Charakterisierung der E3 Ligase HectH9 und deren Einfluss auf cMyc und TopBP1

The proto-oncogene c-myc encodes for the transcription factor cMyc, which acti- vates expression of target genes in a heterodimer with Max, and acts as a repressor in a ternary complex with Max and Miz1. In this work I show that the E3 ligase HectH9 interacts with both Miz1 and cMyc. Unlike cMyc, Miz1 is not ubiquitina- ted by HectH9 but acts as an inhibitor of cMyc ubiquitination by HectH9. This polyubiquitination is inter-linked via Lysine 63 and does not lead to increased degradation of cMyc, but alters its biological properties: Depletion of HectH9 by shRNA leads to reduced induction of cMyc target genes, while repression of cMyc target genes is unaltered. These experiments are supported by experiments with Myc KR6, a cMyc mutant which can not be ubiquitinated by HectH9: Myc KR6 fails to induce cell cycle progression and apoptosis in comparison to wild type cMyc in starved 3T3 fibroblasts. One cause for this lies in the inability of Myc KR6 to interact with the histone acetyltransferase p300: While both, wild type cMyc and Myc KR6 bind similarly to Myc target promoters, only wild type cMyc is capable of recruiting p300 which is needed for efficient binding of general transcription factors. TopBP1 is an essential activator of ATR after DNA damage induction by UV-B exposition. Cells that over-express Miz1 show signs of DNA damage and have increased half life times of TopBP1 and ATR. I could show that over-expression of HectH9 reduces the half life of TopBP1 after DNA damage in comparison to control cells and that HectH9 polyubiquitinates TopBP1 via lysine 48 interlinked chains. This reaction is inhibited by over-expression of Miz1. A TopBP1 mutant, which does not bind to Miz1 anymore, is ubiquitinated stronger by HectH9 than wild type TopBP1. Taken together these results show that HectH9 is an important cell cycle inducer by ubiquitinating and activating cMyc and thereby promoting cell cycle entry and in addition to that reducing DNA damage dependent cell cycle arrest by ubiquitinating and degrading TopBP1. These oncogenic properties are apparent in colon carcinomas: Although in normal colon tissue HectH9 is only detectable in 10% of all samples at a low level, it is highly transcribed in 80% of all analyzed colon carcinoma samples.