Analyzing large-scale chromatin fiber via studying cytology of DNA replication and in vivo immunogold labeling

The structure of interphase chromosomes remains elusive after decades of research by using chemical, physical and biological methods. Although large-scale interphase chromatin has been observed repeatedly, its high-order compaction level is hard to reconcile with its active biological functions, like replication and transcription. Decondensation of such large-scale chromosome structure is intuitively expected to be a prerequisite for biological factors to be able to access chromosomes and fulfil biological function. On the other hand, a “melting polymer” model, based on chemical evidence, has been proposed to explain the interphase chromatin structure and behavior. Furthermore, the interphase chromosome is considered as grouped 10 nm fibers without specific higher order folding motif, thus decondensation is not required for interphase chromosome to perform biological function. In this study, the structure of large-scale interphase chromatin fiber was analyzed by several new methods. A cell line with tagged chromosome regions and replication foci were used to investigate the behavior of large-scale chromatin fiber during replication. A novel pulse-chase condition was applied to track the structural change of newly replicated DNA. Minuscule sized nanobody was labeled with nanogold particle via an innovated labeling method, and then integrated into several in vivo loading techniques to stain specific nuclear targets in live cells, aiming at revealing the detail of interphase chromosome structure. Cytology of replication showed dynamic plasticity existed in large-scale structure of interphase chromosome, which allows the highly compact chromatin fiber to replicate without predecondensation. The potentials of new in vivo immunogold labeling techniques suggested the possibility of studying chromosome structure with specificity by electron microscopy