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Identification of e2f specific target genes


The retinoblastoma gene product pRb and its related proteins p107 and p130 regulate the family of E2F transcription factors (E2F-1 to 5) by binding and subsequent inhibiting these proteins. Although many genes have E2F binding sites in their promotor region, it remains unclear which E2F regulates the expression of which target gene. The purpose of the project described in this proposal is to identify specific target genes of the individual members of the E2F family. Therefore we will perform two approaches : One concerning the differential display reverse transcriptase PCR (DDRTPCR) method with which we will use a timed induction of E2F activity by the tetracyclin induction system and by using a fusion protein of the Oestrogen Receptor and E2F (E2F-ER). Secondly, because the DDRT-PCR technique has the important disadvantage of a relative overexpression of the E2F proteins, we will perform formaldehyde cross-linking of chromatin with subsequent immunoprecipitations of the different E2Fs and/or their DNA-binding heterodimerization partner proteins DP-1 and 2. We will use the isolated promoter region to see whether they are targeted by a specific E2F/DP complex in gel retardation assays and in transient transfection studies using the isolated DNA in reporter constructs. Once we have checked whether the induced or repressed genes are regulated in a cell cycle dependent manner, we will proceed by optaining full length cDNAs, raising antibodies and investigate their function in cell cycle control. The laboratory of Dr. Helin at the ElO will be a unique place to execute the described techniques because all the required reagents for a successful project are readily available. When the direct downstream targets of the different E2Fs are found, we will have a perfect tool to investigate and to unravel the specific differences between pRB, p107 and p130 in controlling E2F activities


Via Ripamonti 435
20141 Milano

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