107 CHANGES OF HIGHER ORDER CHROMATIN ARRANGEMENTS CORRELATE WITH GENOME ACTIVATION DURING EARLY BOVINE DEVELOPMENT

Abstract

In interphase nuclei of higher eukaryotes, the genome is organized within discrete chromosome territories (CT). These CT are arranged in a nonrandom fashion depending on cell type, species, and cell cycle stage. Gene-rich CT locate more internally in the nucleus, whereas gene-poor ones are found more peripherally. So far, CT have been investigated mainly in cultured cells and to some extent in tissues, yet little is known about the origin and fate of CT during early development. To gain insights into the very early organization of CT in embryos, we developed a fluorescence in situ hybridization protocol that yields optimal results with 3 dimensionally preserved bovine embryos. Using this protocol, we investigated CT of bovine chromosome 19 and 20. These chromosomes represent the gene-richest and gene-poorest chromosomes, according to the number of annotated genes at the Ensembl database (www.ensembl.org). We could confirm a gene density-related radial nuclear distribution in bovine fibroblasts and lymphocytes with CT 19 being more interior than CT 20. Because cell cycle-dependent effects were not expected in view of published data in other species, we did not consider the cell cycle stage of the investigated specimen. In early bovine embryos generated by in vitro fertilization, we demonstrated the existence of nonoverlapping CT already in zygotic pronuclei. In day 3 embryos, but interestingly not in day 2 embryos, a gene density-related radial distribution of CT 19 and 20 was observed. Because these stages represent a transition stage during which the embryonic genome is activated, our results indicate a correlation between genome activation and changes of higher order nuclear organization. We are currently investigating the question whether transcription is a prerequisite for the establishment of a gene density-related chromosome positioning, as well as how reprogramming after nuclear transfer affects CT distribution of somatic donor nuclei.

The present work is supported by the Deutsche Forschungsgemeinschaft (ZA 425/1).