Molecular and Cell Biology of the (Epi)genome
Although the nucleus is the hallmark of eukaryotic cells, we know
remarkably little about its composition, structure or function.
Our goal is to elucidate the principles and functional consequences
of the dynamic organization of the nucleus by understanding how
factors are recruited to or excluded from their sites of action.
We focus on how genetic and epigenetic information is replicated
at every cell division and how it is "translated" during
development into different gene expression programs, which define
specific cell types and functions. Elucidation of mechanisms maintaining
or reprogramming the epigenome will lead to new approaches in
disease prevention and regenerative medicine.
|Law and order in the nucleus.
The graphic illustrates several of the different compartments
identified in the nucleus with some of their functions highlighted
and their relation to interphase chromatin organization.
DGZ Forschungsprofile 2004
Duplicating the (epi)genome
DNA replication is a central event
of the cell division cycle and is linked to cell cycle regulation
and the cellular response to DNA damage in many ways. The precise
and coordinated duplication of genetic information is critical
for genome stability and errors in DNA replication may trigger
or promote cancer progression.
Replication of the mammalian genome starts at tens of thousands
of origins that are activated at specific times during S phase.
The spatio-temporal progression of DNA replication is inherited
through consecutive cell division cycles, raising the question
how this replication program is coordinated. We are studying the
coordination of the multiple enzymatic activities involved in
the replication of the genome preceding every mitotic division.
With fluorescent fusion proteins and high-resolution multidimensional
time-lapse microscopy, we showed that replication patterns within
the nucleus change in a characteristic manner throughout S phase.
In addition, these studies have yielded a precise and direct way
to identify cell cycle stages in situ, which opens up new experimental
approaches to study cell cycle-dependent processes and protein
dynamics in living cells.
Cell cycle progression
Chromatin is visualized in living cells with fluorescent
histones (labeled in green) and its duplication is visualized
with fluorescent DNA polymerase clamp PCNA (labeled in red)
and fluorescent DNA methyltransferase 1 (labeled in yellow).
The cell cycle dependent changes of both genome and genome
duplicating machinery are depicted. Furthermore, the identification
of each cell cycle stage and their subdivision is possible
in real time.
We are further investigating
the temporal and spatial dynamics of the replication machinery
components in living mammalian cells by a combination of
biochemical in situ fractionations and fluorescence photobleaching/activation
techniques. Our results suggest that processivity and fidelity
of this complex enzymatic machinery is not achieved by stable
interactions between its components.
data is rather consistent with the existence of a stable
core in vivo composed of the PCNA clamp (proliferating cell
nuclear antigen) bound to the replicating DNA while other
factors transiently associate with this core. We are currently
extending this hypothesis by measuring the kinetics of the
various replication factors and their response to challenges
during genome replication.
In parallel, we are trying to
dissect the mechanisms that control the ordered activation
of replication origins and thus determine the replication
program. A careful examination of the temporal and spatial
assembly of new replication sites indicated that they assembled
at adjacent positions suggesting that activation of neighboring
replication origins may occur by a “domino effect”
possibly involving local changes in chromatin structure
of genome replication.
Spatiotemporal changes of DNA replication were followed
with a GFP-PCNA fusion protein by time lapse microscopy.
The replication machinery starts at multiple sites (green)
and over time it "spreads" in a domino-like manner
activating adjacent sites (red) until all the genome is
from Molecular Cell, Vol. 10, Sporbert et al., 1355-1365,
Copyright (2002) with permission from Elsevier".
Finally, we are analyzing links between
DNA replication and repair, in particular the recruitment of DNA
synthesis factors to DNA damage sites and the consequences for
cell cycle progression and genome stability.
and translation of epigenetic information.
A virtual trip through the nucleus of a living mouse myoblast
cell. Fluorescently tagged proteins highlight sites of DNA
replication (red) and of highly methylated satellite DNA
bound by MeCP2 (green).
needs to be "translated" to define specific cell
types with specific sets of active and inactive genes, collectively
called the epigenome. An example of such a translating activity
is the family of methyl-cytosine binding proteins (MeCP2,
MBD1-4), that recognize and bind to sites of DNA methylation
and then recruit other chromatin modifiers such as histone
MeCP2, the founding member of the
MBD family, is mutated in most Rett syndrome patients, which is
the second most common neurological disorder after Down syndrome.
We have found that MBDs induce large-scale
heterochromatin reorganization during terminal differentiation.
Based on this finding, we are currently dissecting the mechanisms
responsible for this chromatin reorganization by a combination
of in vitro and in vivo approaches including biochemical and photodynamic
assays. Furthermore, we are testing the molecular composition
and role of these heterochromatin compartments in genome expression/silencing
during differentiation and in disease.
This should help to elucidate the role of genome topology in cellular
differentiation, and provide new ways to manipulate the phenotypic
plasticity of cells for application in cell replacement therapies
in regenerative medicine.
Methyl CpG binding proteins
induce clustering of heterochromatin during differentiation.
A 3D view through the nucleus (contour in red) of a living mouse
myoblast cell (left) showing multiple heterochromatin centers
(chromocenters, in green). Increasing the level of MeCP family
members during terminal differentiation (myotubes, right) results
in a large scale reorganization and clustering of the highly methylated
satellite DNA at chromocenters.
Accessing the (epi)genome
Nuclear DNA is organized together
with structural proteins into dynamic higher order chromatin structures,
which reflect and control gene expression during the cell division
cycle and cellular differentiation. Chromatin can be subdivided
into eu- and heterochromatin, depending on its condensation state,
transcriptional activity and the modification of associated chromatin
organizing proteins,. Whereas euchromatin is generally assumed
to be actively transcribed and less condensed, heterochromatin
condensation is thought to be similar to mitotic chromosomes in
which DNA metabolism, (e.g. transcription and replication) has
stopped. It is unclear whether and how changes in the chromatin
compaction state affect the mobility of chromatin organizing proteins
and the access of proteins to chromatin. To address these questions,
we are using a combination of live-cell chromatin labels and high-speed
single molecule tracing microscopy as well as photodynamic assays
in living mammalian cells. In parallel, we are evaluating and
exploiting novel non-invasive methods to introduce molecules into
cells directly via peptide transducing domains that can cross
cellular membranes. These techniques will allow us to compare
the accessibility and mobility of proteins in different subnuclear
compartments. In particular, we are interested in elucidating
the physico-chemical principles regulating genome accessibility
and, thus, controlling nuclear metabolism.
accessibility at the single molecule level.
Fluorescent streptavidin was used as a probe protein to
test the accessibility of constitutive heterochromatin marked
with fluorescent MeCP2 (labeled in dark grey). After injection
streptavidin into the cytoplasm (surrounding light grey
areas), trajectories of single streptavidin molecules were
recorded by high speed fluorescence microscopy (191 Hz).
Tracks were overlaid on the reference heterochromatin image
and color coded. The blue tracks represent particles moving
within the cytoplasm, green ones within MeCP2-labeled heterochromatin,
red indicate particles crossing the heterochromatin borders
and yellow particles moving elsewhere in the nucleoplasm.
This data reveal that physical accessibility per se should
not control genome metabolism but rather local concentration
of reactants and binding sites should play a role.