HIC1 is a new E2F1 transcriptional target involved in cell death Free

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The HIC1 gene (Hypermethylated in Cancer 1) located on chromosome 17 encodes for a transcription factor with five zinc fingers, and an amino-terminally located BTB/POZ repression domain. It chiefly acts as a transcriptional repressor that functionally cooperates with p53 to suppress cancer development. Recently it was shown that inactivation of HIC1 results in upregulated SIRT1, a deacetylase which inactivates p53, allowing cells to bypass apoptosis. Furthermore, we and others showed that HIC1 is a transcriptional target of p53, forming a positive feedback loop.

In an attempt to identify new transcriptional regulators of HIC1 we screened a human HIC1 promoter reporter construct for activation by a set of transcription factors. We identified the transcription factor E2F1 as a strong activator of the full-length HIC1 promoter reporter. Co-transfection of the HIC1 promoter reporter with E2F1 showed a 22-fold induction as compared with the empty vector control. Promoter deletion and mutation constructs identified two E2F responsive elements in the HIC1 promoter region. Mutation of either E2F binding site in the HIC1 promoter significantly reduced the response to E2F1. Moreover, binding of E2F1 to the endogenous HIC1 promoter was shown by chromatin immunoprecipitation assays in normal human TIG3 fibroblasts expressing tamoxifen-inducible E2F1.

Upon activation of E2F1 in TIG3 cells, we found an 11-fold increase of endogenous HIC1 mRNA compared with non-induced control cells. Interestingly, this effect was not seen in U2OS cancer cells upon E2F1 activation most likely since in this cell line the HIC1 promoter is hypermethylated. In line with this hypothesis we found that treating U2OS cells with the demethylating agent 5-aza-2’-deoxycytidine increased HIC1 mRNA levels 7-fold. In addition, shRNA-mediated knock-down of HIC1 in TIG3 cells significantly reduced E2F1-induced cell death compared with cells expressing a control shRNA as shown by tetrazolium salt reduction assays. TIG3 cells with knocked-down p53 were used as control for reduced cell death upon E2F1 activation.

E2F1 can induce cell cycle progression as well as cell death. The latter is caused by dysregulated E2F1 expression and is seen as tumor suppressor mechanism. We identified E2F1 as transcriptional activator of HIC1 and propose that E2F1-induced cell death occurs partially via induction of HIC1. Subsequently, elevated HIC1 levels might suppress the p53 negative regulator SIRT1 allowing for higher p53 activity. The newly found E2F1-HIC1 network delineates yet another E2F1-induced cell death pathway.