Lampbrush chromosomes enable study of cohesin dynamics

Abstract The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organi-zation and plasticity exhibited by lampbrush chromo-somes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/ SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromo-somal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a tech-nique for de novo chromosome assembly, we demon-strate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lamp-brush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes. Keywords lampbrush chromosome. chromatin. cohesin. SMC. oocyte nucleus Abbreviations EGFP enhanced green fluorescent protein FRAP fluorescence recovery after photobleaching LBC lampbrush chromosome SMC structural maintenance of chromosomes RNP ribonucleoprotein RNAPII RNA polymerase II YFP yellow fluorescent protei