of the Max - MDC

Molecular and Cell Biology
of the (Epi)genome
M. Cristina Cardoso
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.
Duplicating the (epi)genome
(C. S. Casas Delucchi, P. Domaing, M. Fillies, S. M.
Görisch, S. Haase, D. Nowak, J. H. Stear)
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 timelapse
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 cycledependent
processes and protein dynamics in living cells.
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
Figure 1. Dynamics of the (epi)genome and its duplication.
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). 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.
Cardiovascular and Metabolic Disease Research 43