2013 ABSTRACT BOOK_THE 1ST ANNUAL SYMPOSIUM FOR RESEARCH ORIENTED ELITE PROGRAM

ROEP Symposium 2013THE 1ST ANNUAL SYMPOSIUM FOR RESEARCH ORIENTED ELITE PROGRAMKeynote LecturesMolecular Mechanisms of Chromosome SegregationHongtao Yu* (University of Texas Southwestern Medical Center, United States)Accurate chromosome segregation requires the coordination between the dissolution of sister-chromatid cohesion and the establishment of proper kinetochoremicrotubule attachment. During mitosis, sister-chromatid cohesion at centromeres enables the bi-orientation of and tension across sister kinetochores. We have shown that the complex between shugoshin and protein phosphatase 2A (Sgo1PP2A) localizes to centromeres in mitosis, binds to cohesin in a reaction requiring Cdk-dependent phosphorylation of Sgo1, dephosphorylates cohesin-bound sororin, and protects a centromeric pool of cohesin from mitotic kinases and the cohesin inhibitor Wapl. The centromeric localization of Sgo1 requires histone H2A phosphorylation (pH2A) by the kinase Bub1. Cohesin and pH2A specify two distinct pools of Sgo1PP2A at inner centromeres and kinetochores, respectively. Kinetochore tension triggers Sgo1 dephosphorylation and redistributes Sgo1 from inner centromeres to kinetochores. Cleavage of centromeric cohesin by separase allows sister chromatids connected to microtubules from opposing poles to be evenly partitioned into daughter cells. Incomplete Sgo1 redistribution causes chromosome nondisjunction. Therefore, the Bub1-mediated pH2A mark installs a tension-sensitive, Sgo1-based molecular switch for chromosome segregation.、Keynote LecturesNeural Plasticity: From Synapse to CognitionMu-ming Poo* (Institute of Neuroscience, Chinese Academy of Sciences, China)The cognitive functions of the brain, such as learning and memory, depend on the ability of neural circuits to change their properties of signal processing after these circuits have been used by the organism. Many of these use-dependent changes (“plasticity”) occur at synapses, where signals are transmitted between nerve cells (neurons). Depending on the pattern of neuronal activities, repetitive synaptic transmission could cause long-term potentiation (LTP) or long-term depression (LTD) of the synapse in its efficacy for future transmission. I will summarize our studies on how the timing of neuronal activities (spikes) in the pre- and post-synaptic neurons determines whether a synapse undergoes LTP or LTD, a phenomenon known as “Spike Timing-Dependent Plasticity” (STDP), and how STDP may provide the mechanism for coding and storing the information on the temporal sequence and interval of sensory signals, two key elements of episodic memory. I will also discuss in general the idea that neural plasticity is the main factor that shapes the development of neural circuits, and that neural plasticity offersthe potential for functional recovery from injuries and diseases of the adult brain. Finally, to argue that higher cognitive functions in humans such self-awareness may originate from experience-dependent neural plasticity, I will present our recent findings showing that mirror self-recognition, a cognitive function known to be limited only to humans and great apes, could be acquired by rhesus monkeys following training for visual-somatosensory association.SESSION OVERVIEWSEPTEMBER 218:30-8:40Welcome Addresses8:40-9:20Keynote Lecture Dr. Hongtao Yu9:20-9:55S1: DNA and Histone Structure9:55-10:55Poster Session 1(Odd Numbers)10:55-11:45S2:Cell Biology11:45-12:00Group Photo13:30-14:25S3:Plant Biology14:40-15:35S4: Metabolism and Stress Response15:50-16:30S5:Synthetic Biology16:30-17:30Poster Session 2(Even Numbers)18:30-20:00Informal talk with Dr. Yu and Dr. Poo20:00-21:00Game TimeSEPTEMBER 228:30-9:10Keynote Lecture Dr. Mu-ming Poo9:10-9:20Presentation of Awards 9:20-10:10Concluding Remarks and Free discussionNotes:Shuttle bus heading to the hotel leaves at 7:00 pm, Sep. 20thfrom the gate of the New Life Science Building.Shuttle bus heading back to school leaves at 10:30am, Sep. 22ndfrom the reception of the hotel.PROGRAM SCHEDULESATURDAY, SEPTEMBER 21, 20137:30-8:30Breakfast8:30-8:40Welcome Addresses8:40-9:20Keynote Lecture Dr. Hongtao YuMolecular mechanisms of chromosome segregation9:20-9:55S1: DNA and Histone StructureXiaowei YanHistone chaperone spt16 regulates sir-mediated epigenetic silencing