Cell nuclei are highly organized structures. In diploid mammalian cells, some 6x109 base pairs of DNA are folded so as to occupy a nucleus measuring roughly 10 µm across. To achieve this, DNA, as a nucleoprotein complex (i.e., chromatin), must fold into a series of higher-order arrays. The nucleus also contains the machinery required for transcription of genes and processing of RNA products, the precise replication of the genome once per cell cycle and the processes of recombination and repair. This general complexity of the nuclear morphology is obvious; however, less clear are the mechanisms forming the threedimensional structure of the nucleus, in spite of the fact that recent evidence points more and more to the higher-order gene structure as an important, if not a key, determnant of activity. We concentrate therefore on a multi-disciplinary approach that allows us to study nuclear activities in relation to the higher-order nuclear structures, e.g. to the nucleoskeleton.
Current main research projects:
- Molecular anatomy of structures directing nuclear compartmentalization
- Nuclear compartmentalization of nuclear DNA helicase II
- Characterization of nuclear myosin I - recently discovered molecular motor
- Epigenetic gene-position effects in gene expression - role of nuclear structure

      Electron microscopic identification of replicating structures in S-phase cell nuclei and a description of the replication dynamics in these structures suggested an alternative model for replication with an enzymatic complex fixed to the nucleoskeleton while the DNA moves through. As a result of the ultrastructural localization of nucleolar transcription in the so-called "dense fibrillar component" (DFC) and at the border between DFC and another nucleolar component, the "fibrillar centres" (FC), a novel model for transcription of ribosomal genes was formulated. We imagine that the template - and not the polymerase - moves, pulled through fixed polymerization complexes as nascent transcripts are extruded.

Fig. 1: Old (A-C) and new (D-F) models for DNA replication. The alternative model involves passage of the template through a fixed complex and extrusion of nascent DNA.

      We also found that cell nuclei contain a nucleoskeleton that is a permanent structure regardless of their transcriptional activity. Lamins form an internal nuclear skeleton and not only the nuclear lamina, as previously thought. Centromeres, telomeres, and ribosomal DNA vary in their anchoring to intranuclear structures and therefore contribute in various ways to the maintenance of nuclear architecture.

      Helicases are proteins involved in maintaining genomic stability and in cellular safeguard mechanisms. We found recently that nuclear DNA helicase II is a component of PML (promyelocytic leukemia) nuclear bodies, and its distribution in the nucleus is dependent upon the physiological status of the cell. In this project we investigate the mechanisms and physiological role of NDH II distribution in the nucleus, identify the genes transcribed at PML nuclear bodies after interferon-stimulation, and question the function of NDH II and PML nuclear bodies in gene expression and cellular senescence.

Fig. 2: A model for transcription of ribosomal genes.

Fig. 3: The distribution of nuclear DNA helicase II dramatically changes upon transcription inhibition.

      Cell nuclei contain the newly discovered specific myosin I, which can form together with nuclear actin a molecular motor. Studies investigating the role of actin and myosin in the transcription of ribosomal and protein-coding genes are currently in progress. Results obtained together with the laboratory of Prof. deLanerolle in Chicago show that both actin and myosin I are required for transcription, possibly providing the driving force necessary for the movement of the DNA template relative to the transcription complex.

Fig. 4: Both actin (green dots) and myosin (red dots) are present in nuclei, sometimes colocalizing.

      The relationship between gene imprinting and the spatial organization of gene transcription in the nucleus is currently investigated. The relative position of active and imprinted (inhibited) genes in relation to the chromosomal axis and the intechromosomal space is followed by in situ hybridization studies and laser confocal microscopy.