Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSexpression. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3. In these cases absenceof H3K27me3 is insufficient for expression, which suggests a requirement for additional factors for gene expression. Taken together, we show that absenceof H3K27me3 allows expression of 14% of all annotated genes, resulting in derepression of predominantly secondary metabolite pathways and otherspecies-specific functions, including potentially secreted pathogenicity factors. This study provides the framework for novel targeted strategies to controlthe “cryptic genome” and specifically secondary metabolite expression.386. Circadian clock-gated cell division cycles in Neurospora crassa. C. Hong 1 , J. Zamborszky 1 , M. Baek 1 , K. Ju 1 , H. Lee 1 , L. Larrondo 2 , A. Goity 2 , A. Csikasz-Nagy 3 . 1) Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH; 2) Departamento de Genética Molecular y Microbiología, Facultad deCiencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile; 3) Randall Division of Cell and Molecular Biophysics, andInstitute for Mathematical and Molecular Biomedicine, King’s College London, London, SE1 UL, UK.Asynchronous nuclear divisions are readily observed in filamentous fungi such as Ashbya gossypii and Neurospora crassa. Our computational simulations,however, predict synchronous circadian clock-gated mitotic divisions if the division cycles of such multinucleated organisms are coupled with circadianrhythms. Based on this hypothesis, we investigate the coupling between the cell cycle and the circadian clock in Neurospora crassa. First, we show WC-1-dependent light-induced expression of stk-29 mRNA (homolog of wee1), which suggests that there exists a conserved coupling between the clock and thecell cycle via STK-29 in Neurospora as in mammals. Second, we demonstrate that G1 and G2 cyclins, CLN-1 and CLB-1, respectively, show circadianoscillations with luciferase bioluminescence reporters. Moreover, clb-1 and stk-29 gene expressions show circadian clock-dependent light-induced phaseshifts, which may alter the timing of divisions. Third, we show circadian clock-dependent synchronized nuclear divisions by tracking nuclear morphologywith histone hH1-GFP reporter. Synchronized divisions occur late in the evening, and they are abolished in the absence of circadian rhythms (frq KO ). Ourfindings demonstrate the importance of circadian rhythms for synchronized mitotic cycles and establish Neurospora crassa as an ideal model system toinvestigate mechanisms that couple the cell cycle and the circadian clock.387. Protein Binding Microarrays and high-throughput real-time reporters studies: Building a four-dimensional understanding of transcriptionalnetworks in Neurospora crassa. A. Montenegro-Montero 1 , A. Goity 1 , C. Olivares-Yañez 1 , A. Stevens-Lagos 1 , M. Weirauch 2 , A. Yang 3 , T. Hughes 3 , L. F.Larrondo 1 . 1) Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, Chile; 2) CAGE, Cincinnati Children`’s Hospital MedicalCenter, University of Cincinnati. U.S.A; 3) Banting and Best Department of Medical Research, University of Toronto, Canada.It has been suggested that ~20% of the Neurospora-transcriptome may be under circadian control. Nevertheless, there is scarce information regardingthe regulators that are involved in the rhythmic expression of clock-controlled genes (ccgs). We are using a high-throughput platform, based on variouscodon-optimized luciferase transcriptional- and translational-reporters, to monitor time-of-day-specific gene expression and to identify key elementsmediating circadian transcriptional control. Thus, we have identified transcription factors -such as SUB-1- that affect the expression of known and novelccgs, among which there are transcriptional regulators that give access to a group of third-tier ccgs. In addition, we are characterizing several rhythmicbZIP-coding genes as potential nodes of circadian regulation. In order to characterize regulatory networks in which these and all Neurospora transcriptionfactors participate, we are using double-stranded DNA microarrays containing all possible 10-base pair sequences to examine their binding specificities andin that way, predict possible targets on a genome-wide manner. Currently, these Protein Binding Microarray studies have provided DNA-bindingspecificities for over 120 