FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

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Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 30histone PTMs in offspring can be affect by the maternal diet, mammary tissue from 130 days oldCO-CO or CA-CO groups F1 mice was isolated. The biological location of the two PTMs is to beinvestigated by Chromatin Immunoprecipitation-based Sequencing (ChIP-Seq) leading to thegeneration of epigenomic maps corresponding to the different diets. ChIP DNA was prepared asdescribed by Rada-Iglesias and colleagues (2005) using a MeCP2 ChIP compatible antibody(Abcam ab2828). The DNA fragment library was to be prepared from separate biological samplesusing the ABI SOLID System 2.0 Fragment Library Preparation protocol, and then sequenced bythe Marshall University Genomics Core Facility using the Illumina HiSeq1000 system. Oncecompleted, this should allow us to identify genes that are differentially regulated by changes inH3K18ac and H3K4me2 signal following maternal consumption of the omega-3 rich diet comparedto maternal consumption of the omega-6 rich diet.Results and Discussion: The ChIP has been performed and libraries have been prepared andsequenced in the Illumina HiSeq1000 system. We are now in the process of analyzing the resultsfrom the deep sequencing runs.NGS Project #3Title: Identification of genomic binding sites of transcriptional repressors Snail and ZEB in epithelialcells undergoing epithelial-mesenchymal transition (EMT) by ChIP-Seq.Subproject investigator: Alexey Ivanov, PhDIntroduction: Epithelial-mesenchymal transition (EMT), the generation of motile mesenchymal-likecells from epithelia, has been increasingly recognized as a major contributor to the metastaticprocess in breast as well as in many other human epithelial cancers. EMT has been associated withcancer cell acquisition of stem cell properties, increased invasion and resistance tochemotherapeutic drugs. Key inducers of EMT are developmentally regulated transcription factorsSnail and ZEB. Snail and ZEB are over-expressed in breast adenocarcinomas, and their increasedexpression is an independent prognostic factor for metastasis, poor survival and disease relapse inpatients with breast carcinoma. At the molecular level, Snail and ZEB function to repress genetranscription by binding to E-box DNA element. The role of Snail and ZEB in triggering EMT is wellestablished. However, the molecular mechanism of their action in this process is not fullyunderstood. It is unknown, for example how Snail or ZEB, being repressors, activate hundreds ofgenes. We have previously characterized gene expression changes in human mammary epithelialcells undergoing Snail/ZEB-induced EMT using microarrays and identified more than threethousand Snail/ZEB-regulated genes.Hypothesis: Most genes down-regulated by Snail/ZEB are likely directly bound and repressed bySnail/ZEB; while genes up-regulated by Snail/ZEB are likely indirect Snail/ZEB targets activatedthrough secondary transcriptional and post-transcriptional events.Specific Aim: We propose to identify Snail and ZEB genomic binding sites using ChIP-Seq in thesame cell culture model where we have analyzed Snail/ZEB-induced transcriptome changes andcorrelate Snail/ZEB promoter occupancy with the identified target genes.Methods: We utilized the same system of tet-inducible expression of Snail1, Snail2 or ZEB1 inHMLE cells, which we have previously used for gene expression profiling. Two ChIP protocols,from Dr. Peggy Farnham’s lab and from Dr. Richard Myer’s lab were compared in assayperformance. Both labs are members of the ENCODE consortium. Next generation sequencing willbe performed at the Genomics Facility of University of Southern California.Results and Discussion: It is of crucial importance to have good, ChIP-validated antibody toPHS 2590 (Rev. 06/09)Continuation Format Page
Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 31perform ChIP-Seq experiments. First, we evaluated the efficiency of several commercially availableantibodies to Snail1 (4 antibodies), Snail2 (2 antibodies) and ZEB1 (5 antibodies) to ChIP positivecontrol E-Cadherin promoter, the best characterized direct target of Snail and ZEB. One antibody forSnail1 and one for ZEB1 have previously been published to work in ChIP. The published Snail1antibody performed poorly and the published ZEB1 antibody performed satisfactory in our antibodyChIP validation. In fact, we found that some other antibodies from our screen perform good (one forSnail1 and one for Snail2) or better (one for ZEB1) compared to the published ones, and will beused in further experiments. Second, we compared the two ChIP protocols and optimized Farnham’slab protocol for our cell line. Currently, we are conducting our large scale ChIP with ZEB1 antibody,and resulting immune precipitated DNA will be sent to the USC Genomics Core for NGS.NGS Project #4Title: Identification of Potential Susceptibility Variants for Obesity and Non-Insulin DependentDiabetes in the TALLYHO MouseIntroduction: The TALLYHO/Jng (TH) mouse is an inbred polygenic model for Type 2 diabetescharacterized by obesity, insulin resistance, hyperlipidemia, and hyperglycemia. Genetic outcrossexperiments with lean non-diabetic strain of C57BL/6 (B6) mice revealed major susceptibility locifor obesity on chromosome 6 (Tabw2) and for diabetes on chromosome 4 (Tanidd4). For both loci,the TH alleles are associated with disease susceptibilities. Currently, the Tabw2 locus has beenfine mapped to 8-Mb interval and the Tanidd4 locus to 15.8-Mb interval.Methods: In order to identify potential susceptibility variants for Tabw2 and Tanidd4 loci, weperformed whole genome sequencing of the TH mouse genome in two 2 x 100 paired end readstrategies using an Illumina HiSeq1000 next generation sequencer. Libraries were prepared andsequenced using standard Illumina protocols.Results and Discussion: We generated ~60 Gb of TH sequence with an average coverage of~25X. Seventy five percent of the readout aligned to the mouse reference B6 genome (Build 37). Inour initial test, we compared the TH genome to the B6 genome using Illumina CASAVA softwareand identified 4,749,554 SNPs. Among the SNPS, 38,147 mapped to the Tabw2 interval and 83,122to the Tanidd4 interval. In order to refine these sets, we compared the TH, which contains Swisslineage, variant set to those variants in three Swiss-derived non-obese, non-diabetic strains (A/J,AKR/J and Balb/cJ). We identified 17,360 SNPs and 28,297 SNPS within the Tabw2 and Tanidd4intervals, respectively, that were not found in any of the three non-diabetic strains.In conclusion, we applied a whole-genome sequencing strategy in combination with mappinginformation to identify a set of candidate genes for the obese and diabetic phenotypes in TH mice.Further defined and systemic filtration of the variants (e.g. based on variant effect of function) willfacilitate identification of the causal genes and variants.NGS Project #5Title: Characterizing the AHR cistrome in human MCF-7 breast cancer cellsSubproject investigator: Travis Salisbury, PhDa) Project summary & Hypothesis:The Aryl Hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediatestranscriptional responses to exogenous and endogenous chemicals. Ligand-activated AHRinduces toxicity, transcription of phase I and phase II drug metabolizing enzymes and changes incellular growth. The AHR is likely to control cellular responses by regulating the expression ofgenes. Obesity is a risk factor for several types of cancer. Prior reports and our data indicate thatadipocytes secrete signaling molecules that induce breast cancer cells to grow rapidly. We havediscovered that blockade of AHR activity in breast cancer cells inhibits cancer cell responsivenessPHS 2590 (Rev. 06/09)Continuation Format Page
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