Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSThursday, March 14 3:00 PM–6:00 PMFred Farr ForumNucleic Acid-Protein Interactions that Impact Transcription and TranslationCo-chairs: Michael Freitag and Mark CaddickChIP-seq: an inexpensive and powerful method for studying genome-wide chromatin remodeling and transcription regulation in fungi. Koon Ho Wong,Kevin Struhl. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA.Chromatin Immuno-precipitation (ChIP) is a commonly used technique for studying protein-DNA interactions. When coupled with the Next GenerationSequencing (NGS) technology, ChIP-seq can map and measure genome-wide locations and occupancies of any protein-of-interest at very high resolution,and is an invaluable technique for studying chromatin-associated processes including transcription regulation. However, owing to the fact that NGSexperiments are expensive, this powerful technique has yet been widely applied to fungal studies. The output of current sequencing technologies vastlyexceeds the sequencing depth requirement of ChIP-seq experiments as well as many NGS applications in fungi. We have developed a multiplex sequencingmethod that allows up to 96 different samples to be included in a single sequencing reaction, providing a means to obtain whole-genome data at a highlyaffordable cost. Using multiplex sequencing, ChIP-seq and a technique called Anchor-Away for conditional depletion of proteins from the nucleus, we havegained important insights into different aspects of transcription regulation including the repression mechanism of the Cyc8-Tup1 co-repressor complex inSaccharomyces cerevisiae. Examples on how ChIP-seq applications may be broadly applied to address common questions regarding transcriptionregulation will also be presented.Regulatory Networks Governing Global Responses to Changes in Light and Time. Jay C. Dunlap, Jennifer J. Loros, & the P01 Consortium**"FunctionalAnalysis and Systems Biology of Model Filamentous Fungi". coordinated from Dept Gen, Geisel School of Medicine at Dartmouth, Hanover, NH.**including PIs Deb Bell-Pedersen, Michael Freitag, James Galagan, Matthew Sachs, Eric Selker, Jeff Townsend, and members of their labs at institutionsnot listed here. Free-living fungi live in a profoundly rhythmic environment characterized by daily changes in light intensity and temperature. Some fungihave well described systems for anticipation of temporal change, circadian systems, and nearly all fungi can respond acutely to changes in light intensity.The nuts and bolts of the regulatory structures underlying circadian regulation and responses to blue light are well known in Neurospora. The circadianclock comprises a negative feedback loop wherein a heterodimer of proteins, WC-1 and WC-2, acts as a transcription factor (TF) to drive expression of frq.FRQ stably interacts with a putative RNA helicase (FRH) and with casein kinase 1, and the complex down-regulates the White Collar Complex (WCC). Withappropriate phosphorylation mediated delays, this feedback loop oscillates once per day (Baker, Loros, & Dunlap, FEMS Microbiol. Reviews 36: 95-106,2012). In turn, blue light is detected by FAD stably bound by WC-1, eliciting photochemistry that drives a conformational change in the WCC resulting inactivation of gene expression from promoters bound by the WCC (Chen, Dunlap & Loros, FGB 47, 922-9, 2010). With this as context, the consortium teamlisted above is using the tools of next generation sequencing, recombineering and luciferase reporters to see how the initial simple steps of clock controland light perception ramify via regulatory networks to elicit development in response to the cues of light and time. Interestingly, the same players andnetworks appear to be involved in many places. For instance, the circadian feedback loop yields rhythmic activation of WCC that regulates many genesincluding transcription factors (TFs). Genes encoding TFs that do not affect the circadian feedback loop itself provide circadian output. In this manner theseTFs act as second order regulators, transducing regulation from light responses or from the core circadian oscillator, to banks of output clock-controlledgenes (ccgs), some of which are in turn other TFs. Assembling the global regulatory networks governing light and clock regulation is now a feasible goal.Protein Binding Microarrays and high-throughput real-time reporters studies: Building a four-dimensional understanding of transcriptional networks inNeurospora 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 Medical Center,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.Ending messages: alternative polyadenylation in filamentous fungi. Julio Rodriguez-Romero, Ane Sesma. CBGP/ Univ Politécnica de Madrid, Pozuelo deAlarcón, Madrid, Spain.The 3' end polyadenylation of pre-mRNAs is a two-step process. First, pre-mRNAs are cleaved at their 3' end. The second step involves the addition of thepolyA tail by RNA polymerases. Presence of multiple potential 3' end cleavage sites is common in eukaryotic genes, and the selection of the right site isregulated during development and in response to cellular cues. This mechanism of alternative (or non-canonical) polyadenylation generates mRNAisoforms with different exon content or 3' UTR lengths and regulates the presence of cis elements in the mRNA. Proteins involved in alternativepolyadenylation (APA) include Cleavage Factor I in metazoans (CFIm), Hrp1 in yeast and Rbp35 in filamentous fungi. The cis elements present in the 3'UTRs such as miRNA target sites modulate gene expression by affecting cytoplasmic polyadenylation, subcellular localization, stability, translation and/ordecay of the mRNA. Therefore, the selection of a proper 3' end cleavage site represents an important step of regulation of gene expression. Using DirectRNA Sequencing (DRS), we are carrying out in the rice blast fungus Magnaporthe oryzae a comprehensive map of genome wide polyadenylation sites and27th Fungal Genetics Conference | 53