Identification of Saccharomyces cerevisiae proteins that bind to nucleosomes at positions other than the N-terminal histone tails

[摘要] English: Mutation studies have shown that the nucleosome surface is extremely important for properDNA replication, transcription, chromatin remodelling and thus correct gene expression.Therefore it is expected that these surfaces of the nucleosome would bind some of thearsenal of regulatory proteins available in the cell. Despite the obvious functional importanceof the nucleosome cores, little is known with regards to possible binding partners.A few interactions with the nucleosome surface has been studied. Silent InformationRegulator protein 3 (SIR3) is grouped in a family of chromatin regulators thought to facilitatetranscriptional silencing via compaction of chromatin. Two viral proteins have also beenshown to interact with chromatin by binding the acidic pocket formed by the H2A-H2B dimer.These are the latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associatedherpesvirus and the 72kDa immediate early 1 (E1) protein of human cytomegalovirus. Thenucleosome core surfaces are thus also assumed to play key roles in infectious cycles ofthese and possibly more viruses. Other known binding partners include members of the highmobility group (HMGN) proteins and regulator of chromosome condensation 1 (RCC1). Theseproteins play vital roles in the regulation of chromatin structure and thus DNA accessibility.By identifying the binding partners of the nucleosome cores we will advance ourunderstanding of the regulatory mechanisms of chromatin which will aid in understanding theonset and progression of many diseased states. This study aimed to identify proteins whichbind to the non-tail parts of the nucleosome to further our understanding of the regulation ofDNA function.To this end Xenopus laevis histones were expressed in Escherichia coli, both as full-length,canonical histones as well as N-terminally truncated globular domains. These werereconstituted with a biotinylated DNA-fragment to pull down any proteins bound to the histoneoctamers following incubation with a nuclear extract from Saccharomyces cerevisiae. Theresulting proteins were identified when analysed by nanoLC-MS/MS and searched against theSwissProt database using Mascot. In total 25 nuclear proteins were identified to bindexclusively to the globular domain of the nucleosomal core particle.The results obtained provide a good starting point for further computer simulation studies aswell as directed approaches to study specific interactions in vivo.

[发布机构] University of the Free State