Location: Avian Disease and Oncology Research2012 Annual Report
1a. Objectives (from AD-416):
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. 3. Compare by qRT-PCR the relative expression of key genes between MD tumors and CD4+ T cells. 4. Isolate RNA from line 6 (MD resistant) and line 7 (MD susceptible) CEF at various time before and after infection with either MDV or MDV lacking Meq, and run on Affymetrix Chicken microarrays.
3. Progress Report:
This project is directly linked to Specific Cooperative Agreements 3635-31320-008-26S and 3635-31320-008-27S titled "Positional Candidate Genes for Resistance to Marek's Disease by Screening for Marek's Disease Virus Meq-Regulated Genes." Genetic resistance to Marek’s disease (MD) is characterized by the lack of tumors or nerve enlargements following exposure to Marek’s disease virus (MDV), a highly-oncogenic alphaherpesvirus. MDV Meq is a transcription factor and the likely MDV oncogene suggesting that one pathway for resistance is the inability of Meq to regulate the transcription of specific genes in individuals resistant to MD, thereby, failing to initiate transformation. Therefore, it is of interest to define DNA-binding sites and the genes that are directly regulated by Meq. Previous work with chromatin immunoprecipitation (ChIP) has shown that Meq binds to specific sites on the MDV genome that are dependent on whether Meq is a homodimer or a heterodimer with c-Jun, another transcription factor and a key regulator for many biological pathways including those involved with cancer. Also, different forms of Meq are expressed and, in general, the full-length form is highly expressed during viral latency and in MD tumors while the Meq-vIL8 variant, which shows no transactivation ability, is expressed at low levels during lytic replication. Recent advancements in ultra-high throughput sequencing and bioinformatics analyses can identify and define the sequences in the chicken genome bound by Meq alone or in combination with c-Jun, the preferential dimerization partner for Meq. The identification of DNA-binding sites combined with DNA chip analyses that profile genes regulated by Meq may reveal positional candidate genes that confer genetic resistance to MD. Furthermore, identification of Meq binding sites may help to explain some of the allele-specific expression (ASE) identified in a related project. Thus far, using chicken cells that do or do not express Meq, ChIP analysis has identified 22,000+ genomic regions that bind Meq. Examining these peaks, there are approximately 3,000 and 2,000 genes within 2 kb of the Meq and c-Jun binding sites, respectively, with about 600 of the peaks in common. Binding of Meq and c-Jun are highly enriched for the promoter regions, especially 500-800 bases upstream of the transcriptional start site. Motif analysis for the Meq-binding sites have confirmed existing motifs (AP-1 and MERE II) as well as identified new ones such as those recognized by CREB, GCN4, Myc, and HAC1. In parallel, the same set of cells was processed on DNA microarrays to reveal 1,829 (FDR=0.01) differentially expressed genes with 1,575 being down-regulated in the presence of Meq, which agrees with the previous conclusion that Meq-Meq homodimers repressed gene expression. Integrating the ChIP seq results that detect chromosomal regions bound by a transcription factor with microarray analysis that examines gene expression differences has revealed 354 and 93 genes that are down- or up-regulated by the directly binding of Meq, respectively. Pathway analysis has suggested genes and molecular mechanisms for MDV-induced transformation.