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