Heat Shock alters the distribution andin vivointeraction of major nuclear structural proteins – Lamin B with DNA Topoisomerase II, and with nucleic acids

Abstract

ABSTRACTLamins and topoisomerases have been studied as major karyoskeletal proteins since early 80-ties. Today, lamins are thought to be responsible not only for maintenance and structural support of cell nuclei but also involved, directly or indirectly, in regulation of chromatin structure, chromatin distribution, gene expression, splicing, transcription and transport. Topoisomerases are involved in maintenance of chromosomal scaffold and structural functions in cell nuclei and topology of chromatin and DNA, and play an important role in regulation of transcription. They are also necessary in mitosis. For studying lamins and topoisomerase II,Drosophila melanogastermodel has been used for many years since flies genome contains only single gene coding for B-type lamins (lamin Dm) and single gene coding for A-type lamins (lamin C). Flies have only single gene coding for topoisomerase II (Top2).We previously reported that lamin Dm (Lam Dm) and topoisomerase II (Top2) bindin vivoboth DNA and RNA and the properties of both proteins have been modulated by specific phosphorylation on particular sites. Here we report the first part of results of the project focused on the demonstration of the role of lamins and Top2 in the regulation of gene expression and chromatin organization upon heat shock (HS) induction and recovery (R). We demonstrated that heat shock significantly induced specific phosphorylation of lamin Dm at least on S25, which affects lamin properties and distribution. Lam Dm and Top2 were relocated and changed properties including solubility. Both proteins interact with each other directly and indirectly and binding was significantly increased under HS. The relocation of Lam Dm and Top2 was associated with the relocation of chromatin as detected in polyploid third instar larvae nuclei.In vivophotocrosslinking and IP (immunoprecipitation) studies indicated a significant increase in binding to chromatin and nucleic acids upon HS induction. The highest binding affinity showed a soluble fraction of lamin Dm and topoisomerase II while the lowest was the insoluble fraction (“nuclear matrix” fraction). All the detected changes in properties and location of proteins returned to “normal” after recovery from heat shock. Based on thi s data and our preliminary interactome data for lamin Dm and Top2 we believe that both proteins play essential roles in the proper response of fly cells to HS by participation in the rearrangement of protein complexes, also associated with DNA and RNA, chromatin remodelling and proper gene expression regulation.