Determining the role of the SUMO-specific, ubiquitin ligase RNF4

The work here is directly relevant to finding out how cellular components are destroyed. Understanding how this is done is important as it is involved in the regulation of basic cellular processes. The subject of this proposal, RNF4 is a unique enzyme that adds chains of ubiquitin molecules onto proteins already modified with another ubiquitin-like protein known as SUMO. This targets the modified proteins for proteasomal degradation. It is RNF4 that mediates the therapeutic effect of arsenic in treatment of Acute Promyelocytic Leukaemia, by inducing degradation of the oncogenic PML-RAR fusion protein responsible for the disease. Understanding the detailed mechanism of RNF4 action could therefore have important implications for the development of future therapeutic agents. The remarkable efficacy of the proteasome inhibitor Velcade in Multiple Myeloma suggests that more substrate specific inhibition of proteasomal degradation could allow the development of drugs with increased efficacy and reduced side effects. Identification of suitable targets for the development of new drugs requires a detailed knowledge of the molecules involved in protein degradation and their mechanisms of action. The work described here is an attempt to obtain a detailed mechanism for the function of RNF4.

Our objective is to determine the role of the SUMO-specific ubiquitin ligase RNF4 in DNA damage and other stress responses. As RNF4 contains multiple SUMO Interaction Motifs (SIMs) and a RING domain our hypothesis is that RNF4 targets proteins bearing poly-SUMO chains for ubiquitination and subsequent degradation via the proteasome. To test this hypothesis and establish the biological pathways that RNF4 participates in, we will ablate RNF4 expression either by siRNA in human cells or by gene knock out in chicken DT40 cells, and determine the phenotypes of the RNF4 depleted cells. As the yeast homologues of RNF4 have a role in DNA damage responses and RNF4 can complement the yeast mutants it is likely that vertebrate RNF4 will have a role in DNA damage responses. We will therefore expose the wild type or RNF4 depleted cells to a variety of genotoxic insults and test a range of DNA damage responses. This should reveal the steps in the DNA damage response that are subject to RNF4 dependent regulation. Quantitative proteomics will be used to identify the cellular substrates targeted for ubiquitination by RNF4. Bioinformatic analysis of the data will reveal the additional biological processes that RNF4 participates in. This will be confirmed by further siRNA analysis. In Acute Promyelocytic Leukaemia the PML gene is fused to the retinoic acid receptor (RAR) gene resulting in the generation of a PML-RAR fusion protein which acts as a potent transcriptional repressor, blocking differentiation and allowing continued proliferation of the leukaemic cells. SUMO modification of PML-RAR is required to maintain transcriptional repression and for the development of disease. Treatment of patients with arsenic is an effective therapy for APL and the arsenic achieves this by inducing degradation of PML. Our preliminary evidence indicates that this is mediated by RNF4. Thus we will seek to prove that RNF4 is recruited to SUMO modified PML after arsenic treatment and that once bound it ubiquitinates PML-RAR and targets it for proteasomal degradation. We will study this process in vivo using siRNA ablation of RNF4 expression and in vitro using well defined recombinant modification components and SUMO modified PML substrates.