Български
Research is sponsored fully by the Bulgarian Science Fund, Grant number DMU 02/8
Summary:
Chromatin structure and dynamics
are prerequisite for the processes of cellular transformation and cancer
propagation. Nevertheless, these changes are still poorly understood.
Saccharomyces cerevisiae has long been considered as a model organism in all kinds
of molecular biology studies. The only homologue of the linker histones in this
organism is Hho1p, which possesses some unique characteristics in comparison to
the linker histones of higher eukaryotic cells. On one hand, this protein is
coded by one gene copy and on the other its molecule has two globular domains
and no carboxyl – end in contrast to the other linker histones. The most
doubtful and yet unproven remain its roles in the chromatin organization and in
the regulation of gene expression. In the current project proposal we intend to
utilize modern molecular biological and biophysical methods (including
Chromatin Yeast Comet assay, AFM (Atomic Force Microscope), ChIP
(Chromatin-Immunoprecipation) and FRAP (Fluorescent Recovery After Photo bleaching)
as well as standard biochemical methods for chromatin studies. The aim is to
study in details the role of Hho1p in the higher-order structuring of yeast
chromatin and its dynamics in the nucleus under normal and stress conditions.
For that reason we intend to delete the gene HHO1 in three different S.
cerevisiae strains and to study the phenotype of these mutants by comparing
them with their isogenic wild type strains. Additionally, we intend to probe
for a relationship between Hho1p and some of the chromatin – remodeling
complexes. Therefore, we plan to create double S. cerevisiae mutants comprising
a deleted gene for the linker histone and point mutations in Act3p/Arp4 (an
important subunit of INO80, SWR1 and NuA4 chromatin modifying complexes). The idea
behind is to thoroughly investigate these mutants with the above mentioned
methods in search for alterations in the chromatin compaction as well as in the
cellular morphology. On the basis of the expected results we could develop a
model, describing the interaction between Hho1p and the chromatin remodeling
complexes in yeast cells. Such a model could be easily applied to higher
eukaryotic cells, where implications in the processes of chromatin remodeling
and gene expression lead to abnormality and malignant transformation.
Project Objectives:
1. Scientific hypothesis:
The
main hypothesis that set off the idea of this grant proposal relies on the
above-mentioned scientific data about Hho1p but also on the previous experience
of the project team. At first place, it should be outlined that the current
grant proposal is a kind of continuation of Milena Georgieva’s PhD thesis in
which it was shown that the yeast linker histone has an influence on the
accessibility of chromatin toward nuclease action, as well as on the ability of
cells to overcome temperature stress. It was also demonstrated that yeast cells
with a single point mutation in the gene for Act3p/Arp4 exhibit serious
alterations in the cellular morphology and changes in chromatin structure. The
created double mutant act3/arp4ts26 delta
hho1p during the time of the experiments in the thesis and the following
studies on it led to the identification of a cellular morphology of these cells
pretty much alike the cellular morphology of the deltahho1 mutant cells. Moreover, the ability of double mutant
cells to overcome temperature stress was similar to the ability of deltahho1 cells in contrast to act3/arp4 single mutants. These preliminary
results of the characterization of yeast cells with altered processes of chromatin
– remodeling (i.e. act3/arp4 mutants)
and without a linker histone (deltahho1p)
prompt interesting compensatory mechanisms in the double mutant cells caused by
the deletion of the gene for the linker histone. Accordingly, we decided to aim
these prompted compensatory mechanisms in the current grant proposal and search
for the linker histone contribution to the higher-order chromatin organization
and the linker histone participation in the remodeling of chromatin structure.
Such functional interplays among chromatin structural and functional proteins
are very important and could reveal crucial regulatory mechanisms for the cell
fate and the development of the whole organism.
Research is sponsored fully by the Bulgarian Science Fund, Grant number DMU 02/8.
