2013 Annual Report
1a.Objectives (from AD-416):
As described in USDA NRI-funded grant, we will:
1. Identify MDV Meq and c-Jun binding sites in the chicken genome.
2. Identify differentially expressed genes following MDV-reactivation in MSB-1 cells.
3. Determine if differentially expressed genes identified in objective 2 also show differentially expression between MD tumors and CD4+ T cells.
4. Identify differentially expressed genes as a function of genetic resistance status or the presence of Meq.
1b.Approach (from AD-416):
1. Utilize chromatin immunoprecipitation (ChIP) using Meq or c-Jun antibodies and determine the DNA sequences via Solexa paired-end reads.
2. Isolate RNA from MSB-1 cells at time 0 and following MDV reactivation, and run on Affymetrix Chicken microarrays.
This project is directly linked to project 3635-31320-009-01R titled "Positional Candidate Genes for Resistance to Marek's Disease by Screening for Marek's Disease Virus Meq-Regulated Genes." Marek’s disease (MD) is an economically significant disease in chickens caused by the highly oncogenic Marek’s disease virus (MDV). Meq, a bZIP transcription factor discovered in the 1990s, is critically involved in viral oncogenicity. Only a few of its host target genes have been described, thus, impeding our understanding of MDV-induced tumorigenesis. Over the course of the project, using ChIP-seq and microarray analysis, a high confidence list of Meq-binding sites in the chicken genome and a global transcriptome of Meq-responsive genes was generated. Meq binding sites were found to be enriched in the promoter regions of up-regulated genes, but not in those of down-regulated genes. ChIP-seq was also performed for c-Jun, a known heterodimeric partner of Meq. Close location of binding sites of Meq and c-Jun was noted, suggesting cooperativity between these two factors in modulating transcription. Pathway analysis indicated that Meq transcriptionally regulates many genes that are part of several signaling pathways which include the ERK/MAPK, Jak-STAT, and ErbB pathways that are critical for oncogenesis and/or include signaling mediators involved in apoptosis. Meq activates oncogenic signaling cascades by transcriptionally activating major kinases in the ERK/MAPK pathway and simultaneously repressing phosphatases, as verified using inhibitors of MEK and ERK1/2 in a cell proliferation assay. This study provides significant insights into the mechanistic basis of Meq-dependent cell transformation.