Neurospora transcription factors granting an unprecedented and powerful tool for transcriptional network studies. Finally, wehave generated graphic tools to explore the spatial differences observed in the temporal control of gene expression. Funding: Conicyt/Fondecyt/regular1090513.388. Glycogen metabolism is regulated by the circadian clock in Neurospora crassa. S. Virgilio, T. Candido, M. C. Bertolini. Instituto de Química, UNESP,Araraquara, São Paulo, Brazil.The fungus Neurospora crassa has been widely used in studies of circadian rhythms and photobiology. Our research group has been using this modelorganism to study the molecular mechanisms involved in glycogen metabolism regulation and recent findings have revealed that circadian rhythms controla variety of physiological and metabolic functions in different organisms. In a screen of a mutant strains set we identified a number of transcriptionfactors/cofactors likely acting as regulators of glycogen metabolism. Among them, several transcription factors were previously described as controlled byregulators of the circadian clock in N. crassa. The result led us to start to investigate whether glycogen metabolism is under control of circadian clock in N.crassa. Experiments were performed to verify whether glycogen was rhythmically accumulated in a wild-type strain. In circadian clock experiments, theglycogen content varied according to the circadian rhythm, with cyclical periods ranging from 22 to 24 h. The glycogen synthase activity (GSN) wasquantified in the presence and absence of the allosteric activator glucose-6-phosphate (G6P). The -/+ G6P ratio is considered as an index ofphosphorylation, higher levels correlating with lower phosphorylation. The GSN phosphorylation was influenced by the biological clock, showing changesin the GSN phosphorylation status along the experiment. The expression of the gsn and gpn (encoding glycogen phosphorylase) genes was evaluated inthe same experiments and in light-induced experiments as well. In circadian clock analysis, the gsn and gpn transcripts showed rhythmic expressionalthough not as pronounced as the levels of the ccg-1 transcript (positive control). In light-induced experiments, the levels of glycogen were kept constantduring different times after exposure to light, however the expression of the gsn and gpn genes showed to be delayed light-induced. The results suggesteda connection between the energy derived from the glycogen metabolism and the circadian clock in N. crassa. Supported by FAPESP and CNPq.389. Genetic and Molecular Dissection of the Neurospora Circadian Oscillatory System. Qijun Xiang 1 , Bin Wang 1 , Chandru Mallappa 1 , Jennifer Hurley 1 ,Arko Dasgupta 1 , Jennifer Loros 2 , Jay Dunlap 1 . 1) Department of Genetics, Dartmouth Medical School, Hanover, NH03755; 2) partment of Biochemistry,Dartmouth Medical School, Hanover, NH03755.Transcription/ translation feedback loops are central to all eukaryotic circadian clocks. In the circadian oscillator, the negative feedback loop drivesperiodic expression of proteins that feed back to reduce their own expression. A heterodimer of proteins, WC-1 and WC-2, acts as a transcription factor todrive expression of the frq gene. Its product FRQ dimerizes and forms a complex with another protein FRH. This complex inhibits the activity of the WCheterodimer creating the negative feedback loop. While canonical clock proteins such as FRQ work exclusively in timing, all systems utilize additionalproteins performing other functions in the cell. Among these in Neurospora is the essential putative RNA helicase, FRH. A novel, unbiased genetic screenfor circadian negative feedback mutants uncovered a point mutation that completely complements the essential functions of FRH yet is totally arrhythmic,thus genetically separating essential functions from clock-associated roles. In other experiments we used mass spectrometry to look for interactors of FRH,FRQ, and to follow posttranslational modifications of these proteins over the day. Although few modifications are found on FRH, FRQ is extensivelymodified with nearly 100 phosphorylations. By examining the phenotypes of strains bearing mutants that have lost these sites individually and in groups,we begin to see how temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability. For over 60% of the27th Fungal Genetics Conference | 215