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

English: Mutation studies have shown that the nucleosome surface is extremely important for proper DNA replication, transcription, chromatin remodelling and thus correct gene expression. Therefore it is expected that these surfaces of the nucleosome would bind some of the arsenal of regulatory proteins available in the cell. Despite the obvious functional importance of the nucleosome cores, little is known with regards to possible binding partners. A few interactions with the nucleosome surface has been studied. Silent Information Regulator protein 3 (SIR3) is grouped in a family of chromatin regulators thought to facilitate transcriptional silencing via compaction of chromatin. Two viral proteins have also been shown 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-associated herpesvirus and the 72kDa immediate early 1 (E1) protein of human cytomegalovirus. The nucleosome core surfaces are thus also assumed to play key roles in infectious cycles of these and possibly more viruses. Other known binding partners include members of the high mobility group (HMGN) proteins and regulator of chromosome condensation 1 (RCC1). These proteins 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 our understanding of the regulatory mechanisms of chromatin which will aid in understanding the onset and progression of many diseased states. This study aimed to identify proteins which bind to the non-tail parts of the nucleosome to further our understanding of the regulation of DNA 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 were reconstituted with a biotinylated DNA-fragment to pull down any proteins bound to the histone octamers following incubation with a nuclear extract from Saccharomyces cerevisiae. The resulting proteins were identified when analysed by nanoLC-MS/MS and searched against the SwissProt database using Mascot. In total 25 nuclear proteins were identified to bind exclusively to the globular domain of the nucleosomal core particle. The results obtained provide a good starting point for further computer simulation studies as well as directed approaches to study specific interactions in vivo.