dictyNews Electronic Edition Volume 38, number 14 May 25, 2012 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= 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 MŸller3, Lynn Tomsho1, Elissa Ward1, Marius Felder4, Cizhong Jiang1,6, Ludwig Eichinger5, Gernot Glšckner2, 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 5Õ ends by poly-T tracts and precisely at their 3Õ 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 [gxc187@psu.edu] -------------------------------------------------------------------------------------- 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 aprAø 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 [rgomer@mail.bio.tamu.edu] -------------------------------------------------------------------------------------- CP55, a novel key component of centrosomal organization in Dictyostelium Oliver Kuhnert, Otto Baumann, Irene Meyer and Ralph GrŠf 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 GrŠf [rgraef@uni-potsdam.de] -------------------------------------------------------------------------------------- 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 [j.g.williams@dundee.ac.uk] -------------------------------------------------------------------------------------- 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 [fukuzawa@cc.hirosaki-u.ac.jp] ============================================================== [End dictyNews, volume 38, number 14]