and promoter activity was analysed by flow cytometry, quantitative real-time PCR and cytotocicity assays (MTT assay) depending on the gene of interest.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]