Characterizing Transcription and Splicing Kinetics by 3D Orbital Tracking

Abstract

Chromosomes segregate into discrete regions of the nucleus during interphase, which are referred to as ''chromosome territories''; however, the level of independence (or conversely, the level of interaction) between the territories is vigorously debated. Chromosome conformation and capture techniques, such as Hi-C, can provide detailed information on the organization of the genome. From an analysis of Hi-C data, we have found that the chromosomes of both mouse and human embryonic stem (ES) cells tend to interact much less than the chromosomes of differentiated cells. DNA FISH experiments with chromosome paints support the Hi-C data showing that chromosome territories in ES cells tend to be farther apart; in fact, a global decrease in inter-chromosomal interactions correlated with an increase in average nuclear size. Surprisingly, the primary transcriptional hardware, RNAPII, did not show clear organizational changes upon cellular differentiation. Direct stochastic optical reconstruction (dSTORM) microscopy in both ES and differentiated cell nuclei showed that RNAPII maintain a constant density and level of clustering. Our data reveal a structural difference in genome organisation between ES cells and differentiated cells and suggest that the genome undergoes a fundamental reorganization after cellular differentiation.