dictyNews
Electronic Edition
Volume 38, number 14
May 25, 2012
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Abstracts
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Unusual combinatorial involvement of poly-A/T tracts in organizing
genes and chromatin in Dictyostelium
Gue Su Chang1, Angelika A Noegel3, Travis N Mavrich1, Rolf Müller3,
Lynn Tomsho1, Elissa Ward1, Marius Felder4, Cizhong Jiang1,6,
Ludwig Eichinger5, Gernot Glöckner2, Stephan Schuster1,
B. Franklin Pugh1#
Genome Res. 2012, published online before print.
Dictyostelium discoideum is an amoebozoa that exists in both a free-living
unicellular and a multi-cellular form. It is situated in a deep branch in the
evolutionary tree, and is particularly noteworthy in having a very A/T-rich
genome. Dictyostelium provides an ideal system to examine the extreme
to which nucleotide bias may be employed in organizing promoters, genes,
and nucleosomes across a genome. We find that Dictyostelium genes are
demarcated precisely at their 5’ ends by poly-T tracts and precisely at their
3’ ends by poly-A tracts. These tracts are also associated with
nucleosome-free regions, and are embedded with precisely positioned TATA
boxes. Homo- and heteropolymeric tracts of A and T demarcate nucleosome
border regions. Together these findings reveal the presence of a variety of
functionally distinct polymeric A/T elements. Strikingly, Dictyostelium
chromatin may be organized in di-nucleosome units, but is otherwise
organized as in animals. This includes a +1 nucleosome in a position that
predicts the presence of a paused RNA polymerase II. Indeed, we find a
strong phylogenetic relationship between the presence of the NELF pausing
factor and positioning of the +1 nucleosome. Pausing and +1 nucleosome
positioning may have co-evolved in animals.
Submitted by Gue Su Chang [[log in to unmask]]
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AprA functions as an endogenous chemorepellant in Dictyostelium discoideum
Jonathan E. Phillips and Richard H. Gomer
PNAS, in press
Chemorepellants may play multiple roles in physiological and pathological
processes. However, few endogenous chemorepellants have been
identified, and how they function is unclear. We found that the autocrine
signal AprA, which is produced by growing Dictyostelium discoideum cells
and inhibits their proliferation, also functions as a chemorepellant. Wild-type
cells at the edge of a colony show directed movement outwards from the
colony, whereas cells lacking AprA do not. Cells show directed movement
away from a source of recombinant AprA and dialyzed conditioned media
from wild-type cells, but not dialyzed conditioned media from aprA¯ cells.
The secreted protein CfaD, the G protein Gα8, and the kinase QkgA
are necessary for the chemorepellant activity of AprA as well as its
proliferation-inhibiting activity, whereas the putative transcription factor
BzpN is dispensable for the chemorepellant activity of AprA but necessary
for inhibition of proliferation. Phospholipase C and PI3-kinases 1 and 2,
which are necessary for the activity of at least one other chemorepellant in
Dictyostelium, are not necessary for rAprA chemorepellant activity. Starved
cells are not repelled by rAprA, suggesting that aggregation-phase cells are
not sensitive to the chemorepellant effect. Cell tracking indicates that AprA
affects the directional bias of cell movement, but not cell velocity or the
persistence of cell movement. Together, our data indicate that the
endogenous signal AprA acts as an autocrine chemorepellant for
Dictyostelium cells.
Submitted by Richard Gomer [[log in to unmask]]
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CP55, a novel key component of centrosomal organization in Dictyostelium
Oliver Kuhnert, Otto Baumann, Irene Meyer and Ralph Gräf
University of Potsdam, Dept. of Cell Biology, Potsdam-Golm, Germany
Cell. Mol. Life Sci. in press
Dictyostelium centrosomes consist of a layered core structure surrounded
by a microtubule-nucleating corona. At the G2/M transition the corona
dissociates, and the core structure duplicates yielding two spindle pole
bodies. Finally, in telophase the spindle poles mature into two new,
complete centrosomes. CP55 was identified in a centrosomal proteome
analysis. It is a component of the centrosomal core structure, and persists
at the centrosome throughout the entire cell cycle. FRAP experiments
revealed that during interphase the majority of centrosomal GFP-CP55 is
immobile, which indicates a structural task of CP55 at the centrosome.
The CP55 null mutant is characterized by increased ploidy, a less
structured, slightly enlarged corona, and by supernumerary, cytosolic
MTOCs, containing only corona proteins and lacking a core structure.
