download report - Istituto Pasteur

P. Ballario - Molecular machines and effectors involved in the regulation of light response and cell cycle in simple Eukaryotes
the paper by Vernarecci et al. (2008), we report that
Gcn5 is physically associated to the centromere and
genetically interacts with several components of
the kinetochore, controlling chromosomes segregation
during cell cycle. Based on these results we
want now to identify the molecular mecchanism
used by Gcn5 for the regulation of structure and
function of the centromeric region/kinetochore.
Next steps will be: i) mapping of genetic interactions
among Gcn5 and centromere/kinetochore
elements, ii) analysis of the level of Gcn5 control
(transcriptional or post translational), iii) validation
of the model emerging for yeast cells in human culture
cells, iv) clarification of the role of Gcn5 in
the activation of the mitotic chekpoint associated
to the spindle.
B) In 2006 (Grimaldi et al., Mol Biol Cell) we demonstrated
the important role of the Neurospora NGF-1
HAT (the homologous of S. cerevisiae Gcn5) in the
transcriptional activation of light regulated genes.
In particular we have observed the transient acetylation
of histone H3 residue K14 in the promoter
regions of light regulated genes (i.e. Al3 and Vvd). In
the last two years we have focused our investigation
on the process of reversible acetylation of the main
protein involved in light transduction: White
Collar1. We have demonstrated an in vitro interaction
between WC-1 and ScGcn5, and also, by ChIP
analysis, that WC-1 is acetylated in vivo both in dark
and in light conditions of growth. However the in
vivo acetylation of WC-1 does not seem to be NGF-
1 dependent because the protein is acetylated also in
an ngf-1 mutant strain. The in vitro acetylation
assays suggest that WC-1 contains an acetyltransferase
activity (in prep.). Now we will search the
other proteins present in the WC-1WC-2Gcn5 complex
by Maldi-Toff and two hybrid analyses.
32
C) We are also interested in comparing our data on
light transduction in Neurospora (WCC white collar
complex) with the light system utilized by Tuber
borchii a filamentous fungus, characterized by an
hypogeous style of life. In the period 2005-2006 we
isolated WC-1 and WC-2 from T. borchii and characterized
light responses in the mycelium but we need
some molecular-genetic tools like transformation
and homologous recombination. We therefore set up
the protocol for Tuber borchii transformation based
on Agrobacterium tumefaciens binary vector system.
We are also participating to an italian-french consortium
for Tuber melanosporum genome sequencing,
annotating genes involved in light transduction
pathways. Several interesting data have been
obtained. We have found the entire velvet system of
red light transduction (previously characterized in A.
nidulans) in T. melanosporum, this finding was quite
unexpected, in fact, characterizing the action spectra
of Tuber spp no red light response has been observed
however the level of response to white and blue light
is not the same in term of mycelia growth. This suggest
the existence of a blue-red cross talk.
Selected publications
Bizzarri B, Chimenti F, Bolasco A, Maccioni E,
Chimenti P, Fioravanti R, Secci D, Ballario P,
Vernarecci S, Filetici P. A novel histone acetyltransferase
inhibitor modulating Gcn5 network:
cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2yl)hydrazone.
J Med Chem. 2008, Epub.
Vernarecci S, Ornaghi P, Bâgu A, Cundari E,
Ballario P, Filetici P. Gcn5p plays an important role
in centromere kinetochore function in budding yeast.
Mol Cell Biol. 2008, 28:988-96.