in Saccharomyces cerevisiaeMo ZhaoSingle nucleotide polymorphism related Dnase hypersensitivity site and alteration using CRISPR-Cas9 system9:55-10:55Poster Session 1(Odd Numbers)10:55-11:45S2:Cell BiologyXin GuAna3 is a novel conserved protein required for centriole duplication or integrityAnzhi Yaomir-28* regulation in b cell lymphomasXiangyu ZhangIdentification of downstream effectors of RIPK3-MLKL dependent necrosis by non-natural amino acid photocrosslinker11:45-12:00Group Photo12:00-13:30Lunch13:30-14:25S3:Plant BiologyYan GongNovel analysis system for dynamic root gravitropismAnRan LiA small molecule disturbs root development in Arabidopsis thalianaYao XiaoIdentification of proteins associated with Ein2 the key protein involved in ethylene signaling pathway14:25-14:40Coffee Break14:40-15:35S4: Metabolism and Stress ResponseTian LuMrap2 is not regulated transcriptionally by energy state, but Agrp pathwaySiyang XiaMeasurement of adrenal activity in five captive primate speciesZidong ZhangSpatial distribution dynamics of p53 in response to stress15:35-15:50Coffee Break15:50-16:30S5:Synthetic BiologyWeiyue JiEngineering microbial consortiaPu Zheng, Shuaixing He, Huan Wang, Haoran XueAromatics busted：A comprehensive and quantitative detection for aromatics16:30-17:30Poster Session 2(Even Numbers)17:30-18:30Dinner18:30-20:00Informal talk with Dr. Yu and Dr. Poo20:00-21:00Game TimeSUNDAY, SEPTEMBER 22, 20137:30-8:30Breakfast8:30-9:10Keynote Lecture Dr. Mu-ming PooNeural plasticity: From synapse to cognition9:10-9:20Presentation of Awards9:20-10:10Concluding Remarks and Free discussion10:10-10:30Checking OutStudent Session: DNA and Histone Structure Histone Chaperone Spt16 Regulates Sir-Mediated Epigenetic Silencing inSaccharomyces cerevisiaeXiaowei Yan (严筱溦)* (College of Life Sciences, Peking University, China), Qing Li (College of Life Sciences, Peking University, China)Epigenetic inheritance of the silent chromatin, also termed as heterochromatin, is critical for the maintenance of gene expression patterns and genome integrity. In Saccharomyces cerevisiae, the formation and spreading of the heterochromatin at the HM mating loci are dependent on the stepwise polymerization of four silent information regulator proteins (Sir1p, Sir2p, Sir3p and Sir4p). However, how the Sir proteins are recruited during the assembly of heterochromatin remains largely unknown. Here we show that histone chaperone SPT16 is required for the heterochromatin silencing in the absence of SIR1. We found that mutations in SPT16 severely exacerbate the silencing defects in sir1Dmutant cells. Moreover, the binding of Sir2-4 proteins to HM loci is significantly reduced in spt16 sir1D double mutant cells. Agree with that, the acetylation of lysine 16 on H4 (H4K16Ac) is significantly increased in these regions. Interestingly, mutations in SPT16 in combination with deletions of CAC1 and RTT106, two other histone chaperones, lead to completely loss of silencing at the HM loci. These results reveal a novel role for Spt16 in heterochromatin silencing and demonstrate that histone chaperones, a group of key players in nucleosome assembly, are required for the recruitment of Sir2-4 proteins onto chromatin in the formation of heterochromatin. Single Nucleotide Polymorphism Related Dnase Hypersensitivity Site and Alteration Using CRISPR-Cas9 SystemMo Zhao (赵墨)* (College of Life Sciences, Peking University, China), Jonathan Flint (Nuffield Department of Medicine, University of Oxford, United Kingdom)In eukaryotes genome, there are certain regions that show higher sensitivity to DNase I. These hypersensitive regions are thus called “DNase Hypersensitivity Site”(DHS). DHS are markers of transcriptional activity and cis-regulatory elements including enhancers, insulators and silencers. Genomic sequencing and DNase sequencing data showed that, in 8 inbred strains of Mus musculus, DHS vary between strains. Moreover, a small portion of variable DHS is correlated with single nucleotide polymorphism (SNP) within them. Based on these facts, we proposed that the SNP might be the causal factor of DHS variation. To verify this hypothesis, we plan to use the clustered regularly interspaced palindromic repeats/ CRISPR-associated proteins (CRISPR-Cas9) system to change SNP in mice ES cells, and test if DHS appear/disappear accordingly.My contributions to this project are as