Dynamic organization of chromosomes in the mammalian cell nucleus

Uncovering the motifs of a higher order nuclear architecture and
its implications on nuclear function has raised increasing interest
in the past decade. The nucleus of higher eukaryotes is considered
to display a highly dynamic interaction of DNA and protein factors.
There is an emerging view that there are hierarchical levels of
gene regulation, reaching from epigenetic modifications at the DNA-
and histone level to a higher order functional nuclear topology, in
the context of which gene-activating and -repressing processes
influence the gene expression profile of an individual cell beyond
the sequence information of the DNA. The present work focuses on
the analysis of the dynamic aspects of higher order nuclear
architecture in living cells. As a prerequisite, an in vivo
replication labeling strategy was developed, that enabled the
simultaneous visualization of early and mid-to-late replicating
chromatin as well as single chromosome territories on the basis of
a labeling/segregation approach. The presented scratch replication
labeling protocol combines a high labeling efficiency with reduced
“damaging” effects and can be successfully applied to a number of
adherently growing cell lines, including primary human fibroblasts.
In addition, a live cell observation system was developed that
facilitates time-lapse confocal (4D) microscopy over elongated time
periods which made it possible to follow a complete cell cycle or
more. To address possible long-range movements of chromosome
territories (CTs) during an entire interphase, fluorescence
labeling of a small number of CTs was performed in living HeLa
cells stably expressing histone H2B-GFP. This was achieved by in
vivo scratch replication labeling with fluorescent nucleotides.
Labeled cells were cultivated for several cell cycles until labeled
chromatids had segregated. Such cells were followed by time-lapse
confocal microscopy over time-scales of up to 20 hours covering
major parts or the complete cell cycle. Positional changes of the
intensity gravity centers of labeled CTs in the order of several µm
were observed in early G1, thereafter, the positions remained
within a range of ~1 µm till the end of G2. In conclusion, CT
arrangements were highly constrained from mid G1 to late G2 / early
prophase, whereas major changes of CT neighborhoods occurred from
one cell cycle to the next. More extended movements observed in
early G1 might play a role when CTs “home in” to establish a
non-random radial CT arrangement. To analyze possible changes of
chromosome arrangements from one cell cycle to the next, nuclei
were photobleached in G2 maintaining a contiguous zone of
unbleached chromatin at one nuclear pole. This zone was stably
preserved until the onset of prophase whereas unbleached chromosome
segments were often observed to become located at distant sites in
the metaphase plates. Accordingly, chromatin patterns observed in
daughter nuclei differed significantly from the mother cell
nucleus, indicating that CT neighborhoods were not preserved during
mitosis. The variability of CT neighborhoods during clonal growth
was further confirmed by 3D-FISH experiments. A series of
experiments of a more preliminary character looked at the influence
of the nuclear lamina in constraining and determining a higher
order nuclear architecture by selectively interacting with
mid-to-late replicating chromatin. Simultaneous immunodetection of
lamin B on two-color replication labeled neuroblastoma cell nuclei
revealed specific attachment of the mid-to-late replicating
chromatin compartment not only along the periphery but also inside
the nucleus along invaginations of the lamina. 4D-live cell
observation of lamin C-GFP expressing CHO cells with mid-to-late
replicating chromatin labeled simultaneously revealed concomitant
movements of replication foci attached to lamin invaginations.
Moreover, a functional essay was employed which uses injection of a
dominant negative lamin A mutant protein (ΔNLA) to cause a
reversible disruption of the nuclear lamina. Initial results point
to concomitant distortion of the mid-to-late replication pattern
and a preferential attachment of the respective chromatin sites to
partially disrupted lamin B (as compared to lamin A and nuclear
pore complex). Finally, a model is presented on the chromosome
positioning in mammalian nuclei depending on cell cycle and nuclear
shape.