Live cell imaging showed that supernumerary MTOCs arise in telophase.
Lack of CP55 also caused premature recruitment of the corona organizer
CP148 to mitotic spindle poles, already in metaphase instead of telophase.
Forces transmitted through astral microtubules may expel prematurely
acquired or loosely attached corona fragments into the cytosol, where they
act as independent MTOCs. CP55null cells were also impaired in growth,
most probably due to difficulties in centrosome splitting during prophase.
Furthermore, although they were still capable of phagocytosis, they
appeared unable to utilize phagocytosed nutrients. This inability may be
attributed to their partially disorganized Golgi apparatus.
Submitted by Ralph Gräf [[log in to unmask]]
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Identification of the kinase that activates a non-metazoan STAT gives
insights into the evolution of phosphotyrosine-SH2 domain signaling
Tsuyoshi Araki1, Takefumi Kawata2 and Jeffrey G. Williams+1
1 College of Life Sciences, Welcome Trust Biocentre, University of Dundee,
Dow St., Dundee, DD1 5EH, UK
2 Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama,
Funabashi, Chiba 274-8510, Japan
PNAS in press
SH2 domains are integral to many animal signaling pathways. By
interacting with specific phosphotyrosine residues, they provide regulatable
protein-protein interaction domains. Dictyostelium is the only non-metazoan
with functionally characterised SH2 domains but the cognate tyrosine
kinases are unknown. There are no orthologues of the animal tyrosine
kinases but there are very many tyrosine kinase-like kinases (TKLs); a group
of kinases which, despite their family name, are mainly classified as
serine-threonine kinases. STATs are transcription factors that dimerise via
phosphotyrosine-SH2 domain interactions. STATc is activated, by
phosphorylation on Tyr922, when cells are exposed to the prestalk inducer
DIF-1: a chlorinated hexaphenone. We show that in a null mutant for Pyk2,
a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc.
Conversely, over-expression of Pyk2 causes constitutive STATc activation.
Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc
both in vitro and in vivo. This demonstration that a TKL directly activates a
STAT has significant implications for understanding the evolutionary origins
of SH2 domain-phosphotyrosine signaling. It also has mechanistic
implications. Our previous work suggested that a predictedly constitutive
STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1
regulated activity of PTP3: a Tyr922 phosphatase. Here we show that the
STATc-Pyk2 complex is formed constitutively, by an interaction between
the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are
generated by autophosphorylation. Also Pyk2 is, as predicted, constitutively
active as a STATc kinase. This observation provides further evidence for
this highly atypical, possibly ancestral, STAT regulation mechanism.
Submitted by Jeff Williams [[log in to unmask]]
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An orthologue of the Myelin-gene Regulatory Transcription Factor
regulates Dictyostelium prestalk differentiation
Hiroshi Senoo, Hong Yu Wang, Tsuyoshi Araki, Jeffrey G. Williams
and Masashi Fukuzawa
Int. J. of Dev. Biol., in press
Background The prestalk region of the Dictyostelium slug is comprised
of an anterior population of pstA cells and a posterior population of pstO
cells. They are distinguished by their ability to utilize different parts of the
promoter of the ecmA gene. Methods We identify, by mutational analysis
and DNA transformation, CA-rich sequence elements within the ecmA
promoter that are essential for pstA-specific expression and sufficient to
direct pstA-specific expression when multimerised. The CA-rich region
was used in affinity chromatography with nuclear extracts and bound
proteins were identified by mass spectrometry. Results The CA-rich
elements purify MrfA, a protein with extensive sequence similarity to
animal Myelin-gene Regulatory Factor (MRF)-like proteins. The MRF-like
proteins and MrfA also display more spatially limited but significant
sequence similarity with the DNA binding domain of the yeast Ndt80
sporulation-specific transcription factor. Furthermore, the ecmA CA- rich
elements show sequence similarity to the core consensus Ndt80 binding
site (the MSE) and point mutation of highly conserved arginine residues
in MrfA, that in Ndt80 make critical contacts with the MSE, ablate binding
of MrfA to its sites within the ecmA promoter. MrfA null strains are delayed
in multicellular development and highly defective in pstA-specific gene
expression. Conclusions These results provide a first insight into the
intracellular signaling pathway that directs pstA differentiation and identify
a non-metazoan orthologue of a family of molecularly uncharacterised
transcription factors.
Submitted by Masashi Fukuzawa [[log in to unmask]]
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[End dictyNews, volume 38, number 14]
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