the following: Before I joined in, more than 100 candidates of DHS site that correlate with nearby/within SNP were detected by computational method. Using UCSC genome browser and our own sequencing database, I validated and shortlisted these candidates. After being validated, I designed and ordered specific oligos for the CRISPR-Cas9 system to work. I also did the construction and validation of all the plasmids (8 in total). The significance of my work is to provide deeper understanding of transcriptional factor binding and regulation of transcriptional activity. Student Session: Cell BiologyAna3 Is a Novel Conserved Protein Required for Centriole Duplication or IntegrityXin Gu (古欣)* (College of Life Sciences, Peking University, China), David M. Glovers lab (Department of Genetics, University of Cambridge, United Kingdom)Centrioles are microtubule-based structures that are essential for the formation of centrosomes and cilia. Centriole number is tightly regulated and genetic screen has revealed several proteins in worms are essential for centriole duplication. Ana3 is one of the novel proteins recently identified important for centriole duplication, however, its cellular localization and exact function are largely unknown. Ana3 depletion led to a significant reduction of the number of centrioles in cultured Drosophila cells. Similar results were found for testes cells of Ana3 mutant flies, indicating its essential function in centriole duplication or integrity. Live-imaging of the embryos and immune-staining of the testes of GFP-Ana3 transgenic flies revealed Ana3 chimeric protein localized in centrosome during all phases of mitosis and meiosis. Using super-resolution microscopy, the localization of exon1 of Ana3 was found to lie in the “core” region of centrosome, consistent with its essential functions in centriole duplication. Interestingly, the C terminus of Ana3 human homologue, Rotatin, is located out of the “core” region, suggesting distinct functions for different domains. Mass spectral analysis has identified Ana3 existing in a complex with Reduction of Cnn 4(Rcd4) from cultured Drosophila cells. Functional knocking down of Rcd4 and Ana3 both led to reduction of centriole number, albeit Ana3 knocking down has a more dramatic effect, indicating Ana3 might work upstream of Rcd4. mir-28* Regulation in B Cell LymphomasAnzhi Yao (姚安之)* (Yuanpei College, Peking University, China), Catherine Yan (Beth Israel Deaconess Medical Center, and the Harvard Stem Cell Institute, United States)Oncogenic c-Myc expression contributes to many types of cancers, including B-lymphoma. MicroRNAs (miRs) which can down-regulate c-Myc expression may be the potential targets of cancer therapies. miR-28 is expressed in normal human follicular and centroblastic B cell subsets, and is shown to be downregulated in diffuse large B cell lymphoma (DLBCL). Its overexpression in vivo has been shown to delay tumor development yet promote tumor metastasis. Recently, we generated a conditional approach in mice to specifically inactivate c-Myc in nave normal and lymphoma-prone B cells. Preliminarily, using a qPCR based miRNA library screen, we identified miR-28* to be constitutively upregulated in naive B cells deficient for c-Myc, and is conversely down-regulated in c-Myc-activated B cells and B lymphomas, suggestive of a not previously define potential inverse relationship between miR28* and c-Myc expression or action. The focus of my project is to investigate the molecular interplay between miR-28* and c-Myc, which focus on the impact of miR28* on c-Myc expression, and the proliferative survival and migration/metastasis of c-Myc activated B cells and B lymphomas. Identification of Downstream Effectors of RIPK3-MLKL Dependent Necrosis by Non-Natural Amino Acid PhotocrosslinkerXiangyu Zhang(张翔宇)* (College of Life Sciences, Peking University, China), Zhirong Shen (National Institute of Biological Sciences, China), Xiaodong Wang (National Institute of Biological Sciences, China)Necrosis, like apoptosis, can be executed by regulated mechanisms. The receptor-interacting serine-threonine kinase 3(RIP3) and the mixed lineage kinase domain-like protein (MLKL) are two key signaling molecules in the TNF- induced necrosis pathway. In order to investigate how RIPK3/MLKL complex functions to cause downstream excution of necrosis, we apply genetically