Higher-order nuclear organization

Beyound any doubt, there are different levels of order within the cell nucleus (Haaf and Schmid 1991). Although the nucleus has no membrane-bound compartments, all basic nuclear processes such as transcription, transcript-processing and transport through the nucleoplasm, chromatin replication, and DNA recombination and repair (see below) seem to be confined to particular intranuclear locations (Haaf et al. 1995). The interphase arrangement of chromosomes is highly defined (cell-type specific), but changes dramatically in the cell cycle, during differentiation, or with the pathophysiological state of the cell. Higher-order nuclear architecture is thought to provide a structural framework that may affect the accessibility of nuclear genes to regulatory factors and, thus, may serve an essential role in the regulation of gene expression beyond that at the single-gene level. We use FISH and other immunofluorescence techniques to simultaneously visualize specific chromosomal regions and functional nuclear domains and to elucidate the relationship between specific chromosome arrangements and nuclear functions. In particular, we are interested in the extent to which changes in higher-order nuclear organization are implicated in the etiopathogenesis of human genetic disease (Stout et al. 1999, Riesselmann and Haaf 1999).

Data from classical genetics, nuclear transplantation experiments and human imprinting disorders suggest that normal mammalian development requires the participation of both a maternal and a paternal genome. The one-cell embryo is formed by two very different sets of transcriptionally quiescent chromatin: the highly methylated sperm nucleoprotamine, which arrives in almost crystalline structure, and the relatively undermethylated nucleosomal egg chromatin. During preimplantation development, these different gametic marks are largely reset into their functional forms. Until now, the general belief was that the paternal and the maternal chromosome complements are mixed together to form the zygotic genome after the breakdown of the pronuclear envelope during its progression through the first metaphase. We have developed different techniques which allow us to distinguish paternal and maternal chromosomes (Hardt et al. 1999). Mouse eggs fertilized with BrdU-labeled sperm and detection of BrdU-labeled sperm-DNA strands in embryos of the next generation, as well as differential heterochromatin staining in mouse interspecific hybrids are used to study the behaviour of paternal and maternal during early mammalian development (Mayer et al 2000a). Compartmentalization of the nucleus according to the parental origin may make it easier for the cellular machinery of the fertilized egg to reprogram and revive the paternal chromosomes and to control maternal gene expression. Active demethylation of the paternal zygotic genome (Mayer et al. 2000b) has important implications for genomic imprinting and the establishment of transcriptional totipotency in fertilized eggs and somatic nuclei during mammalian cloning. Epigenetic reprogramming defects in the early diploid embryo may also be responsible for spontaneous abortions during normal development and the loss of embryos after in vitro fertilization. Epigenetic phenomena, i.e. global methylation and acetylation patterns, are studied by the use of specific antibodies.