Research

 

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 markers.

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.

 

 

Our 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 and accessibility.

 

Propagation 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 duplicated.

"Reprinted 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.

 

Replication 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).

 

Epigenetic information 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 deacetylase complexes.

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.

 

Probing (epi)genome 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.

 

 

 
     
  Cardoso Lab, Technische Universität Darmstadt Back to TUD