encoded, highly efficient protein photocrosslinking probe, DiZPK, to profile the in vivo downstream effectors of RIPK3 and MLKL. For RIPK3 crosslink assay, I constructed Hela stable cell line over-expressed with DiZPK-tRNA and DiZPK-tRNA synthase. Transient co-transfection of RIP3 mutants construct together with Pyl-tRS (DiZPK-tRNA and DiZPK-tRNA synthase) shows a gel shift of RIP3 protein, which indicates that there are potential novel interacting proteins were crosslinked with RIP3. For MLKL crosslink assay, I constructed X21 MLKL knockout cell line via CRISPR/Cas 9 system, which could be transfected with MLKL-DiZPK mutants to screen for potential interacting proteins of MLKL. If the subsequent experiment results of Inmunnoprecipitate and Mass spectrometry presented functional candidates, it would provide a much deeper understanding of signaling and executive mechanism of RIP3/MLKL in necrosis. Student Session: Plant BiologyNovel Analysis System for Dynamic Root GravitropismYan Gong (龚龑)* (College of Life Sciences, Peking University, China), Takehiko Ogura (Gregor Mendel Institute, Austria), Wolfgang Busch (Gregor Mendel Institute, Austria)Plant root gravitropism response is essential for water and nutrients absorption. To date, by traditional forward genetics using model plant Arabidopsis thaliana, the outline of the gene regulatory network for gravitropism response has been depicted, but the details are still unclear. We aim to understand the dynamic process of root gravitropismand identify novel components of gravitropism response in roots by combining automatic imaging system and genome wide associate study (GWAS). In cooperation with Dr. Edgar Spalding, we performed high-throughput automatic imaging to analysis root gravitropism response utilizing 232 A. thaliana natural accessions. After gravity reverse, about 40,000 images of single plant were created by a duration of 8 hours at 4 minutes interval. Those images are applied to automated image processing to accurately quantify root tip angle change. These data had been used to conduct GWAS to find genomic regions that were associated with root gravitropism response. We found several genes in proximity of significantly associated regions. Using public databases of large scale transcriptome, we selected highly confident genes showing consistent expression profiles with the known gravitropism responsive genes, including an alpha/beta-hydrolases superfamily protein. We are currently investigating these genes by phenotypic study of mutant lines and natural accessions, expression examination and biochemical characterization.A Small Molecule Disturbs Root Development in Arabidopsis thalianaAnran Li (李安然)* (College of Life Sciences, Peking University, China), Hongwei Guo (College of Life Sciences, Peking University, China)Using a chemical biology screening approach, our lab identified a small chemical molecule, Ket, which can specifically inhibit the ethylene triple response in the root of etiolated seedlings of Arabidopsis thaliana . According to primary result, Ket can effectively promote the root elongation of eto1-2(ethylene overproduction 1-2) which demonstrate a constitutively ethylene triple response in the absence of exogenous ethylene. Next, we will take a deep look into the mechanism of how this small chemical affects the root elongation using both molecular and biochemical methods. Whats more, Ket was reported to be a non-steroidal anti-inflammatory drug (NSAID) in human, which can inhibit jasmonate biosynthesis in plant. So further research need to be done to reveal the role of Ket on ethylene-induced inhibition of root growth and jasmonate biosynthesis.Identification of Proteins Associated with EIN2 The Key Protein Involved in Ethylene Signaling PathwayYao Xiao (肖瑶)* (College of Life Sciences, Peking University, China), Hongwei Guo (College of Life Sciences, Peking University, China)EIN2 (Ethylene-insensitive 2) is an essential transducer in ethylene signaling pathway, bridging the signaling from ER to nucleus to regulate the downstream transcriptional factor EIN3/EIL1.Loss of function of EIN2causes totally insensitive to ethylene treatment while double mutantein3eil1remains partial responses to ethylene, which reminds us to investigate proteins associated with EIN2(E2APs) to expand our knowledge of ethylene signaling transduction. Currently, identification o