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Characterization of the human Ki67 promoter for high‐throughput functional genomic screens of G1‐S phase cell‐cycle regulation
Author(s) -
Zambon Alexander C.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.636.3
Subject(s) - biology , e2f , cell cycle , promoter , functional genomics , gene , microbiology and biotechnology , transcription factor , cell growth , transcription (linguistics) , genetics , gene expression , genomics , genome , linguistics , philosophy
Recent studies have shown how a gene's promoter can be used to drive expression of a selectable marker for functional genomic screens to identify cDNAs that can activate transcription of a target gene [Takahashi et. al, Cell, 2006 126 : 1–14]. Our goal was to identify a promoter that was specifically active as cells enter the cell cycle, but that was inactive in arrested or quiescent (G0) cells. We chose the human Ki67 gene because it is a commonly used marker for cellular proliferation and used PCR to clone the Ki67 1.5 kb proximal promoter (−1233–+291). Comparative genomic analysis of this fragment reveals several highly conserved E2F transcription factor binding sites, suggesting that it is under control of E2F transcription factors, which are known to regulate the G1‐S phase of the cell cycle. Expression of GFP driven by the 1.5 kb Ki67 promoter results in cellular colocalization of GFP with endogenous Ki67 protein in HT1080 cells. Furthermore Ki67 promoter activity is attenuated by serum starvation or treatment with mitomycin C, evidence in support of its specificity for proliferating cells. In conclusion, the Ki67 promoter provides a useful molecular tool that could have a variety of applications, including rapid functional genomics screens that seek to identify novel oncogenes and tumor suppressors as well as for in vivo identification of cellular proliferation in transgenic animals (Supported by UCSD IRACDA training fellowship).